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Energy and Climate

Temperature Controls eDNA Persistence across Physicochemical Conditions in Seawater
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  • Luke J. McCartin
    Luke J. McCartin
    Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015-3027, United States
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  • Samuel A. Vohsen
    Samuel A. Vohsen
    Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015-3027, United States
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  • Susan W. Ambrose
    Susan W. Ambrose
    Department of Earth and Environmental Sciences, Lehigh University, Bethlehem, Pennsylvania 18015-3027, United States
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  • Michael Layden
    Michael Layden
    Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015-3027, United States
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  • Catherine S. McFadden
    Catherine S. McFadden
    Department of Biology, Harvey Mudd College, Claremont, California 91711, United States
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  • Erik E. Cordes
    Erik E. Cordes
    Department of Biology, Temple University, Philadelphia, Pennsylvania 19122-6008, United States
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  • Jill M. McDermott
    Jill M. McDermott
    Department of Earth and Environmental Sciences, Lehigh University, Bethlehem, Pennsylvania 18015-3027, United States
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  • Santiago Herrera*
    Santiago Herrera
    Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015-3027, United States
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0001-7204-9434
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Environmental Science & Technology

Cite this: Environ. Sci. Technol. 2022, 56, 12, 8629–8639
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https://doi.org/10.1021/acs.est.2c01672
Published June 3, 2022

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Abstract

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Environmental DNA (eDNA) quantification and sequencing are emerging techniques for assessing biodiversity in marine ecosystems. Environmental DNA can be transported by ocean currents and may remain at detectable concentrations far from its source depending on how long it persist. Thus, predicting the persistence time of eDNA is crucial to defining the spatial context of the information derived from it. To investigate the physicochemical controls of eDNA persistence, we performed degradation experiments at temperature, pH, and oxygen conditions relevant to the open ocean and the deep sea. The eDNA degradation process was best explained by a model with two phases with different decay rate constants. During the initial phase, eDNA degraded rapidly, and the rate was independent of physicochemical factors. During the second phase, eDNA degraded slowly, and the rate was strongly controlled by temperature, weakly controlled by pH, and not controlled by dissolved oxygen concentration. We demonstrate that marine eDNA can persist at quantifiable concentrations for over 2 weeks at low temperatures (≤10 °C) but for a week or less at ≥20 °C. The relationship between temperature and eDNA persistence is independent of the source species. We propose a general temperature-dependent model to predict the maximum persistence time of eDNA detectable through single-species eDNA quantification methods.

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Copyright © 2022 The Authors. Published by American Chemical Society

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Synopsis

Organisms shed trace amounts of DNA into the environment (eDNA). Quantification and sequencing of eDNA can inform the distribution and diversity of biological communities. Estimating the temporal persistence of eDNA is fundamental to constraining its source. This study (1) finds that temperature strongly controls the maximum persistence time of eDNA in the ocean and (2) proposes a model to estimate the maximum persistence time of eDNA as it relates to seawater temperature.

Introduction

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Anthropogenic impacts are causing species extinctions and population declines globally. (1−3) Molecular biology methods accelerate the pace at which vulnerable biological communities are characterized to inform conservation and ecosystem restoration efforts. (4,5) Environmental DNA (eDNA) sequencing and quantification complement conventional methods for assessing biodiversity and the distribution of invasive and ecologically important species in marine habitats. (6−10) However, the utility of eDNA methods hinges on understanding the factors that control the distribution and abundance of eDNA across spatiotemporal scales. (11)
Aqueous eDNA is DNA that is dissolved in solution or associated with larger suspended particles, such as cells, organelles, or aggregates. (12) Animal eDNA may be shed into the environment through several processes, and the shedding rate depends on factors like biomass, metabolic rate, ontogeny, and activity. (13−19) Once shed from an animal, eDNA changes from intracellular to subcellular states. (20−22) eDNA degrades at rates influenced by physicochemical and biotic factors, and the relative influence of these factors may depend on its state. (12,22−25) Before completely degrading into short fragments that molecular methods cannot detect, marine eDNA can be transported away from its source by ocean currents. (25,26) Determining how the persistence of eDNA is controlled by physicochemical conditions is vital to constraining when and where it was shed from a source organism. This knowledge is essential to define the spatiotemporal resolution of the ecological information derived from eDNA quantification and sequencing.
Experimental studies using freshwater and marine animals have established that eDNA degrades beyond the detection limit of PCR-based methods over timescales spanning hours to weeks. These studies have indicated that temperature, pH, and dissolved oxygen concentration may influence the degradation rate and, thus, the persistence of environmental DNA. Temperature controls the degradation rate of eDNA shed from diverse organisms. (17,21,24,27−33) Higher temperatures increase the kinetics of many processes responsible for DNA degradation, including cell and organelle lysis, hydrolysis and oxidation of DNA molecules, and breakdown by extracellular enzymes. The stability of the DNA molecules is affected by pH. (34−36) Consistently, experiments have indicated that the rate of eDNA degradation is also affected by pH. (27,29,37,38) However, these experimental inquiries have been limited to freshwater systems where pH ranges widely, from 5.0 to 9.0. No study has experimentally tested the effect of pH on eDNA degradation in seawater, which has a narrower range between 7.5 and 8.3.
Dissolved oxygen concentration (hereafter abbreviated as [DO]) is another potentially important physicochemical parameter that has received comparatively little attention. Dissolved oxygen concentration is biologically relevant due to its importance to microbial metabolism. Oxygen is also chemically relevant due to the susceptibility of DNA molecules to oxidation. Only one study has experimentally manipulated [DO] to determine its effect on eDNA decay in seawater. (23) That study found that the degradation rate of eDNA was slower at a dissolved oxygen saturation of 20% versus 55%. To fully determine the effect of [DO] on eDNA degradation, it is necessary to perform experiments across the range of oxygenation states in marine ecosystems, from fully oxygenated to nearly anoxic.
Temperature, pH, and [DO] vary considerably with location and depth in the open ocean. Temperature decreases from ∼30 °C in the tropical surface ocean to near freezing in the deep sea, and pH typically changes with depth from approximately 8.3 to 7.5. (39) Seawater is usually nearly saturated with oxygen in the surface ocean. Oxygen saturation decreases to a minimum at mesopelagic depths and increases with higher solubility at lower temperatures in the deep sea. (40) Considering the potentially interactive effects of these abiotic factors on the persistence of marine eDNA is necessary when applying eDNA methodologies to study ocean biodiversity.
Here, we test the relative effects of temperature, pH, and [DO] on the degradation rate of eDNA across conditions that reflect the subtropical open ocean, spanning near-surface waters to the deep sea. We developed an experimental setup that allows tight control of physicochemical conditions and produces reproducible results. We used the deep-sea coral Desmophyllum pertusum (Linnaeus 1758) as the source of eDNA. D. pertusum is hereafter referred to as Lophelia for consistency with abundant literature that uses its synonymized name Lophelia pertusa. (41) Lophelia is an ecologically important reef-forming coral with a cosmopolitan distribution in cold and deep waters of the Atlantic and Pacific Oceans. (42,43) We hypothesized that temperature, pH, and [DO] would have interactive effects controlling the degradation rate of eDNA.

Materials and Methods

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Experimental Conditions

To test our hypothesis, we conducted eDNA degradation experiments under 11 fixed combinations of temperature (set to either 4, 10, or 20 °C), [DO] (set to either nearly anoxic, 0.1 mg/L; half saturated, 3.6 mg/L; or fully saturated, 7.2 mg/L), and pH (set to either 7.6, 7.9, or 8.2). These discrete sets of physicochemical conditions were chosen to reflect the range of conditions found in the subtropical open ocean, spanning from near-surface waters to the deep sea. Each set of conditions was investigated in duplicate experiments (22 experiments in total) to assess the reproducibility in estimating the eDNA degradation rate. We maintained these conditions at target values throughout the experiments with accuracy (Figure 1).

Figure 1

Figure 1. (A) Matrix of target conditions for 11 combinations of temperature, pH, and [DO] investigated to determine the persistence of eDNA from the coral Lophelia among a range of marine physicochemical states. Two replicate experiments were conducted for each combination of temperature, pH, and [DO]. At 20 °C temperature, half- and fully saturated oxygen experiments (shaded light blue) represent conditions characteristic of subtropical near-surface environments. At 4 °C temperature, half-saturated experiments (shaded dark blue) represent conditions characteristic of the deep-sea environment. The remaining cells (shaded blue) represent other conditions characteristic of the global open and deep ocean. (B) Schematic example of experimental conditions. Compressed air and nitrogen gas flow rates were adjusted to reach the target [DO]. Small doses of 0.5 M HCl were automatically administered to the tanks when target pH values were exceeded. Physicochemical measurements were monitored by suspending probes in the tanks. Caps were secured with O-rings to control the oxygen concentration in the above headspace. All tubing and probes were placed through small openings in these caps. A photograph of the experimental setup is presented in Figure S1. (C) Measurements of temperature, pH, and [DO] over the 22 eDNA degradation experiments. Points indicate individual measurements, and paths are drawn through daily averages. All measurements were recorded daily and at each sampling time point. Temperature measurements were made using a laser thermometer. pH and [DO] measurements were made with probes.

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Experimental Setup

Experiments were conducted in a controlled-temperature room (KE2 Therm Solutions Inc., Washington, MO, USA) set to the target temperature. The ambient temperature room was monitored using a HOBO data logger (Onset Computer Corporation, Bourne, MA, USA). The temperature of each tank was also measured daily and at each sampling time point using an Etekcity infrared laser thermometer (Vesync Co., Anaheim, CA, USA).
Experiments were conducted in opaque polypropylene carboys in the controlled-temperature room with no windows, and artificial fluorescent lights were on only during sampling. These conditions removed the potential effect of UV light on the eDNA degradation rate in the experiments. Natural levels of UV radiation from sunlight have not been found to affect marine eDNA degradation significantly. (44) Therefore, eliminating UV light should have a negligible effect on interpreting results from experiments that may reflect conditions in the surface ocean (high-pH and oxygenated), where UV radiation penetrates. (45)
We continuously monitored the pH using a BlueLab pH Controller Connect System (BlueLab, New Zealand) with a single-junction pH electrode (Oakton, Vernon Hills, IL, USA) and an RTD temperature probe (BlueLab) for automatic temperature compensation. Target pH values were maintained by automated dosing with small volumes of 0.5 M hydrochloric acid whenever the controller pH reading exceeded 0.1 pH unit over the target value (approximately hourly). We manually added small volumes (<5 mL) of 0.5 M sodium hydroxide if the pH reading dropped below 0.1 pH unit of the target value. Duplicate, small-volume samples (∼50 mL) were removed at each sampling time point to obtain independent pH measurements using an Orion PerpHect Log R meter (Thermo Fisher Scientific, Waltham, MA, USA) with a single-junction pH electrode (Oakton). The pH monitoring systems were calibrated at the start of each experiment using a three-point calibration with pH 4.01, 7.00, and 10.01 solutions (Oakton). The pH meter used for daily sampling measurements was calibrated before each measurement using two-point calibration with pH 7.00 and 10.01 solutions (Oakton). pH measurements were temperature-compensated manually by setting the monitor to the ambient temperature of the room.
We controlled the [DO] by manipulating the flow rates of compressed air and high-purity nitrogen gas. Compressed air and nitrogen flow rates were set with Aalborg GFC Mass Flow Controllers (Aalborg, Orangeburg, NY, USA). Before each experiment, gas flow rates were independently verified using a soap film bubble flow meter. Gasses were delivered to each experimental tank using independent gas lines capped with air stones placed at the bottom (Figure 1). Dissolved oxygen concentrations and oxidative reductive potential (ORP) were monitored within the experimental tanks and recorded daily and at each sampling time point with Pinpoint II Dissolved Oxygen and Pinpoint ORP monitors, respectively (American Marine, Ridgefield, CT, USA). Dissolved oxygen concentration and ORP monitors were calibrated at the start of each experiment, following the manufacturer’s instructions. The [DO] monitors were calibrated to atmospheric oxygen concentration. The ORP monitors were calibrated using the manufacturer-supplied 400 ORP AgCl reference standard (American Marine).

eDNA Degradation Experiments

For each experiment, 42 L of artificial seawater at 35 PSU salinity were prepared in a polypropylene carboy (Foxx Life Sciences, Salem, NH, USA) using the B-Ionic Artificial Seawater (ASW) System (ESV Aquarium Products, Hicksville, NY, USA). Seawater was prepared with ultrapure deionized water using a Milli-Q system with an attached LC-Pak Polisher (Millipore Sigma, Burlington, MA, USA). The LC-Pak Polisher removes trace organics from water. We chose to make ASW from Milli-Q water to restrict organic material sources to the B-Ionic ASW components and our eDNA source.
We prepared a tissue homogenate from freshly frozen Lophelia fragments as the source of eDNA. Fragments approximately 10 cm in length with 8–10 polyps were collected at two sites at ∼700 and ∼760 m depth at the Richardson Reef Complex off the coast of South Carolina, USA (31.98°N, 77.41°W and 31.88°N, 77.37°W, respectively). Collection details are presented in the Supplementary Methods. Lophelia tissue was removed from the skeleton with a strong stream of ASW using a water jet dental flosser. The resulting mixture of tissue and ASW was collected in a sterile Whirl-Pak. This mixture was homogenized using a FisherBrand 150 rotor–stator homogenizer with a 5 mm fine-sawtooth probe (Thermo Fisher Scientific, Waltham, MA, USA). The homogenate was aliquoted into 15 mL sterile centrifuge tubes (Celltreat, Pepperell, MA, USA), frozen, and stored at −80 °C. One aliquot was thawed on ice immediately before each experiment. Details about the preparation and choice of source eDNA are presented in the Supplementary Methods.
Between 12 and 15 mL of tissue homogenate was added to each tank at the beginning of each experiment. The concentration was adjusted based on the measured concentration of coral eDNA in each preparation to keep starting concentrations consistent among experiments (see Supplementary Methods). From this point onwards, seawater from each experiment was serially sampled at increasing time intervals to quantify the concentration of coral eDNA over time. The first sample was taken between 30 and 45 min after adding the homogenate to allow mixing. After conducting pilot experiments, we determined that sampling over 8 days was sufficient for experiments at 20 °C since the limit of quantification of our qPCR assay was reached in less than a week. However, this timeframe was insufficient to capture the entire degradation process at lower temperatures. Thus, sampling for experiments at 10 and 4 °C was conducted less frequently over the first 2 days and extended to 17 days. Sampling time points for each experiment are shown in Figure 2. We sampled coral eDNA by filtering approximately 1 L of water (mean = 1008 mL; SD = 74 mL; range = 404 to 1145 mL) through a 0.22 μm pore-size polyethersulfone Sterivex filter (Millipore-Sigma). Water was pumped through the filter using an L/S peristaltic pump, Easy-Load II pump heads, and L/S 15 high-performance precision tubing (Masterflex, Vernon Hills, IL, USA). The pump was set to a rate of 100 RPM. The tubing was primed with tank water before a filter was attached. The effluent from each filtered sample was weighed using a balance to adjust measured concentrations of eDNA by mass (in g) of water filtered. Any water remaining in the tubing was pumped back into the experimental tanks once filtration was complete and the filter was removed. Following filtration, Sterivex filters were stored in sterile Whirl-Pak bags and frozen at −80 °C immediately after eDNA filtration until DNA extraction, which occurred within 1 month.

Figure 2

Figure 2. Degradation of Lophelia eDNA in 22 experiments among a range of marine physicochemical states. eDNA concentration was measured as the concentration of a 154 base pair fragment of the Lophelia mitochondrial COI gene. The concentration of eDNA (y-axis) is plotted against time (x-axis). The y-axis is natural-log scaled. Different colors represent the two experimental replicates at each experimental condition. Points represent the average of three qPCR replicate measurements for two samples at a given time point. Lines represent the fit to a biphasic model. Thin lines connecting points to the line of best fit represent the distance from the observed to the fitted values at each time point (the residuals). Panels are arranged by temperature (descending top to bottom) and [DO] (increasing left to right). Values shown on top of each panel indicate the target experimental conditions. Points below the limit of quantification of the qPCR assay (77.8 copies/reaction or 13 copies/mL seawater filtered) are not plotted. The dashed line indicates the limit of quantification.

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DNA Extraction and Quantitative PCR

DNA was extracted from each filter using a Qiagen DNEasy Blood & Tissue Kit (Hilden, Germany) following methods initially developed by Spens et al. with modifications described in Govindarajan et al. (46,47) The DNA was eluted in two separate volumes of 50 μL of AE buffer for a total volume of 100 μL per extraction. The total concentration of DNA in each extract was quantified using the Qubit 1X High-Sensitivity Double-Stranded DNA Assay (Thermo Fisher Scientific, Waltham, MA, USA). The average DNA concentration was 3.40 ng/μL, and the range was from <0.1 (too low to quantify) to 52.0 ng/μL. After this assay, the DNA was stored frozen at −20 °C.
We developed a new qPCR assay to amplify and quantify a 154 base pair fragment of the Lophelia mitochondrial COI gene (Lop-COI) using primers that match the COI gene of caryophylliid corals. The forward primer sequence is 5′-CTGGGGGACGATCATCTTTA-3′, and the reverse primer sequence is 5′-TGTTTAATCGGGGGAAAGC-3′. Primers were synthesized by Eurofins Genomics (Louisville, KY, USA), purified by standard desalting, and normalized at 100 μM in TE buffer (pH 8.0). Quantitative PCR reactions were performed in 20 μL volumes using the Quantabio PerfecTa SYBR Green Fastmix (Beverly, MA, USA) and a Qiagen RotorGene 6000 Thermal Cycler with either the 72-well or 36-well ring. We used clear Qiagen 0.1 mL four-strip tubes (for a 72-well ring) or 0.2 mL single tubes (for a 36-well ring) designed for the RotorGene. DNA extracts were diluted 10-fold in Buffer AE (Qiagen, Germany), and 6 μL of this dilution was used in each reaction (effectively 0.6 μL of DNA template). Primer stocks in TE buffer were diluted to 10 μM using molecular-grade water, and 6 pmol of both the forward and reverse primers were added to each reaction for a final concentration of 300 nM for each primer. We used molecular-grade water to complete the 20 μL volume of each reaction. Cycling conditions were as follows: initial denaturation at 95 °C for 10 min, then 35 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, and elongation at 72 °C for 20 s. Amplification was followed by melting curve analysis and conducted with pre-melt conditioning at 72 °C for 90 s followed by a ramp from 72 to 95 °C with holds of 5 s per 0.5 °C increase in temperature. The details regarding primer design and qPCR assay optimization are presented in the Supplementary Methods. The qPCR assay’s quantification (LOQ) and detection limits (LOD) were calculated and visualized using code adapted from Klymus et al. (48) (see the Supplementary Methods for further details). The LOQ was determined to be 77.8 copies per reaction, equivalent to 13 copies/mL of seawater filtered using our sampling and eDNA extraction methods. The LOD with three qPCR replicates is 3.4 copies per reaction or 0.6 copies/mL of seawater filtered.
Each qPCR run consisted of 19 samples in triplicate, 4 10-fold dilutions of purified plasmids containing the target fragment (778,000 to 778 copies per reaction or 130,000 to 130 copies/mL seawater filtered) in triplicate, and 3 PCR negative controls. On average, each replicate qPCR reaction analyzed 6 mL (SD = 1.3 mL) of seawater. A sample was determined to have amplified if the characteristic exponential amplification curve was visualized in the log(RFU) and Cq value graph and if there was a distinct peak in the melting curve at 78 °C, which corresponded to the peak of the plasmid standard PCR products. The Cq threshold was set using the auto-threshold setting in RotorGene software. The amplified samples’ concentrations were determined by fitting the line of best fit to the standard curve in each run. The standard curves’ average R2 and doubling efficiencies for the 33 qPCR runs were 0.99236 ± 0.01502 (SD) and 85.4 ± 4.7% (SD), respectively. The high R2 of these calibration curves demonstrates that the linear dynamic range spanned the full range of DNA concentrations examined, from the highest DNA concentrations of our standards to the LOQ. The DNA concentrations from all experimental samples above the LOQ were bracketed by this range. The average slope of the standard curves was −3.7391 ± 0.155 (SD) Cq per order of magnitude decrease in concentration, and the average intercept was 7.4540 ± 2.5069 (SD) Cq.

Contamination Controls

Before each experiment, tanks were sterilized with a solution of 10% household bleach (6% sodium hypochlorite) and deionized water and were subsequently rinsed with Milli-Q water. Sampling tubes (Cole Parmer, Antylia Scientific, Vernon Hills, IL, USA) were sterilized by pumping a 10% bleach solution through them for 15 min and subsequently rinsed with at least 1 L of Milli-Q water. Luer fittings, air stones, and air stone tubings were sterilized by soaking in 10% bleach for at least an hour and rinsed by soaking in Milli-Q water. Temperature, pH, [DO], and ORP probes were rinsed thoroughly with Milli-Q water. All tubing and probe wiring exteriors were wiped with 10% bleach followed by Milli-Q water before placing them in the tanks. Experimental controls (42 L of ASW) without added tissue homogenate were conducted simultaneously with each set of degradation experiments (up to four at once). The physicochemical conditions in these controls were set and maintained in the same way as the degradation experiments. This tank was sampled at each time point alongside the experiments, serving as a sampling negative control to monitor for contamination during sampling. Sampling negative control samples were processed similarly to the experimental samples in all subsequent laboratory steps. All laboratory work was conducted in a laboratory adjacent to but separated from the controlled-temperature room where the experiments were conducted. DNA extractions were conducted at a dedicated bench where no tissue DNA extractions had occurred. Pipettes for eDNA extraction were wiped with 10% bleach solution and UV-irradiated before each set of extractions. In each set of extractions, an extraction negative control was also processed to monitor for contamination at this step. Quantitative PCR (qPCR) preparation was conducted in a My-PCR Prep Station (Mystaire, Creedmoor, NC, USA) hood with positive airflow, air filtration through a HEPA filter, and an overhead UV lamp. Sterile filter tips were used at all stages of laboratory work. qPCR was conducted at a bench for qPCR cycling and post-PCR work. qPCR products were disposed of in this lab area, and PCR products were not brought into any other part of the lab.
Of all sampling negative controls, extraction negative controls, and PCR negative controls measured, a single qPCR replicate from one extraction negative control and one PCR negative control amplified (both below the LOQ). The estimated concentrations of these two samples were 9.6 and 5.6 copies per reaction, or 1.6 and 0.9 copies/mL seawater filtered, respectively. No replicates from any other triplicates of the 204 negative control samples amplified. This results in a false positive rate of just 0.9% if the amplification of a single qPCR triplicate at concentrations below the LOQ is considered a positive signal. Thus, we have high confidence that our experiments were free of contamination that would affect the interpretation of our results.

Statistical Analyses

All data analyses and visualizations were conducted using R version 4.1.0 (R Core Team) in RStudio. Plots were created with ggplot2 (49) and edited in Adobe Illustrator. Decay models were fitted to the mean of qPCR triplicates for each eDNA sample to determine decay rate constants for each experiment. All three replicates were averaged in all cases, and we did not remove any potential outlying qPCR replicates. We only analyzed experimental time points where the mean concentration of the three qPCR triplicates was above the LOQ.
A simple exponential model was fit as a linear model to natural log-transformed data from each experiment using the lm function. The linear model is defined as LN(Y) = – kt + LN(Y0). Here, the slope is mathematically equivalent to an exponential model’s decay rate constant, k. A decreasing rate of eDNA degradation over time has been observed in other studies. (28,32,50,51) Therefore, we also fit a biphasic model to natural log-transformed data as two linear models, allowing for a change in the decay rate constant over time. The biphasic linear model is defined as , where k1 is the initial decay rate constant and k2 is the second decay rate constant defined for time points, t, after a breakpoint, b, in the model. The natural log of Y1 and Y2 are the intercepts of each model (in natural log eDNA copies per reaction) during the initial and second phases of eDNA decay, respectively. The optimal breakpoint time for each model was estimated using the R package segmented. (52) The number of breakpoints to be estimated was set to 1, and the starting value for estimation was set to 24 h.
To avoid overparameterization, we chose to fit a biphasic model over other more complex nonlinear models. This model choice also allows for direct comparisons of the decay rate constant estimates from each phase among experiments and the decay rate constants reported in other studies. For each experiment, we calculated the log-likelihood and Bayesian information criterion (BIC) of the linear and biphasic models. A likelihood ratio test and comparison of BICs were conducted to determine which model was better at describing the data.
We fit a linear mixed model (LMM) on eDNA concentrations from each phase separately and considered the influence of temperature, pH, [DO], and relevant covariates on eDNA degradation. One outlier experiment, which exhibited a breakpoint in the biphasic model that was substantially later than the others (discussed more in depth in the Results and Discussion section), was omitted from these analyses. Linear mixed models were fit to the data using the R package lme4. (53) The significance of each coefficient was assessed with t tests using Satterthwaite’s method to determine degrees of freedom as implemented in the package lmertest. (54) We also determined the effect size of each model term from the Type III sum of squares of an ANOVA with Satterthwaite’s method using the package effectsize. (55)
Linear mixed models were formulated to test a priori hypotheses regarding the relative influence of temperature, pH, and [DO] on the eDNA degradation rate. The response variables in all LMMs were the natural log-transformed Lophelia eDNA concentrations (triplicate qPCR averages in copies per reaction) for each of the duplicate eDNA samples taken at each time point. The experiments were considered as random intercepts to account for the non-independence of concentration measurements from the same experiment. Time (in days) was included as a random slope to permit variation in the degradation rate (the slope of the relationship between eDNA concentration and time) across the experimental groups. (56)
Fixed effects included time and the average temperature, pH, [DO], and ORP over the course of each experiment when eDNA concentrations were quantifiable. In addition, the natural-log plus one transformed concentration of total DNA was included as a fixed effect in the second-phase models. In some experiments, total DNA concentration (presumably of microbial origin) increased over time (Figure S3). The interactions of time with average temperature, pH, [DO], ORP, and total DNA concentration were included to determine the effect of these factors on eDNA degradation.
Two additional LMMs were also fit to second-phase data from subsets of experiments to control for the unbalanced nature of our experimental conditions. One model only included the subset of experiments conducted at 4 and 10 °C to determine the influence of pH while eliminating the potentially confounding effects of experiments conducted at 20 °C, which were all conducted at pH 8.2. Temperature, [DO], ORP, total DNA concentration, and the interactions of these variables with time were included as fixed effects. The other model only included experiments conducted at 20 and 4 °C, and pH 8.2, to further investigate the influence of [DO]. This data subset represents a fully factorial design nested within our larger experimental framework.

Literature Search and Comparison to Other Studies

To place our results in a broader context, we compared our eDNA persistence time estimates to those from other marine eDNA studies that have fit a simple exponential model to their data. A literature search was conducted in the Web of Science database and the search terms (“eDNA” OR “environmental DNA”) AND (“persist*” OR “decay” OR “degrad*”). This search was also supplemented by reviewing all publications from the journal Environmental DNA. Only studies that conducted experiments with marine organisms and reported decay rate constants derived from time series of individual eDNA degradation experiments using exponential models were included in the analysis. In total, 11 studies fit the search criteria. (8,23,31,32,44,57−62) To compare across studies with variable starting eDNA concentrations, we calculated persistence time as the time until degradation of 99.9% of eDNA using the formula , in the case of the simple exponential model, where k is the decay rate constant. In the case of the biphasic model, we used the formula , where Y1 and Y2 are concentrations corresponding to the intercepts of each phase of the biphasic model and k2 is the decay rate constant for the second phase. This 1000-fold decrease in initial eDNA concentrations reflects the approximate decrease from the eDNA starting concentration to the LOQ or LOD typical of eDNA persistence studies and is thus ecologically and methodologically relevant.

Results and Discussion

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Modeling eDNA Degradation

eDNA degradation did not show a trend consistent with a simple exponential decay process in our experiments. Instead, it showed a general trend of decreasing degradation rate over time. The simple exponential model consistently underestimated measured eDNA concentrations at earlier time points and overestimated them at intermediate time points (Figure S2). Lower BIC and significant likelihood ratio tests indicated that the biphasic model better fit the data from 20 of the 22 experiments (Table S1). We report the results of the biphasic model for the two experiments in which the biphasic model did not have a significantly better fit because the slopes of the initial and second phases were similar. The biphasic model’s average breakpoint, parameter b, was 38.9 ± 18.6 h (Table S2). In all experiments, the decay rate constant was greater during the initial phase (steeper slope) than during the second phase (Figure 2).
A decreasing rate of eDNA degradation may be explained by a decrease in the size of particles that the eDNA is associated with over time. (20−22) Field and experimental studies show that eDNA is present primarily in relatively large size fractions, between 1 and 10 μm, including whole cells and mitochondria. (58,63,64) Experimental work indicates that the proportion of eDNA in large-size fractions decreases rapidly. However, the proportion of eDNA in size fractions smaller than 3 μm increases over time, and its degradation is slower than the degradation of eDNA in larger size fractions. (20,21) Our observations indicating that the degradation rate decreased over time may reflect a shift in the association of eDNA with larger particles (e.g., cells or organelles) to smaller particles. (20−22)
Since tissue homogenization and freezing likely lysed Lophelia cells, most of the eDNA early on in our experiments may not have been exclusively contained within cells but likely included free DNA or DNA in mitochondria. We assume that it is unlikely that eDNA was adsorbed to foreign particulates in our experiments because we used filtered artificial seawater. It seems likely that free DNA would pass through the 0.22 μm non-polar filter membrane we used. However, a recent study found that a 0.45 μm non-charged membrane can recover measurable concentrations free DNA molecules from seawater. (65) In our experiments, the observed rapid initial decrease of eDNA may be explained not only by the degradation of DNA in mitochondria but also by the lysis of mitochondria and the loss of free DNA that passes through the filter. Persistent, low concentrations of eDNA at later time points may reflect remaining whole mitochondria, free DNA captured on the filter, or free DNA associated with material from the Lophelia tissue.
In addition to spawning, it is likely that eDNA from corals is also shed through mucus production. Lophelia maintains a protective mucus layer, sheds mucus in response to disturbance, and may also produce mucus nets as a strategy to capture prey. (66,67) Previous studies have quantified eDNA shedding for Lophelia and two species of hydrozoans. (8,32,59) However, the state of eDNA that Lophelia and other cnidarians shed through processes such as mucus secretion remains unknown. Experiments that concurrently quantify coral eDNA shedding and mucus secretion would be informative, especially if the state of shed eDNA is determined.

Effects of Temperature, pH, and Oxygen Concentration on eDNA Degradation

Temperature

Our findings support the hypothesis that temperature is the most significant controlling factor of eDNA degradation rate in seawater. The effect of temperature was substantial in the second phase of eDNA degradation but not in the initial phase. During the initial phase of eDNA degradation, the estimated decay rate constants among experiments conducted at 20 °C (mean ± SD = 0.084 ± 0.016 h–1) were, on average, higher than those conducted at 10 °C (0.070 ± 0.014 h–1) and 4 °C (0.073 ± 0.013 h–1; Figure 3A). However, there was no significant effect of any predictor variable when fitting an LMM to the initial phase dataset (Table S3). Therefore, we find no evidence that eDNA degradation is influenced by temperature, pH, or [DO] early in the experiments before the model breakpoint.

Figure 3

Figure 3. Decay rate constant (k) estimates for the initial (A) and second (B) phases of eDNA degradation for 22 experiments conducted across 11 combinations of temperature, pH, and [DO]. Decay rate constants were estimated by fitting an exponential decay equation with initial and second degradation phases with different rates (biphasic). Decay rate constants are arranged on the x axis by the average pH over the course of each experiment. Vertical error bars represent 95% confidence intervals for the decay rate constants. The color of each point represents the average [DO] over each experiment.

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Second phase decay rate constants in experiments conducted at 20 °C (0.036 ± 0.017 h–1) were substantially higher than those at 10 °C (0.011 ± 0.002 h–1) and 4 °C (0.018 ± 0.004 h–1; Figure 3B). In the second phase, the interaction between temperature and time (in days) was the most significant predictor of eDNA concentration (P = 0.003; Table S4). Consistently, the interaction between temperature and time was the only significant predictor (P = 0.035) of eDNA concentration among the subset of pH 8.2 experiments conducted at 4 and 20 °C (Table S5). In addition, the effect size for the interaction of temperature and time was greater than the effect size for any other model term (Tables S4 and S5). These results imply that the temperature and time since the addition of eDNA are the strongest predictors of eDNA concentration in our experiments. We interpret that temperature controls the persistence of eDNA more strongly than pH or [DO].
The fact that temperature strongly controlled eDNA persistence only during the second phase of the experiments suggests that this effect may depend on the eDNA state. As stated previously, we propose that faster degradation during the initial phase of our experiments may reflect the lysis of mitochondria. Degradation of eDNA in smaller size fractions may be dominant in the second phase. If this is the case, our results contrast with a recent meta-analysis that found that the effect of temperature on eDNA degradation rate is stronger in studies using filter pore sizes of >0.7 μm than pore sizes of <0.45 μm. (22) Our results suggest that the persistence of eDNA in smaller size fractions may have been more affected by temperature than the persistence in larger size fractions.

pH

Our findings tentatively support the hypothesis that pH may influence the eDNA degradation rate in seawater. In the second phase, pH was a marginally significant predictor of eDNA concentration (P = 0.048; Table S4). The effect size of pH was substantially smaller than the effect size of the interaction between time and temperature (Table S4). The LMM fit to the subset of the second-phase data that excluded experiments conducted at 20 °C indicated that the interaction of time and pH is a nearly significant predictor of eDNA concentration (P = 0.069; Table S6). Indeed, higher pH among experiments conducted at 4 °C was associated with higher decay rate constants (Pearson’s r = 0.601, P = 0.039; Figure 3B). We infer from these results that pH may affect the persistence of eDNA in the range of marine conditions that we tested. However, this effect is small compared to that of temperature.
Previous work in freshwater systems has demonstrated that eDNA degrades faster at more acidic pH values. Lance et al. investigated a pH range similar to our study’s (7.5 and 8.0) (27,29,37,38) and found that lower pH was associated with faster eDNA degradation in freshwater. (29) Here, we present data suggesting that relatively more acidic seawater with a pH near 7.6 may prolong the persistence of eDNA compared to seawater at higher pH. Our results suggest that the influence of pH on eDNA degradation in seawater may need to be further considered.

Dissolved Oxygen Concentration

Our findings do not support the hypothesis that [DO] is a significant controlling factor of eDNA degradation in seawater. We conducted experiments in a fully factorial design with a full range of [DO] at two temperatures (4 and 20 °C) and a pH of 8.2. In the LMMs fit to this subset of the data and the full second-phase dataset, neither average [DO], average ORP, nor the interaction of these factors with time were significant predictors of eDNA concentration (Tables S4 and S5). From these results, we infer that [DO] and the oxidative state of seawater have the least influence on eDNA degradation rate and persistence among the conditions we tested. Thus, eDNA degradation may be driven by oxygen-independent processes.

Temperature Dependence of eDNA Degradation in the Marine Environment

Environmental DNA persistence times estimated from our study are comparable to previous marine studies performed at similar temperatures regardless of the source species (Figure 4). For example, eDNA persistence times from a skate at 4 °C (T99.9% = 14.4 and 26.2 days) (23) fall within our calculated persistence times for Lophelia (T99.9% range = 12.5 to 28.8 days). Further, persistence times from our experiments at 10 °C (T99.9% range = 16.0 to 24.0 days) are comparable to the persistence time calculated using data from a previous study of Lophelia eDNA degradation using natural seawater and live coral at 8 °C (T99.9% = 16.9 days). (8)

Figure 4

Figure 4. eDNA persistence time (time until degradation of 99.9% of starting eDNA concentration), estimated using decay rate constants from our study and other published marine studies, as a function of temperature. The linear model was only fit to decay rate constants calculated from simple exponential models. The best fit line is indicated by the dashed line, and the shaded region represents the 95% confidence interval for the slope. Points are dodged slightly from actual recorded temperatures to improve visualization. (Inset) Comparison of calculated persistence times when fitting a single exponential versus a biphasic model to the data in this study. Data are reported in Table S7.

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We fit a linear model to eDNA persistence time (time until degradation of 99.9% of starting eDNA concentration), estimated using decay rate constants from our study and other published marine studies, and temperature (Figure 4). We found that this model has a satisfactory fit (adjusted R2 = 0.4792, P < 0.001). The model predicts that eDNA persistence decreases by ∼0.74 days for each increase in degrees Celsius. Our results align with findings by Allan et al. They demonstrated a positive linear relationship between temperature and decay rate constants in most studies investigating eDNA degradation at multiple temperatures. (32) Similarly, Mauvisseau et al. found a linear relationship between increased temperature and faster decay rate constant in studies of freshwater and marine fish. (12) Further investigations at intermediate (∼8 to 12 °C) and high temperatures (greater than ∼25 °C), as well as more studies of invertebrate taxa, would improve confidence in the generality of this model. This model does not consider other factors that may influence eDNA quantification or detection in the ocean, including microbial activity, pH, advection, or diffusion. However, it serves as a resource to approximate maximum eDNA persistence times, constrained by temperature, in the marine environment.

Implications for eDNA Quantification and Sequencing in the Marine Environment

Investigating the spatiotemporal scale of eDNA in the marine environment is critical to defining the spatiotemporal resolution of ecological information derived from eDNA quantification or detection. Recent studies have found that eDNA abundance can exhibit strong vertical stratification, from just 5 m below the surface to 2000 m deep in the mesopelagic open ocean and within the first 10 m below the surface in nearshore habitats. (47,68,69) Further, model simulations have suggested that eDNA in the ocean remains within tens of meters from the depth where it is shed. (70) Field data from eDNA sampling along short, horizontal transects has revealed discrete eDNA community profiles at a fine scale in diverse shallow-water habitats, including coral reefs and kelp forests. (6,71,72) Together, these results suggest that eDNA community profiles, obtained using techniques such as meta-barcoding, can accurately reflect biological communities structured by depth. However, the ability of eDNA meta-barcoding to distinguish communities in deep-sea environments is less clear. eDNA sequencing efforts in the deep sea have resulted in benthic community composition profiles that agree with expected differences due to habitat at sites ∼15 km apart. (73) However, eDNA sampling paired with video analyses revealed remarkable disparities between video observations and sequence read abundances from deep-sea corals. (7) Some coral species were observed in video surveys but were not detected in eDNA, and many sequence reads were recovered from species not observed in videos.
Modeling studies suggest that the extent of horizontal transport of eDNA at concentrations detectable through single-species quantitative methods can be substantial, on the order of tens of kilometers, in the surface ocean and at depth. (8,26) These models provide valuable insights for understanding the horizontal spatial scale of eDNA in the ocean. However, studies modeling eDNA transport have utilized decay rate constants derived from simple exponential models. (8,26) We found that at low temperatures of ≤10 °C, a simple exponential model can substantially underestimate the persistence of eDNA. Environmental DNA persistence time, defined as the time until degradation of 99.9% of the starting eDNA concentration, was up to 75% longer when fitting the data with the biphasic model rather than the simple exponential model (Figure 4 and Table S7). Thus, in cold marine environments including the deep sea, detectable eDNA may be transported over longer distances than predicted by simulations that assumed a simple exponential decay.
The interpretation of field eDNA data from deep-sea corals is complicated because eDNA may persist for longer durations at cold temperatures and may be shed infrequently or at low concentrations. We conducted field sampling within 1 m of a large aggregation of live Lophelia and at altitudes of 5, 10, and 20 m above the seafloor in the Gulf of Mexico. We found that caryophylliid eDNA could be detected (positive qPCR amplification) within 10 m above the seafloor. However, copy number estimates were below the LOQ of our qPCR assay (Table S8; see the Supplementary Methods). Kutti et al. also measured relatively low concentrations of Lophelia eDNA in the Norwegian Sea (up to 1000 s of copies per L of seawater filtered), demonstrating that Lophelia eDNA is present at low concentrations in its environment. (8)
If eDNA in nature is shed at low concentrations, it may be diluted rapidly and degrade past the detectable limit more quickly than in an experimental setting. Thus, an eDNA sample may provide an accurate signal of the proximate biological community. However, our experiments demonstrate that eDNA can persist for weeks at low temperatures, so detectable concentrations may be transported over large distances. When using single-species quantitative methods, such as qPCR or digital-droplet PCR, considerable care should be taken when interpreting the source of a positive eDNA amplification. We suggest that a range of possible sources must be considered for an observed eDNA signal. This range can be constrained by coupling estimated persistence times and hydrodynamic models.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.est.2c01672.

  • Supplementary methods; supplementary figures and tables referenced in the main text; supplementary table supporting qPCR inhibition testing as described in the Supplementary Methods (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Luke J. McCartin - Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015-3027, United States
    • Samuel A. Vohsen - Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015-3027, United States
    • Susan W. Ambrose - Department of Earth and Environmental Sciences, Lehigh University, Bethlehem, Pennsylvania 18015-3027, United States
    • Michael Layden - Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015-3027, United States
    • Catherine S. McFadden - Department of Biology, Harvey Mudd College, Claremont, California 91711, United States
    • Erik E. Cordes - Department of Biology, Temple University, Philadelphia, Pennsylvania 19122-6008, United States
    • Jill M. McDermott - Department of Earth and Environmental Sciences, Lehigh University, Bethlehem, Pennsylvania 18015-3027, United States
  • Funding

    This research was funded by the National Oceanic and Atmospheric Administration’s Oceanic and Atmospheric Research, Office of Ocean Exploration and Research, under award NA18OAR0110289 to S.H. and J.M.M. at Lehigh University, and sub-awards to E.E.C at Temple University and C.S.M. at Harvey Mudd College.

  • Notes
    The authors declare no competing financial interest.

    All data and R-code are available at 10.6084/m9.figshare.19775608.

Acknowledgments

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We would like to thank members of the Herrera lab for their assistance with aspects of the laboratory work, Dylan Faltine-Gonzalez for his advice and help with the qPCR assay, Elizabeth Andruszkiewicz Allan for her advice regarding experimental methods, and Alexis Weinnig and Adam Hallaj for their help in providing Lophelia samples. We also thank the captains and crew of the NOAA Ship Ronald Brown and the R/V Point Sur and the ROV Jason and ROV Global Explorer teams. We thank the editor, Prof. Alexandria Boehm, and four anonymous reviewers for their insightful and constructive feedback on this manuscript. We thank Liliana Monroy, Mauricio Herrera and Linda McDermott for their support.

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    The environmental DNA (eDNA) technique is expected to become a powerful, non-invasive tool for estg. the distribution and biomass of organisms. This technique was recently shown to be applicable to aquatic vertebrates by collecting extraorganismal DNA floating in the water or absorbed onto suspended particles. However, basic information on eDNA release rate is lacking, despite it being essential for practical applications. In this series of expts. with bluegill sunfish (Lepomis macrochirus), we examd. the effect of fish developmental stage on eDNA release rate. EDNA concn. reached equil. 3 days after the individual fish were introduced into the sep. containers, enabling calcn. of the eDNA release rate (copies h-1) from individual fish on the assumption that the no. of eDNA released from the fish per unit time equals total degrdn. in the container (copies h -1). The eDNA release rate was 3-4 times higher in the adult (body wt.: 30-75 g) than in the juvenile group (0.5-2.0 g). Such pos. relationship between fish size and eDNA release rate support the possibility of biomass rather than d. estn. using eDNA techniques. However, the eDNA release rate per fish body wt. (copies h -1 g -1) was slightly higher in the juvenile than the adult group, which is likely because of the ontogenetic redn. in metabolic activity. Therefore, quant. eDNA data should be carefully interpreted to avoid overestimating biomass when the population is dominated by juveniles, because the age structure of the focal population is often variable and unseen in the field. EDNA degrdn. rates (copies l-1 h -1), calcd. by curve fitting of time-dependent changes in eDNA concns. after fish removal, were 5.1-15.9% per h (half-life: 6.7 h). This suggests that quant. eDNA data should be cor. using a degrdn. curve attained in the target field.
  14. 14
    Klymus, K. E.; Richter, C. A.; Chapman, D. C.; Paukert, C. Quantification of eDNA Shedding Rates from Invasive Bighead Carp Hypophthalmichthys nobilis and Silver Carp Hypophthalmichthys molitrix. Biol. Conserv. 2015, 183, 77– 84,  DOI: 10.1016/j.biocon.2014.11.020
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    Lacoursière-Roussel, A.; Côté, G.; Leclerc, V.; Bernatchez, L. Quantifying Relative Fish Abundance with EDNA: A Promising Tool for Fisheries Management. J. Appl. Ecol. 2016, 53, 1148– 1157,  DOI: 10.1111/1365-2664.12598
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    15
    Quantifying relative fish abundance with eDNA: a promising tool for fisheries management
    Lacoursiere-Roussel, Anais; Cote, Guillaume; Leclerc, Veronique; Bernatchez, Louis
    Journal of Applied Ecology (2016), 53 (4), 1148-1157CODEN: JAPEAI; ISSN:0021-8901. (Wiley-Blackwell)
    Summary : Assessment and monitoring of exploited fish populations are challenged by costs, logistics and neg. impacts on target populations. These factors therefore limit large-scale effective management strategies. Evidence is growing that the quantity of eDNA may be related not only to species presence/absence, but also to species abundance. In this study, the concns. of environmental DNA (eDNA) from a highly prized sport fish species, Lake Trout Salvelinus namaycush (Walbaum 1792), were estd. in water samples from 12 natural lakes and compared to abundance and biomass data obtained from standardized gillnet catches as performed routinely for fisheries management purposes. To reduce environmental variability among lakes, all lakes were sampled in spring, between ice melt and water stratification. The eDNA concn. did not vary significantly with water temp., dissolved oxygen, pH and turbidity, but was significantly pos. correlated with relative fish abundance estd. as catch per unit effort (CPUE), whereas the relationship with biomass per unit effort (BPUE) was less pronounced. The value of eDNA to inform about local aquatic species distribution was further supported by the similarity between the spatial heterogeneity of eDNA distribution and spatial variation in CPUE measured by the gillnet method. Synthesis and applications. Large-scale empirical evidence of the relationship between the eDNA concn. and species abundance allows for the assessment of the potential to integrate eDNA within fisheries management plans. As such, the eDNA quant. method represents a promising population abundance assessment tool that could significantly reduce the costs assocd. with sampling and increase the power of detection, the spatial coverage and the frequency of sampling, without any neg. impacts on fish populations.
  16. 16
    Sansom, B. J.; Sassoubre, L. M. Environmental DNA (eDNA) Shedding and Decay Rates to Model Freshwater Mussel EDNA Transport in a River. Environ. Sci. Technol. 2017, 51, 14244– 14253,  DOI: 10.1021/acs.est.7b05199
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    16
    Environmental DNA (eDNA) Shedding and Decay Rates to Model Freshwater Mussel eDNA Transport in a River
    Sansom, Brandon J.; Sassoubre, Lauren M.
    Environmental Science & Technology (2017), 51 (24), 14244-14253CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)
    Freshwater mussels are vital components of stream ecosystems, yet remain threatened. Thus, timely and accurate species counts are crit. for proper conservation and management. Mussels live in stream sediments and can be challenging to survey given constraints related to water depth, flow, and time of year. The use of environmental DNA (eDNA) to monitor mussel distributions and diversity is a promising tool. Before it can be used as a monitoring tool, however, we need to know how much eDNA mussels shed into their environment and how long the eDNA persists. We present a novel application of eDNA to est. both the presence/absence and abundance of a freshwater mussel species, Lampsilis siliquoidea. The eDNA shedding and decay rates reported within are the 1st for freshwater mussels. We detd. that eDNA shedding was statistically similar across mussel densities, but that 1st-order decay consts. varied between exptl. treatments. We effectively modeled downstream transport of eDNA and present a model that can be used as a complementary tool to est. mussel d. Our results suggest that eDNA has the potential to be a complementary tool to survey mussels and enhance current efforts to monitor and protect freshwater mussel biodiversity.
  17. 17
    Jo, T.; Arimoto, M.; Murakami, H.; Masuda, R.; Minamoto, T. Estimating Shedding and Decay Rates of Environmental Nuclear DNA with Relation to Water Temperature and Biomass. Environ. DNA 2020, 2, 140– 151,  DOI: 10.1002/edn3.51
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    Ostberg, C. O.; Chase, D. M. Ontogeny of eDNA Shedding during Early Development in Chinook Salmon (Oncorhynchus tshawytscha). Environ. DNA 2022, 339,  DOI: 10.1002/edn3.258
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    18
    Ontogeny of eDNA shedding during early development in Chinook Salmon (Oncorhynchus tshawytscha)
    Ostberg, Carl O.; Chase, Dorothy M.
    Environmental DNA (2022), 4 (2), 339-348CODEN: EDNNAZ; ISSN:2637-4943. (John Wiley & Sons, Inc.)
    Knowledge of the timing of major life history events in aquatic species is important for informing conservation and resource management planning. Accordingly, surveys of environmental DNA (eDNA) have been performed to det. the efficacy of eDNA for providing information on life history events, primarily focusing on the timing of events assocd. with spawning, and these studies have proved successful. However, spawning represents only one part of the life history, and therefore, information on eDNA shedding during other life history stages is needed to fill gaps in knowledge. Here, we explored eDNA shedding during early life history (from fertilized eggs until near yolk sac absorption) in Chinook Salmon (Oncorhynchus tshawytscha) at three biomasses in a lab. environment. We found that fertilized eggs shed little eDNA prior to hatching. Hatching coincided with a spike in eDNA, and we obsd. a significant and pos. relationship between eDNA concn. and the no. of hatched eggs. The concn. of eDNA shed by larvae after hatching was not consistent across post-hatch sampling days, suggesting developmental and behavioral changes assocd. with larval ontogeny may affect eDNA shedding rate. These results indicate that eDNA data may be used to identify hatch timing and verify successful reprodn. in oviparous aquatic fishes. The application of eDNA to early life history broadens the capacity of eDNA-based methods for assessing population status and trends.
  19. 19
    Thalinger, B.; Rieder, A.; Teuffenbach, A.; Pütz, Y.; Schwerte, T.; Wanzenböck, J.; Traugott, M. The Effect of Activity, Energy Use, and Species Identity on Environmental DNA Shedding of Freshwater Fish. Front. Ecol. Evol. 2021, 9, 1– 13,  DOI: 10.3389/fevo.2021.623718
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    Jo, T.; Arimoto, M.; Murakami, H.; Masuda, R.; Minamoto, T. Particle Size Distribution of Environmental DNA from the Nuclei of Marine Fish. Environ. Sci. Technol. 2019, 53, 9947– 9956,  DOI: 10.1021/acs.est.9b02833
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    Particle Size Distribution of Environmental DNA from the Nuclei of Marine Fish
    Jo, Toshiaki; Arimoto, Mio; Murakami, Hiroaki; Masuda, Reiji; Minamoto, Toshifumi
    Environmental Science & Technology (2019), 53 (16), 9947-9956CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)
    Environmental DNA (eDNA) analyses have enabled a more efficient surveillance of species distribution and compn. than conventional methods. However, the characteristics and dynamics of eDNA (e.g., origin, state, transport, and fate) remain unknown. This is esp. limited for the eDNA derived from nuclei (nu-eDNA), which has recently been used in eDNA analyses. Here, we compared the particle size distribution (PSD) of nu-eDNA from Japanese Jack Mackerel (Trachurus japonicus) with that of mt-eDNA (eDNA derived from mitochondria) reported in previous studies. We repeatedly sampled rearing water from the tanks under multiple temps. and fish biomass levels, and quantified the copy nos. of size-fractioned nu-eDNA. We found that the concn. of nu-eDNA was higher than that of mt-eDNA at 3-10 μm size fraction. Moreover, at the 0.8-3 μm and 0.4-0.8 μm size fractions, eDNA concns. of both types increased with higher temp. and their degrdn. tended to be suppressed. These results imply that the prodn. of eDNA from large to small size fractions could buffer the degrdn. of small-sized eDNA, which could improve its persistence in water. Our findings will contribute to refine the difference between nu- and mt-eDNA properties, and assist eDNA analyses as an efficient tool for the conservation of aquatic species.
  21. 21
    Jo, T.; Murakami, H.; Yamamoto, S.; Masuda, R.; Minamoto, T. Effect of Water Temperature and Fish Biomass on Environmental DNA Shedding, Degradation, and Size Distribution. Ecol. Evol. 2019, 9, 1135– 1146,  DOI: 10.1002/ece3.4802
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    21
    Effect of water temperature and fish biomass on environmental DNA shedding, degradation, and size distribution
    Jo Toshiaki; Yamamoto Satoshi; Minamoto Toshifumi; Murakami Hiroaki; Masuda Reiji; Yamamoto Satoshi
    Ecology and evolution (2019), 9 (3), 1135-1146 ISSN:2045-7758.
    Environmental DNA (eDNA) analysis has successfully detected organisms in various aquatic environments. However, there is little basic information on eDNA, including the eDNA shedding and degradation processes. This study focused on water temperature and fish biomass and showed that eDNA shedding, degradation, and size distribution varied depending on water temperature and fish biomass. The tank experiments consisted of four temperature levels and three fish biomass levels. The total eDNA and size-fractioned eDNA from Japanese Jack Mackerels (Trachurus japonicus) were quantified before and after removing the fish. The results showed that the eDNA shedding rate increased at higher water temperature and larger fish biomass, and the eDNA decay rate also increased at higher temperature and fish biomass. In addition, the small-sized eDNA fractions were proportionally larger at higher temperatures, and these proportions varied among fish biomass. After removing the fish from the tanks, the percentage of eDNA temporally decreased when the eDNA size fraction was >10 μm, while the smaller size fractions increased. These results have the potential to make the use of eDNA analysis more widespread in the future.
  22. 22
    Jo, T.; Minamoto, T. Complex Interactions between Environmental DNA (eDNA) State and Water Chemistries on eDNA Persistence Suggested by Meta-analyses. Mol. Ecol. Resour. 2021, 21, 1490– 1503,  DOI: 10.1111/1755-0998.13354
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    Complex interactions between environmental DNA (eDNA) state and water chemistries on eDNA persistence suggested by meta-analyses
    Jo, Toshiaki; Minamoto, Toshifumi
    Molecular Ecology Resources (2021), 21 (5), 1490-1503CODEN: MEROCJ; ISSN:1755-098X. (Wiley-Blackwell)
    Understanding the processes of environmental DNA (eDNA) persistence and degrdn. is essential to det. the spatiotemporal scale of eDNA signals and accurately est. species distribution. The effects of environmental factors on eDNA persistence have previously been examd.; however, the influence of the physiochem. and mol. states of eDNA on its persistence is not completely understood. Here, we performed meta-anal. including 26 previously published papers on the estn. of first-order eDNA decay rate consts., and assessed the effects of filter pore size, DNA fragment size, target gene, and environmental conditions on eDNA decay rates. Almost all supported models included the interactions between the filter pore size and water temp., between the target gene and water temp., and between the target gene and water source, implying the influence of complex interactions between the eDNA state and environmental conditions on eDNA persistence. These findings were generally consistent with the results of a reanal. of a previous tank expt. which measured the time-series changes in marine fish eDNA concns. in multiple size fractions after fish removal. Our results suggest that the mechanism of eDNA persistence and degrdn. cannot be fully understood without knowing not only environmental factors but also cellular and mol. states of eDNA in water. Further verification of the relationship between eDNA state and persistence is required by obtaining more information on eDNA persistence in various exptl. and environmental conditions, which will enhance our knowledge on eDNA persistence and support our findings.
  23. 23
    Weltz, K.; Lyle, J. M.; Ovenden, J.; Morgan, J. A. T.; Moreno, D. A.; Semmens, J. M. Application of Environmental DNA to Detect an Endangered Marine Skate Species in the Wild. PLoS One 2017, 12, e0178124  DOI: 10.1371/journal.pone.0178124
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    23
    Application of environmental DNA to detect an endangered marine skate species in the wild
    Weltz, Kay; Lyle, Jeremy M.; Ovenden, Jennifer; Morgan, Jessica A. T.; Moreno, David A.; Semmens, Jayson M.
    PLoS One (2017), 12 (6), e0178124/1-e0178124/16CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Environmental DNA (eDNA) techniques have only recently been applied in the marine environment to detect the presence of marine species. Species-specific primers and probes were designed to detect the eDNA of the endangered Maugean skate (Zearaja maugeana) from as little as 1 L of water collected at depth (10-15 m) in Macquarie Harbor (MH), Tasmania. The identity of the eDNA was confirmed as Z. maugeana by sequencing the qPCR products and aligning these with the target sequence for a 100% match. This result has validated the use of this eDNA technique for detecting a rare species, Z. maugeana, in the wild. Being able to investigate the presence, and possibly the abundance, of Z. maugeana in MH and Bathurst harbor (BH), would be addressing a conservation imperative for the endangered Z. maugeana. For future application of this technique in the field, the rate of decay was detd. for Z. maugeana eDNA under ambient dissolved oxygen (DO) levels (55% satn.) and lower DO (20% satn.) levels, revealing that the eDNA can be detected for 4 and 16 h resp., after which eDNA concn. drops below the detection threshold of the assay. With the rate of decay being influenced by starting eDNA concns., it is recommended that samples be filtered as soon as possible after collection to minimize further loss of eDNA prior to and during sample processing.
  24. 24
    Collins, R. A.; Wangensteen, O. S.; O’Gorman, E. J.; Mariani, S.; Sims, D. W.; Genner, M. J. Persistence of Environmental DNA in Marine Systems. Commun. Biol. 2018, 1, 1– 11,  DOI: 10.1038/s42003-018-0192-6
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    24
    Persistence of environmental DNA in marine systems
    Collins, Rupert A.; Wangensteen, Owen S.; O'Gorman, Eoin J.; Mariani, Stefano; Sims, David W.; Genner, Martin J.
    Communications Biology (2018), 1 (1), 1-11CODEN: CBOIDQ; ISSN:2399-3642. (Nature Research)
    As environmental DNA (eDNA) becomes an increasingly valuable resource for marine ecosystem monitoring, understanding variation in its persistence across contrasting environments is crit. Here, we quantify the breakdown of macrobial eDNA over a spatio-temporal axis of locally extreme conditions, varying from ocean-influenced offshore to urban-inshore, and between winter and summer. We report that eDNA degrades 1.6 times faster in the inshore environment than the offshore environment, but contrary to expectation we find no difference over season. Anal. of environmental covariables show a spatial gradient of salinity and a temporal gradient of pH, with salinity-or the biotic correlates thereof-most important. Based on our estd. inshore eDNA half-life and naturally occurring eDNA concns., we est. that eDNA may be detected for around 48 h, offering potential to collect ecol. community data of high local fidelity. We conclude by placing these results in the context of previously published eDNA decay rates.
  25. 25
    Harrison, J. B.; Sunday, J. M.; Rogers, S. M. Predicting the Fate of eDNA in the Environment and Implications for Studying Biodiversity. Proc. R. Soc. B 2019, 286, 20191409,  DOI: 10.1098/rspb.2019.1409
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    Predicting the fate of eDNA in the environment and implications for studying biodiversity
    Harrison, Jori B.; Sunday, Jennifer M.; Rogers, Sean M.
    Proceedings of the Royal Society B: Biological Sciences (2019), 286 (1915), 20191409CODEN: PRSBC7 ISSN:. (Royal Society)
    A review. Environmental DNA (eDNA) applications are transforming the std. of characterizing aquatic biodiversity via the presence, location and abundance of DNA collected from environmental samples. As eDNA studies use DNA fragments as a proxy for the presence of organisms, the ecol. properties of the complex and dynamic environments from which eDNA is sampled need to be considered for accurate biol. interpretation. In this review, we discuss the role that differing environments play on the major processes that eDNA undergoes between organism and collection, including shedding, decay and transport. We focus on a mechanistic understanding of these processes and highlight how decay and transport models are being developed towards more accurate and robust predictions of the fate of eDNA. We conclude with five recommendations for eDNA researchers and practitioners, to advance current best practices, as well as to support a future model of eDNA spatio-temporal persistence.
  26. 26
    Andruszkiewicz, E. A.; Koseff, J. R.; Fringer, O. B.; Ouellette, N. T.; Lowe, A. B.; Edwards, C. A.; Boehm, A. B. Modeling Environmental DNA Transport in the Coastal Ocean Using Lagrangian Particle Tracking. Front. Mar. Sci. 2019, 6, 1– 14,  DOI: 10.3389/fmars.2019.00477
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    Strickler, K. M.; Fremier, A. K.; Goldberg, C. S. Quantifying Effects of UV-B, Temperature, and pH on eDNA Degradation in Aquatic Microcosms. Biol. Conserv. 2015, 183, 85– 92,  DOI: 10.1016/j.biocon.2014.11.038
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    Eichmiller, J. J.; Best, S. E.; Sorensen, P. W. Effects of Temperature and Trophic State on Degradation of Environmental DNA in Lake Water. Environ. Sci. Technol. 2016, 50, 1859– 1867,  DOI: 10.1021/acs.est.5b05672
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    Effects of Temperature and Trophic State on Degradation of Environmental DNA in Lake Water
    Eichmiller, Jessica J.; Best, Sendrea E.; Sorensen, Peter W.
    Environmental Science & Technology (2016), 50 (4), 1859-1867CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)
    Degrdn. of environmental DNA (eDNA) in aquatic habitats can affect the interpretation of eDNA data and the ability to detect aquatic organisms. The effect of temp. and trophic state on the decay of Common Carp (Cyprinus carpio) eDNA was evaluated using lake water microcosms and quant. PCR for a Common Carp-specific genetic marker in 2 expts. The 1st expt. tested the effect of temp. on Common Carp eDNA decay. Common Carp eDNA exhibited exponential decay that increased with temp. The slowest decay rate was obsd. at 5°, with a T90 value (time to 90% redn. from initial concn.) of 6.6 days, as opposed to ∼1 day at higher temps. In a 2nd expt., decay was compared across waters from lakes of different trophic states. In this expt., Common Carp eDNA exhibited biphasic exponential decay, characterized by rapid decay for 3-8 days followed by slow decay. Decay rate was slowest in dystrophic water and fastest in oligotrophic water, and decay rate was neg. correlated to dissolved org. C concn. The overall rapid decay of eDNA and the effects of temp. and water quality should be considered in protocols for water sample storage and field sampling design.
  29. 29
    Lance, R.; Klymus, K.; Richter, C.; Guan, X.; Farrington, H.; Carr, M.; Thompson, N.; Chapman, D.; Baerwaldt, K. Experimental Observations on the Decay of Environmental DNA from Bighead and Silver Carps. MBI 2017, 8, 343– 359,  DOI: 10.3391/mbi.2017.8.3.08
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    Tsuji, S.; Ushio, M.; Sakurai, S.; Minamoto, T.; Yamanaka, H. Water Temperature-Dependent Degradation of Environmental DNA and Its Relation to Bacterial Abundance. PLoS One 2017, 12, 1– 13,  DOI: 10.1371/journal.pone.0176608
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    Kasai, A.; Takada, S.; Yamazaki, A.; Masuda, R.; Yamanaka, H. The Effect of Temperature on Environmental DNA Degradation of Japanese Eel. Fish. Sci. 2020, 86, 465– 471,  DOI: 10.1007/s12562-020-01409-1
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    The effect of temperature on environmental DNA degradation of Japanese eel
    Kasai, Akihide; Takada, Shingo; Yamazaki, Aya; Masuda, Reiji; Yamanaka, Hiroki
    Fisheries Science (Tokyo, Japan) (2020), 86 (3), 465-471CODEN: FSCIEH; ISSN:0919-9268. (Springer Japan)
    Abstr.: The environmental DNA (eDNA) technique is a convenient and powerful tool to detect rare species. Knowledge of the degrdn. rate of eDNA in water is important for understanding how degrdn. influences the presence and/or est. biomass of aquatic animals. We developed a new set of species-specific primers and probe to detect eDNA of Japanese eel Anguilla japonica, which is a com. important and endangered species, and then conducted a lab. expt. to quantify the temp.-dependent degrdn. of emitted eDNA. Eels were held in tanks at five different temp. levels from 10 to 30°C and water from each tank was sampled and kept in bottles at each temp. over 6 days. The concn. of eDNA was measured every day and the results showed that temp. (T) had a significant and pos. effect on the degrdn. rate (k) as k = 0.02T + 0.18. Improved understanding of the effect of temp. on degrdn. rates would help data interpretations and adjustments would increase the reliability of eDNA anal. in future studies.
  32. 32
    Allan, E. A.; Zhang, W. G.; Lavery, A. C.; Govindarajan, A. F. Environmental DNA Shedding and Decay Rates from Diverse Animal Forms and Thermal Regimes. Environ. DNA 2021, 3, 492– 514,  DOI: 10.1002/edn3.141
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    Caza-Allard, I.; Laporte, M.; Côté, G.; April, J.; Bernatchez, L. Effect of Biotic and Abiotic Factors on the Production and Degradation of Fish Environmental DNA: An Experimental Evaluation. Environ. DNA 2022, 4, 453– 468,  DOI: 10.1002/edn3.266
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    Effect of biotic and abiotic factors on the production and degradation of fish environmental DNA: An experimental evaluation
    Caza-Allard, Isabeau; Laporte, Martin; Cote, Guillaume; April, Julien; Bernatchez, Louis
    Environmental DNA (2022), 4 (2), 453-468CODEN: EDNNAZ; ISSN:2637-4943. (John Wiley & Sons, Inc.)
    Environmental DNA (eDNA) is a very promising approach to facilitate and improve the aquatic species monitoring, which is crucial for their management and conservation. In comparison with the plethora of monitoring studies in the fields, relatively few studies have focused on exptl. investigating the "ecol." of eDNA, in particular pertaining to processes influencing the detection of eDNA. The paucity of knowledge about its ecol. hampers the use of eDNA anal. to its full potential. In this study, we exptl. evaluated the impact of several biotic and abiotic factors on the rate of prodn. and degrdn. of eDNA. Individuals of three freshwater fish species (brown bullhead, tench, and yellow perch) with distinct ecol. were placed in two types of water from the St. Lawrence River (Quebec, Canada) with very distinct physicochem. characteristics and at three different temps. Water samples were then filtered at predetd. time intervals, and quant. PCR was used to quantify the eDNA in each sample. We found that temp., species, water types, and some interactions between these factors had a strong effect on the prodn. and degrdn. of eDNA. The results of this study enhance our knowledge about the ecol. of eDNA, thus improving eDNA data interpretation.
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    Lindahl, T.; Nyberg, B. Rate of Depurination of Native Deoxyribonucleic Acid. Biochemistry 1972, 11, 3610– 3618,  DOI: 10.1021/bi00769a018
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    Rate of depurination of native deoxyribonucleic acid
    Lindahl, Tomas; Nyberg, Barbro
    Biochemistry (1972), 11 (19), 3610-18CODEN: BICHAW; ISSN:0006-2960.
    The rate of depurination of double-stranded Bacillus subtilis DNA, radioactively labeled in the purine residues, has been followed as a function of temp., pH, and ionic strength. In a Mg2+-contg. buffer of physiol. ionic strength, the rate const. for depurination of DNA is 4 × 10-9 sec-1 at 70° and pH 7.4. The activation energy of the reaction is 31 ± 2 kcal/mole. These data strongly indicate that depurination of DNA occurs at a physiol. significant rate under in vivo conditions and consequently that the lesions introduced in this fashion must be repaired.
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    Cheng, Y.-K.; Pettitt, B. M. Stabilities of Double- and Triple-Strand Helical Nucleic Acids. Prog. Biophys. Mol. Biol. 1992, 58, 225– 257,  DOI: 10.1016/0079-6107(92)90007-s
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    Stabilities of double- and triple-strand helical nucleic acids
    Cheng, Yuen Kit; Pettitt, B. Montgomery
    Progress in Biophysics & Molecular Biology (1992), 58 (3), 225-57CODEN: PBIMAC; ISSN:0079-6107.
    A review, with ∼200 refs., centered around the authors' interest in DNA double- and triple-helix formation and stability.
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    Lindahl, T. Instability and Decay of the Primary Structure of DNA. Nature 1993, 362, 709– 715,  DOI: 10.1038/362709a0
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    Instability and decay of the primary structure of DNA
    Lindahl, Tomas
    Nature (London, United Kingdom) (1993), 362 (6422), 709-15CODEN: NATUAS; ISSN:0028-0836.
    A review with 84 refs. Although DNA is the carrier of genetic information, it has limited chem. stability. Hydrolysis oxidn. and nonenzymic methylation of DNA occur at significant rates in vivo, and are counteracted by specific DNA repair processes. The spontaneous decay of DNA is likely to be a major factor in mutagenesis, carcinogenesis and aging, and also sets limits for the recovery of DNA fragments from fossils.
  37. 37
    Seymour, M.; Durance, I.; Cosby, B. J.; Ransom-Jones, E.; Deiner, K.; Ormerod, S. J.; Colbourne, J. K.; Wilgar, G.; Carvalho, G. R.; de Bruyn, M.; Edwards, F.; Emmett, B. A.; Bik, H. M.; Creer, S. Acidity Promotes Degradation of Multi-Species Environmental DNA in Lotic Mesocosms. Commun. Biol. 2018, 1, 1– 8,  DOI: 10.1038/s42003-017-0005-3
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    Acidity promotes degradation of multi-species environmental DNA in lotic mesocosms
    Seymour, Mathew; Durance, Isabelle; Cosby, Bernard J.; Ransom-Jones, Emma; Deiner, Kristy; Ormerod, Steve J.; Colbourne, John K.; Wilgar, Gregory; Carvalho, Gary R.; de Bruyn, Mark; Edwards, Francois; Emmett, Bridget A.; Bik, Holly M.; Creer, Simon
    Communications Biology (2018), 1 (1), 1-8CODEN: CBOIDQ; ISSN:2399-3642. (Nature Research)
    Accurate quantification of biodiversity is fundamental to understanding ecosystem function and for environmental assessment. Mol. methods using environmental DNA (eDNA) offer a non-invasive, rapid, and cost-effective alternative to traditional biodiversity assessments, which require high levels of expertise. While eDNA analyses are increasingly being utilized, there remains considerable uncertainty regarding the dynamics of multispecies eDNA, esp. in variable systems such as rivers. Here, we utilize four sets of upland stream mesocosms, across an acid-base gradient, to assess the temporal and environmental degrdn. of multispecies eDNA. Sampling included water column and biofilm sampling over time with eDNA quantified using qPCR. Our findings show that the persistence of lotic multispecies eDNA, sampled from water and biofilm, decays to non-detectable levels within 2 days and that acidic environments accelerate the degrdn. process. Collectively, the results provide the basis for a predictive framework for the relationship between lotic eDNA degrdn. dynamics in spatio-temporally dynamic river ecosystems.
  38. 38
    van Bochove, K.; Bakker, F. T.; Beentjes, K. K.; Hemerik, L.; Vos, R. A.; Gravendeel, B. Organic Matter Reduces the Amount of Detectable Environmental DNA in Freshwater. Ecol. Evol. 2020, 10, 3647– 3654,  DOI: 10.1002/ece3.6123
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    Organic matter reduces the amount of detectable environmental DNA in freshwater
    van Bochove Kees; Bakker Freek T; van Bochove Kees; Beentjes Kevin K; Vos Rutger A; Gravendeel Barbara; Beentjes Kevin K; Gravendeel Barbara; Hemerik Lia; Gravendeel Barbara
    Ecology and evolution (2020), 10 (8), 3647-3654 ISSN:2045-7758.
    Environmental DNA (eDNA) is used for monitoring the occurrence of freshwater organisms. Various studies show a relation between the amount of eDNA detected and target organism abundance, thus providing a potential proxy for reconstructing population densities. However, environmental factors such as water temperature and microbial activity are known to affect the amount of eDNA present as well. In this study, we use controlled aquarium experiments using Gammarus pulex L. (Amphipoda) to investigate the relationship between the amount of detectable eDNA through time, pH, and levels of organic material. We found eDNA to degrade faster when organic material was added to the aquarium water, but that pH had no significant effect. We infer that eDNA contained inside cells and mitochondria is extra resilient against degradation, though this may not reflect actual presence of target species. These results indicate that, although estimation of population density might be possible using eDNA, measured eDNA concentration could, in the future, be corrected for local environmental conditions in order to ensure accurate comparisons.
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    Paulmier, A.; Ruiz-Pino, D. Oxygen Minimum Zones (OMZs) in the Modern Ocean. Prog. Oceanogr. 2009, 80, 113– 128,  DOI: 10.1016/j.pocean.2008.08.001
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    Addamo, A. M.; Vertino, A.; Stolarski, J.; García-Jiménez, R.; Taviani, M.; Machordom, A. Merging Scleractinian Genera: The Overwhelming Genetic Similarity between Solitary Desmophyllum and Colonial Lophelia. BMC Evol. Biol. 2016, 16, 108,  DOI: 10.1186/s12862-016-0654-8
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    Merging scleractinian genera: the overwhelming genetic similarity between solitary Desmophyllum and colonial Lophelia
    Addamo, Anna Maria; Vertino, Agostina; Stolarski, Jaroslaw; Garcia-Jimenez, Ricardo; Taviani, Marco; Machordom, Annie
    BMC Evolutionary Biology (2016), 16 (), 108/1-108/17CODEN: BEBMCG; ISSN:1471-2148. (BioMed Central Ltd.)
    Background: In recent years, several types of mol. markers and new microscale skeletal characters have shown potential as powerful tools for phylogenetic reconstructions and higher-level taxonomy of scleractinian corals. Nonetheless, discrimination of closely related taxa is still highly controversial in scleractinian coral research. Here we used newly sequenced complete mitochondrial genomes and 30 microsatellites to define the genetic divergence between two closely related azooxanthellate taxa of the family Caryophylliidae: solitary Desmophyllum dianthus and colonial Lophelia pertusa. Results: In the mitochondrial control region, an astonishing 99.8 % of nucleotides between L. pertusa and D. dianthus were identical. Variability of the mitochondrial genomes of the two species is represented by only 12 non-synonymous out of 19 total nucleotide substitutions. Microsatellite sequence (37 loci) anal. of L. pertusa and D. dianthus showed genetic similarity is about 97 %. Our results also indicated that L. pertusa and D. dianthus show high skeletal plasticity in corallum shape and similarity in skeletal ontogeny, micromorphol. (septal and wall granulations) and microstructural characters (arrangement of rapid accretion deposits, thickening deposits). Conclusions: Molecularly and morphol., the solitary Desmophyllum and the dendroid Lophelia appear to be significantly more similar to each other than other unambiguous coral genera analyzed to date. This consequently leads to ascribe both taxa under the generic name Desmophyllum (priority by date of publication). Findings of this study demonstrate that coloniality may not be a robust taxonomic character in scleractinian corals.
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    Cordes, E. E.; McGinley, M. P.; Podowski, E. L.; Becker, E. L.; Lessard-Pilon, S.; Viada, S. T.; Fisher, C. R. Coral Communities of the Deep Gulf of Mexico. Deep Sea Res., Part I 2008, 55, 777– 787,  DOI: 10.1016/j.dsr.2008.03.005
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    Andruszkiewicz, E. A.; Sassoubre, L. M.; Boehm, A. B. Persistence of Marine Fish Environmental DNA and the Influence of Sunlight. PLoS One 2017, 12, e0185043  DOI: 10.1371/journal.pone.0185043
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    Persistence of marine fish environmental DNA and the influence of sunlight
    Andruszkiewicz, Elizabeth A.; Sassoubre, Lauren M.; Boehm, Alexandria B.
    PLoS One (2017), 12 (9), e0185043/1-e0185043/18CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Harnessing information encoded in environmental DNA (eDNA) in marine waters has the potential to revolutionize marine biomonitoring. Whether using organism-specific quant. PCR assays or metabarcoding in conjunction with amplicon sequencing, scientists have illustrated that realistic organism censuses can be inferred from eDNA. The next step is establishing ways to link information obtained from eDNA analyses to actual organism abundance. This is only possible by understanding the processes that control eDNA concns. The present study uses mesocosm expts. to study the persistence of eDNA in marine waters and explore the role of sunlight in modulating eDNA persistence. We seeded solute-permeable dialysis bags with water contg. indigenous eDNA and suspended them in a large tank contg. seawater. Bags were subjected to two treatments: half the bags were suspended near the water surface where they received high doses of sunlight, and half at depth where they received lower doses of sunlight. Bags were destructively sampled over the course of 87 h. eDNA was extd. from water samples and used as template for a Scomberjaponicus qPCR assay and a marine fish-specific 12S rRNA PCR assay. The latter was subsequently sequenced using a metabarcoding approach. S. japonicus eDNA, as measured by qPCR, exhibited first order decay with a rate const. ∼0.01 h-1 with no difference in decay rate consts. between the two exptl. treatments. eDNA metabarcoding identified 190 organizational taxonomic units (OTUs) assigned to varying taxonomic ranks. There was no difference in marine fish communities as measured by eDNA metabarcoding between the two exptl. treatments, but there was an effect of time. Given the differences in UVA and UVB fluence received by the two exptl. treatments, we conclude that sunlight is not the main driver of fish eDNA decay in the expts. However, there are clearly temporal effects that need to be considered when interpreting information obtained using eDNA approaches.
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    Tedetti, M.; Sempéré, R. Penetration of Ultraviolet Radiation in the Marine Environment. A Review. Photochem. Photobiol. 2006, 82, 389– 397,  DOI: 10.1562/2005-11-09-ir-733
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    Penetration of ultraviolet radiation in the marine environment. A review
    Tedetti, Marc; Sempere, Richard
    Photochemistry and Photobiology (2006), 82 (Mar./Apr.), 389-397CODEN: PHCBAP; ISSN:0031-8655. (American Society for Photobiology)
    A review concerning underwater instruments used to measure UVR (UV radiation), presenting data dealing with UVR depth penetration in different oceanic areas, is given. Topics discussed include: introduction; materials and methods (radiometers, dosimeters); and results and discussion (UVR penetration in different ocean areas [open ocean, Antarctic water, coastal water], relationship between UV-B penetration and DNA damage ED).
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    Spens, J.; Evans, A. R.; Halfmaerten, D.; Knudsen, S. W.; Sengupta, M. E.; Mak, S. S. T.; Sigsgaard, E. E.; Hellström, M. Comparison of capture and storage methods for aqueous macrobial eDNA using an optimized extraction protocol: advantage of enclosed filter. Methods Ecol. Evol. 2017, 8, 635– 645,  DOI: 10.1111/2041-210x.12683
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    Govindarajan, A. F.; Francolini, R. D.; Jech, J. M.; Lavery, A. C.; Llopiz, J. K.; Wiebe, P. H.; Zhang, W. G. Exploring the Use of Environmental DNA (eDNA) to Detect Animal Taxa in the Mesopelagic Zone. Front. Ecol. Evol. 2021, 9, 1– 17,  DOI: 10.3389/fevo.2021.574877
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    Klymus, K. E.; Merkes, C. M.; Allison, M. J.; Goldberg, C. S.; Helbing, C. C.; Hunter, M. E.; Jackson, C. A.; Lance, R. F.; Mangan, A. M.; Monroe, E. M.; Piaggio, A. J.; Stokdyk, J. P.; Wilson, C. C.; Richter, C. A. Reporting the Limits of Detection and Quantification for Environmental DNA Assays. Environ. DNA 2020, 2, 271– 282,  DOI: 10.1002/edn3.29
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    Shogren, A. J.; Tank, J. L.; Egan, S. P.; August, O.; Rosi, E. J.; Hanrahan, B. R.; Renshaw, M. A.; Gantz, C. A.; Bolster, D. Water Flow and Biofilm Cover Influence Environmental DNA Detection in Recirculating Streams. Environ. Sci. Technol. 2018, 52, 8530– 8537,  DOI: 10.1021/acs.est.8b01822
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    Water Flow and Biofilm Cover Influence Environmental DNA Detection in Recirculating Streams
    Shogren, Arial J.; Tank, Jennifer L.; Egan, Scott P.; August, Olivia; Rosi, Emma J.; Hanrahan, Brittany R.; Renshaw, Mark A.; Gantz, Crysta A.; Bolster, Diogo
    Environmental Science & Technology (2018), 52 (15), 8530-8537CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)
    The increasing use of environmental DNA (eDNA) for detn. of species presence in aquatic ecosystems is an invaluable technique for both ecol. as a field and for the management of aquatic ecosystems. We examd. the degrdn. dynamics of fish eDNA using an exptl. array of recirculating streams, also using a "nested" primer assay to est. degrdn. among eDNA fragment sizes. We introduced eDNA into streams with a range of water velocities (0.1-0.8 m s-1) and substrate biofilm coverage (0-100%) and monitored eDNA concns. over time (∼10 d) to assess how biophys. conditions influence eDNA persistence. We found that the presence of biofilm significantly increased initial decay rates relative to previous studies conducted in nonflowing microcosms, suggesting important differences in detection and persistence in lentic vs lotic systems. Lastly, by using a nested primer assay that targeted different size eDNA fragments, we found that fragment size altered both the estd. rate const. coeffs., as well as eDNA detectability over time. Larger fragments (>600 bp) were quickly degraded, while shorter fragments (<100 bp) remained detectable for the entirety of the expt. When using eDNA as a stream monitoring tool, understanding environmental factors controlling eDNA degrdn. will be crit. for optimizing eDNA sampling strategies.
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    Bylemans, J.; Furlan, E. M.; Gleeson, D. M.; Hardy, C. M.; Duncan, R. P. Does Size Matter? An Experimental Evaluation of the Relative Abundance and Decay Rates of Aquatic Environmental DNA. Environ. Sci. Technol. 2018, 52, 6408– 6416,  DOI: 10.1021/acs.est.8b01071
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    Does Size Matter? An Experimental Evaluation of the Relative Abundance and Decay Rates of Aquatic Environmental DNA
    Bylemans, Jonas; Furlan, Elise M.; Gleeson, Dianne M.; Hardy, Christopher M.; Duncan, Richard P.
    Environmental Science & Technology (2018), 52 (11), 6408-6416CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)
    Environmental DNA (eDNA) is increasingly used to monitor aquatic macrofauna. Typically, short mitochondrial DNA fragments are targeted because these should be relatively more abundant in the environment as longer fragments will break into smaller fragments over time. However, longer fragments may permit more flexible primer design and increase taxonomic resoln. for eDNA metabarcoding analyses, and recent studies have shown that long mitochondrial eDNA fragments can be extd. from environmental water samples. Nuclear eDNA fragments have also been proposed as targets, but little is known about their persistence in the aquatic environment. Here we measure the abundance of mitochondrial eDNA fragments of different lengths and of short nuclear eDNA fragments, originating from captive fish in exptl. tanks, and we test whether longer mitochondrial and short nuclear fragments decay faster than short mitochondrial fragments following fish removal. We show that when fish are present, shorter mitochondrial fragments are more abundant in water samples than both longer mitochondrial fragments and short nuclear eDNA fragments. However, the rate of decay following fish removal was similar for all fragment types, suggesting that the differences in abundance resulted from differences in the rates at which different fragment types were produced rather than differences in their decay rates.
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    Estimating regression models with unknown break-points
    Muggeo Vito M R
    Statistics in medicine (2003), 22 (19), 3055-71 ISSN:0277-6715.
    This paper deals with fitting piecewise terms in regression models where one or more break-points are true parameters of the model. For estimation, a simple linearization technique is called for, taking advantage of the linear formulation of the problem. As a result, the method is suitable for any regression model with linear predictor and so current software can be used; threshold modelling as function of explanatory variables is also allowed. Differences between the other procedures available are shown and relative merits discussed. Simulations and two examples are presented to illustrate the method.
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    Bates, D.; Mächler, M.; Bolker, B.; Walker, S. Fitting Linear Mixed-Effects Models Using Lme4. J. Stat. Softw. 2015, 67, 1– 48,  DOI: 10.18637/jss.v067.i01
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    Harrison, X. A.; Donaldson, L.; Correa-Cano, M. E.; Evans, J.; Fisher, D. N.; Goodwin, C. E. D.; Robinson, B. S.; Hodgson, D. J.; Inger, R. A Brief Introduction to Mixed Effects Modelling and Multi-Model Inference in Ecology. Peerj 2018, 6, e4794  DOI: 10.7717/peerj.4794
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    Thomsen, P. F.; Kielgast, J.; Iversen, L. L.; Møller, P. R.; Rasmussen, M.; Willerslev, E. Detection of a Diverse Marine Fish Fauna Using Environmental DNA from Seawater Samples. PLoS One 2012, 7, e41732  DOI: 10.1371/journal.pone.0041732
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    Detection of a diverse marine fish fauna using environmental DNA from seawater samples
    Thomsen, Philip Francis; Kielgast, Jos; Iversen, Lars Loensmann; Moller, Peter Rask; Rasmussen, Morten; Willerslev, Eske
    PLoS One (2012), 7 (8), e41732CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    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 com. species, and restricted to particular areas with favorable 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 1/2-L 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). Addnl., 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 expt. addressing eDNA degrdn. 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.
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    Sassoubre, L. M.; Yamahara, K. M.; Gardner, L. D.; Block, B. A.; Boehm, A. B. Quantification of Environmental DNA (eDNA) Shedding and Decay Rates for Three Marine Fish. Environ. Sci. Technol. 2016, 50, 10456– 10464,  DOI: 10.1021/acs.est.6b03114
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    Quantification of Environmental DNA (eDNA) Shedding and Decay Rates for Three Marine Fish
    Sassoubre, Lauren M.; Yamahara, Kevan M.; Gardner, Luke D.; Block, Barbara A.; Boehm, Alexandria B.
    Environmental Science & Technology (2016), 50 (19), 10456-10464CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)
    Anal. of environmental DNA (eDNA) to identify macroorganisms and biodiversity has the potential to significantly augment spatial and temporal biol. monitoring in aquatic ecosystems. Current monitoring methods relying on the phys. identification of organisms can be time consuming, expensive, and invasive. Measuring eDNA shed from organisms provides detailed information on the presence and abundance of communities of organisms. However, little is known about eDNA shedding and decay in aquatic environments. In the present study, we designed novel Taqman qPCR assays for three ecol. and economically important marine fish-Engraulis mordax (Northern Anchovy), Sardinops sagax (Pacific Sardine), and Scomber japonicas (Pacific Chub Mackerel). We subsequently measured fish eDNA shedding and decay rates in seawater mesocosms. eDNA shedding rates ranged from 165 to 3368 pg of DNA per h per g of biomass. First-order decay rate consts. ranged from 0.055 to 0.101 per h. We also examd. the size fractionation of eDNA and concluded eDNA is both intra- and extracellular. Finally, we derived a simple mass-balance model to est. fish abundance from eDNA concn. The mesocosm-derived shedding and decay rates inform the interpretation of eDNA concns. measured in environmental samples and future use of eDNA as a monitoring tool.
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    Minamoto, T.; Fukuda, M.; Katsuhara, K. R.; Fujiwara, A.; Hidaka, S.; Yamamoto, S.; Takahashi, K.; Masuda, R. Environmental DNA Reflects Spatial and Temporal Jellyfish Distribution. PLoS One 2017, 12, e0173073  DOI: 10.1371/journal.pone.0173073
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    Environmental DNA reflects spatial and temporal jellyfish distribution
    Minamoto, Toshifumi; Fukuda, Miho; Katsuhara, Koki R.; Fujiwara, Ayaka; Hidaka, Shunsuke; Yamamoto, Satoshi; Takahashi, Kohji; Masuda, Reiji
    PLoS One (2017), 12 (2), e0173073/1-e0173073/15CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Recent development of environmental DNA (eDNA) anal. allows us to survey underwater macro-organisms easily and cost effectively; however, there have been no reports on eDNA detection or quantification for jellyfish. Here we present the first report on an eDNA anal. of marine jellyfish using Japanese sea nettle (Chrysaora pacifica) as a model species by combining a tank expt. with spatial and temporal distribution surveys. We performed a tank expt. monitoring eDNA concns. over a range of time intervals after the introduction of jellyfish, and quantified the eDNA concns. by quant. real-time PCR. The eDNA concns. peaked twice, at 1 and 8 h after the beginning of the expt., and became stable within 48 h. The estd. release rates of the eDNA in jellyfish were higher than the rates previously reported in fishes. A spatial survey was conducted in June 2014 in Maizuru Bay, Kyoto, in which eDNA was collected from surface water and sea floor water samples at 47 sites while jellyfish near surface water were counted on board by eye. The distribution of eDNA in the bay corresponded with the distribution of jellyfish inferred by visual observation, and the eDNA concn. in the bay was ∼13 times higher on the sea floor than on the surface. The temporal survey was conducted from March to Nov. 2014, in which jellyfish were counted by eye every morning while eDNA was collected from surface and sea floor water at three sampling points along a pier once a month. The temporal fluctuation pattern of the eDNA concns. and the nos. of obsd. individuals were well correlated. We conclude that an eDNA approach is applicable for jellyfish species in the ocean.
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    Wood, S. A.; Biessy, L.; Latchford, J. L.; Zaiko, A.; von Ammon, U.; Audrezet, F.; Cristescu, M. E.; Pochon, X. Release and Degradation of Environmental DNA and RNA in a Marine System. Sci. Total Environ. 2020, 704, 135314  DOI: 10.1016/j.scitotenv.2019.135314
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    Release and degradation of environmental DNA and RNA in a marine system
    Wood, Susanna A.; Biessy, Laura; Latchford, Janie L.; Zaiko, Anastasija; von Ammon, Ulla; Audrezet, Francois; Cristescu, Melania E.; Pochon, Xavier
    Science of the Total Environment (2020), 704 (), 135314CODEN: STENDL; ISSN:0048-9697. (Elsevier B.V.)
    Over the last decade, there has been growing interest in the anal. of environmental DNA (eDNA) to infer the presence of organisms in aquatic environments. The efficacy of eDNA/eRNA based tools are highly depend on the turnover rate of the mol. (their release and degrdn.). Environmental DNA has been shown to persist for days, weeks or years in environmental samples. Environmental RNA (eRNA) is thought to degrade faster than eDNA, however to our knowledge, no exptl. studies have explored this. Here we present an aquarium study to investigate eDNA and eRNA shedding rates and degrdn. for two sessile marine invertebrates. The copy nos. for eDNA and eRNA were assessed using droplet digital PCR targeting the mitochondrial Cytochrome c Oxidase subunit 1 (COI) gene. Environmental RNA persisted after organism removal for much longer than expected with detections for up to 13 h. In contrast, eDNA was detected is samples collected up to 94 h after organism removal. There was no evidence that the decay rates consts. for eDNA and eRNA were different (p = 0.6, Kruskal-Wallis tests). Both eDNA and eRNA was detected in biofilms collected at the end of the expt. (day 21). This suggests binding with org. or inorg. compds. or stabilization of these mols. in the biofilm matrix. The finding of the prolonged persistence of eRNA may provide new opportunities for improved biodiversity surveys through reducing false positives caused by legacy DNA and could also facilitate new research on environmental transcriptomics.
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    Kwong, S. L. T.; Villacorta-Rath, C.; Doyle, J.; Uthicke, S. Quantifying Shedding and Degradation Rates of Environmental DNA (eDNA) from Pacific Crown-of-Thorns Seastar (Acanthaster cf. solaris). Mar. Biol. 2021, 168, 1– 10,  DOI: 10.1007/s00227-021-03896-x
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    Kirtane, A.; Wieczorek, D.; Noji, T.; Baskin, L.; Ober, C.; Plosica, R.; Chenoweth, A.; Lynch, K.; Sassoubre, L. Quantification of Environmental DNA (eDNA) Shedding and Decay Rates for Three Commercially Harvested Fish Species and Comparison between EDNA Detection and Trawl Catches. Environ. DNA 2021, 3, 1142– 1155,  DOI: 10.1002/edn3.236
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    Zaiko, A.; von Ammon, U.; Stuart, J.; Smith, K. F.; Yao, R.; Welsh, M.; Pochon, X.; Bowers, H. A. Assessing the Performance and Efficiency of Environmental DNA/RNA Capture Methodologies under Controlled Experimental Conditions. Methods Ecol. Evol. 2022,  DOI: 10.1111/2041-210x.13879
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    Murray, F.; De Clippele, L. H.; Hiley, A.; Wicks, L.; Roberts, J. M.; Hennige, S. Multiple Feeding Strategies Observed in the Cold-Water Coral Lophelia pertusa. J. Mar. Biol. Assoc. U. K. 2019, 99, 1281– 1283,  DOI: 10.1017/s0025315419000298
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    Canals, O.; Mendibil, I.; Santos, M.; Irigoien, X.; Rodríguez-Ezpeleta, N. Vertical Stratification of Environmental DNA in the Open Ocean Captures Ecological Patterns and Behavior of Deep-sea Fishes. Limnol. Oceanogr. Lett. 2021, 6, 339– 347,  DOI: 10.1002/lol2.10213
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    Vertical stratification of environmental DNA in the open ocean captures ecological patterns and behavior of deep-sea fishes
    Canals, Oriol; Mendibil, Inaki; Santos, Maria; Irigoien, Xabier; Rodriguez-Ezpeleta, Naiara
    Limnology & Oceanography Letters (2021), 6 (6), 339-347CODEN: LOLIBT; ISSN:2378-2242. (John Wiley & Sons Ltd.)
    Establishing the foundations for a sustainable use of deep-sea resources relies on increasing knowledge on this inaccessible ecosystem, which is challenging with traditional methods. The anal. of environmental DNA (eDNA) emerges as an alternative, but it has been scarcely applied to deep-sea fish. Here, we have analyzed the fish eDNA contained in oceanic vertical profile samples (up to 2000 m depth) collected throughout the continental slope of the Bay of Biscay. We detected 52 different fish species, of which 25 were classified as deep-sea fish. We found an increase of deep-sea fish richness and abundance with depth, and that eDNA reflects day-night community patterns and species-specific vertical distributions that are consistent with the known diel migratory behavior of many mesopelagic fishes. These findings highlight the potential of eDNA to improve knowledge on the fish species inhabiting the dark ocean before this still pristine ecosystem is further exploited.
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    Monuki, K.; Barber, P. H.; Gold, Z. eDNA Captures Depth Partitioning in a Kelp Forest Ecosystem. PLoS One 2021, 16, e0253104  DOI: 10.1371/journal.pone.0253104
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    eDNA captures depth partitioning in a kelp forest ecosystem
    Monuki, Keira; Barber, Paul H.; Gold, Zachary
    PLoS One (2021), 16 (11), e0253104CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Environmental DNA (eDNA) metabarcoding is an increasingly important tool for surveying biodiversity in marine ecosystems. However, the scale of temporal and spatial variability in eDNA signatures, and how this variation may impact eDNA-based marine biodiversity assessments, remains uncertain. To address this question, we systematically examd. variation in vertebrate eDNA signatures across depth (0 m to 10 m) and horizontal space (nearshore kelp forest and surf zone) over three successive days in Southern California. Across a broad range of teleost fish and elasmobranchs, results showed significant variation in species richness and community assemblages between surface and depth, reflecting microhabitat depth preferences of common Southern California nearshore rocky reef taxa. Community assemblages between nearshore and surf zone sampling stations at the same depth also differed significantly, consistent with known habitat preferences. Addnl., assemblages also varied across three sampling days, but 69% of habitat preferences remained consistent. Results highlight the sensitivity of eDNA in capturing fine-scale vertical, horizontal, and temporal variation in marine vertebrate communities, demonstrating the ability of eDNA to capture a highly localized snapshot of marine biodiversity in dynamic coastal environments.
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    Allan, E. A.; DiBenedetto, M. H.; Lavery, A. C.; Govindarajan, A. F.; Zhang, W. G. Modeling Characterization of the Vertical and Temporal Variability of Environmental DNA in the Mesopelagic Ocean. Sci. Rep. 2021, 11, 21273,  DOI: 10.1038/s41598-021-00288-5
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    Modeling characterization of the vertical and temporal variability of environmental DNA in the mesopelagic ocean
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    Scientific Reports (2021), 11 (1), 21273CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
    Increasingly, researchers are using innovative methods to census marine life, including identification of environmental DNA (eDNA) left behind by organisms in the water column. However, little is understood about how eDNA is distributed in the ocean, given that organisms are mobile and that phys. and biol. processes can transport eDNA after release from a host. Particularly in the vast mesopelagic ocean where many species vertically migrate hundreds of meters diurnally, it is important to link the location at which eDNA was shed by a host organism to the location at which eDNA was collected in a water sample. Here, we present a one-dimensional mechanistic model to simulate the eDNA vertical distribution after its release and to compare the impact of key biol. and phys. parameters on the eDNA vertical and temporal distribution. The modeled vertical eDNA profiles allow us to quantify spatial and temporal variability in eDNA concn. and to identify the most important parameters to consider when interpreting eDNA signals. We find that the vertical displacement by advection, dispersion, and settling has limited influence on the eDNA distribution, and the depth at which eDNA is found is generally within tens of meters of the depth at which the eDNA was originally shed from the organism. Thus, using information about representative vertical migration patterns, eDNA concn. variability can be used to answer ecol. questions about migrating organisms such as what depths species can be found in the daytime and nighttime and what percentage of individuals within a species diurnally migrate. These findings are crit. both to advance the understanding of the vertical distribution of eDNA in the water column and to link eDNA detection to organism presence in the mesopelagic ocean as well as other aquatic environments.
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    West, K. M.; Stat, M.; Harvey, E. S.; Skepper, C. L.; DiBattista, J. D.; Richards, Z. T.; Travers, M. J.; Newman, S. J.; Bunce, M. eDNA Metabarcoding Survey Reveals Fine-Scale Coral Reef Community Variation across a Remote, Tropical Island Ecosystem. Mol. Ecol. 2020, 29, 1069– 1086,  DOI: 10.1111/mec.15382
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    eDNA metabarcoding survey reveals fine-scale coral reef community variation across a remote, tropical island ecosystem
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    Environmental DNA metabarcoding, a technique for retrieving multispecies DNA from environmental samples, can detect a diverse array of marine species from filtered seawater samples. There is a growing potential to integrate eDNA alongside existing monitoring methods in order to establish or improve the assessment of species diversity. We investigated the utility of eDNA metabarcoding as a high-resoln., multitrophic biomonitoring tool at the Cocos Islands, Australia (CKI)-a remote tropical coral reef atoll situated within the eastern Indian Ocean. Metabarcoding assays targeting the mitochondrial 16S rRNA and CO1 genes, as well as the 18S rRNA nuclear gene, were applied to 252 surface seawater samples collected from 42 sites within a 140 km2 area. Our assays successfully detected a wide range of bony fish and elasmobranchs (244 taxa), crustaceans (88), molluscs (37) and echinoderms (7). Assemblage compn. varied significantly between sites, reflecting habitat partitioning across the island ecosystem and demonstrating the localisation of eDNA signals, despite extensive tidal and oceanic movements. In addn., we document putative new occurrence records for 46 taxa and compare the efficiency of our eDNA approach to visual survey techniques at CKI. Our study demonstrates the utility of a multimarker metabarcoding approach in capturing multitrophic biodiversity across an entire coral reef atoll and sets an important baseline for ongoing monitoring and management.
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    Lamy, T.; Pitz, K. J.; Chavez, F. P.; Yorke, C. E.; Miller, R. J. Environmental DNA Reveals the Fine-Grained and Hierarchical Spatial Structure of Kelp Forest Fish Communities. Sci. Rep. 2021, 11, 1– 13,  DOI: 10.1038/s41598-021-93859-5
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    Laroche, O.; Kersten, O.; Smith, C. R.; Goetze, E. Environmental DNA Surveys Detect Distinct Metazoan Communities across Abyssal Plains and Seamounts in the Western Clarion Clipperton Zone. Mol. Ecol. 2020, 29, 4588– 4604,  DOI: 10.1111/mec.15484
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    Environmental DNA surveys detect distinct metazoan communities across abyssal plains and seamounts in the western Clarion Clipperton Zone
    Laroche, Olivier; Kersten, Oliver; Smith, Craig R.; Goetze, Erica
    Molecular Ecology (2020), 29 (23), 4588-4604CODEN: MOECEO; ISSN:0962-1083. (Wiley-Blackwell)
    The deep seafloor serves as a reservoir of biodiversity in the global ocean, with >80% of invertebrates at abyssal depths still undescribed. These diverse and remote deep-sea communities are critically under-sampled and increasingly threatened by anthropogenic impacts, including future polymetallic nodule mining. Using a multigene environmental DNA (eDNA) metabarcoding approach, we characterized metazoan communities sampled from sediments, polymetallic nodules and seawater in the western Clarion Clipperton Zone (CCZ) to test the hypotheses that deep seamounts (a) are species richness hotspots in the abyss, (b) have structurally distinct communities in comparison to other deep-sea habitats, and (c) that seafloor particulate org. carbon (POC) flux and polymetallic nodule d. are pos. correlated with metazoan diversity. eDNA metabarcoding was effective at characterizing distinct biotas known to occur in assocn. with different abyssal substrate types (e.g., nodule- and sediment-specific fauna), with distinct community compn. and few taxa shared across substrates. Seamount faunas had higher overall taxonomic richness, and different community compn. and biogeog. than adjacent abyssal plains, with seamount communities displaying less connectivity between regions than comparable assemblages on the abyssal plains. Across an estd. gradient of low to moderate POC flux, we find lowest taxon richness at the lowest POC flux, as well as an effect of nodule size on community compn. Our results suggest that while abyssal seamounts are important reservoirs of metazoan diversity in the CCZ, given limited taxonomic overlap between seamount and plains fauna, conservation of seamount assemblages will be insufficient to protect biodiversity and ecosystem function in regions targeted for mining.

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  38. Ngoc-Loi Nguyen, Dhanushka Devendra, Natalia Szymańska, Mattia Greco, Inès Barrenechea Angeles, Agnes K. M. Weiner, Jessica Louise Ray, Tristan Cordier, Stijn De Schepper, Jan Pawłowski, Joanna Pawłowska. Sedimentary ancient DNA: a new paleogenomic tool for reconstructing the history of marine ecosystems. Frontiers in Marine Science 2023, 10 https://doi.org/10.3389/fmars.2023.1185435
  39. Tyson J. Bessell, Sharon A. Appleyard, Rick D. Stuart‐Smith, Olivia J. Johnson, Scott D. Ling, Freddie J. Heather, Tim P. Lynch, Neville S. Barrett, Jemina Stuart‐Smith. Using eDNA and SCUBA surveys for detection and monitoring of a threatened marine cryptic fish. Aquatic Conservation: Marine and Freshwater Ecosystems 2023, 33 (5) , 431-442. https://doi.org/10.1002/aqc.3944
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Cite this: Environ. Sci. Technol. 2022, 56, 12, 8629–8639
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  • Abstract

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    Figure 1

    Figure 1. (A) Matrix of target conditions for 11 combinations of temperature, pH, and [DO] investigated to determine the persistence of eDNA from the coral Lophelia among a range of marine physicochemical states. Two replicate experiments were conducted for each combination of temperature, pH, and [DO]. At 20 °C temperature, half- and fully saturated oxygen experiments (shaded light blue) represent conditions characteristic of subtropical near-surface environments. At 4 °C temperature, half-saturated experiments (shaded dark blue) represent conditions characteristic of the deep-sea environment. The remaining cells (shaded blue) represent other conditions characteristic of the global open and deep ocean. (B) Schematic example of experimental conditions. Compressed air and nitrogen gas flow rates were adjusted to reach the target [DO]. Small doses of 0.5 M HCl were automatically administered to the tanks when target pH values were exceeded. Physicochemical measurements were monitored by suspending probes in the tanks. Caps were secured with O-rings to control the oxygen concentration in the above headspace. All tubing and probes were placed through small openings in these caps. A photograph of the experimental setup is presented in Figure S1. (C) Measurements of temperature, pH, and [DO] over the 22 eDNA degradation experiments. Points indicate individual measurements, and paths are drawn through daily averages. All measurements were recorded daily and at each sampling time point. Temperature measurements were made using a laser thermometer. pH and [DO] measurements were made with probes.

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    Figure 2

    Figure 2. Degradation of Lophelia eDNA in 22 experiments among a range of marine physicochemical states. eDNA concentration was measured as the concentration of a 154 base pair fragment of the Lophelia mitochondrial COI gene. The concentration of eDNA (y-axis) is plotted against time (x-axis). The y-axis is natural-log scaled. Different colors represent the two experimental replicates at each experimental condition. Points represent the average of three qPCR replicate measurements for two samples at a given time point. Lines represent the fit to a biphasic model. Thin lines connecting points to the line of best fit represent the distance from the observed to the fitted values at each time point (the residuals). Panels are arranged by temperature (descending top to bottom) and [DO] (increasing left to right). Values shown on top of each panel indicate the target experimental conditions. Points below the limit of quantification of the qPCR assay (77.8 copies/reaction or 13 copies/mL seawater filtered) are not plotted. The dashed line indicates the limit of quantification.

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    Figure 3

    Figure 3. Decay rate constant (k) estimates for the initial (A) and second (B) phases of eDNA degradation for 22 experiments conducted across 11 combinations of temperature, pH, and [DO]. Decay rate constants were estimated by fitting an exponential decay equation with initial and second degradation phases with different rates (biphasic). Decay rate constants are arranged on the x axis by the average pH over the course of each experiment. Vertical error bars represent 95% confidence intervals for the decay rate constants. The color of each point represents the average [DO] over each experiment.

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    Figure 4

    Figure 4. eDNA persistence time (time until degradation of 99.9% of starting eDNA concentration), estimated using decay rate constants from our study and other published marine studies, as a function of temperature. The linear model was only fit to decay rate constants calculated from simple exponential models. The best fit line is indicated by the dashed line, and the shaded region represents the 95% confidence interval for the slope. Points are dodged slightly from actual recorded temperatures to improve visualization. (Inset) Comparison of calculated persistence times when fitting a single exponential versus a biphasic model to the data in this study. Data are reported in Table S7.

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      Palaeontologists characterize mass extinctions as times when the Earth loses more than three-quarters of its species in a geol. short interval, as has happened only five times in the past 540 million years or so. Biologists now suggest that a sixth mass extinction may be under way, given the known species losses over the past few centuries and millennia. Here we review how differences between fossil and modern data and the addn. of recently available palaeontol. information influence our understanding of the current extinction crisis. Our results confirm that current extinction rates are higher than would be expected from the fossil record, highlighting the need for effective conservation measures.
    2. 2
      Dirzo, R.; Young, H. S.; Galetti, M.; Ceballos, G.; Isaac, N. J. B.; Collen, B. Defaunation in the Anthropocene. Science 2014, 345, 401– 406,  DOI: 10.1126/science.1251817
      2
      Defaunation in the Anthropocene
      Dirzo, Rodolfo; Young, Hillary S.; Galetti, Mauro; Ceballos, Gerardo; Isaac, Nick J. B.; Collen, Ben
      Science (Washington, DC, United States) (2014), 345 (6195), 401-406CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      We live amid a global wave of anthropogenically driven biodiversity loss: species and population extirpations and, critically, declines in local species abundance. Particularly, human impacts on animal biodiversity are an under-recognized form of global environmental change. Among terrestrial vertebrates, 322 species have become extinct since 1500, and populations of the remaining species show 25% av. decline in abundance. Invertebrate patterns are equally dire: 67% of monitored populations show 45% mean abundance decline. Such animal declines will cascade onto ecosystem functioning and human well-being. Much remains unknown about this "Anthropocene defaunation"; these knowledge gaps hinder our capacity to predict and limit defaunation impacts. Clearly, however, defaunation is both a pervasive component of the planet's sixth mass extinction and also a major driver of global ecol. change.
    3. 3
      McCauley, D. J.; Pinsky, M. L.; Palumbi, S. R.; Estes, J. A.; Joyce, F. H.; Warner, R. R. Marine Defaunation: Animal Loss in the Global Ocean. Science 2015, 347, 1255641,  DOI: 10.1126/science.1255641
      3
      Marine defaunation: animal loss in the global ocean
      McCauley Douglas J; Joyce Francis H; Warner Robert R; Pinsky Malin L; Palumbi Stephen R; Estes James A
      Science (New York, N.Y.) (2015), 347 (6219), 1255641 ISSN:.
      Marine defaunation, or human-caused animal loss in the oceans, emerged forcefully only hundreds of years ago, whereas terrestrial defaunation has been occurring far longer. Though humans have caused few global marine extinctions, we have profoundly affected marine wildlife, altering the functioning and provisioning of services in every ocean. Current ocean trends, coupled with terrestrial defaunation lessons, suggest that marine defaunation rates will rapidly intensify as human use of the oceans industrializes. Though protected areas are a powerful tool to harness ocean productivity, especially when designed with future climate in mind, additional management strategies will be required. Overall, habitat degradation is likely to intensify as a major driver of marine wildlife loss. Proactive intervention can avert a marine defaunation disaster of the magnitude observed on land.
    4. 4
      Thomsen, P. F.; Willerslev, E. Environmental DNA - An Emerging Tool in Conservation for Monitoring Past and Present Biodiversity. Biol. Conserv. 2015, 183, 4– 18,  DOI: 10.1016/j.biocon.2014.11.019
      There is no corresponding record for this reference.
    5. 5
      Breed, M. F.; Harrison, P. A.; Blyth, C.; Byrne, M.; Gaget, V.; Gellie, N. J. C.; Groom, S. V. C.; Hodgson, R.; Mills, J. G.; Prowse, T. A. A.; Steane, D. A.; Mohr, J. J. The Potential of Genomics for Restoring Ecosystems and Biodiversity. Nat. Rev. Genet. 2019, 20, 615– 628,  DOI: 10.1038/s41576-019-0152-0
      5
      The potential of genomics for restoring ecosystems and biodiversity
      Breed, Martin F.; Harrison, Peter A.; Blyth, Colette; Byrne, Margaret; Gaget, Virginie; Gellie, Nicholas J. C.; Groom, Scott V. C.; Hodgson, Riley; Mills, Jacob G.; Prowse, Thomas A. A.; Steane, Dorothy A.; Mohr, Jakki J.
      Nature Reviews Genetics (2019), 20 (10), 615-628CODEN: NRGAAM; ISSN:1471-0056. (Nature Research)
      Billions of ha of natural ecosystems have been degraded through human actions. The global community has agreed on targets to halt and reverse these declines, and the restoration sector faces the important but arduous task of implementing programs to meet these objectives. Existing and emerging genomics tools offer the potential to improve the odds of achieving these targets. These tools include population genomics that can improve seed sourcing, meta-omics that can improve assessment and monitoring of restoration outcomes, and genome editing that can generate novel genotypes for restoring challenging environments. We identify barriers to adopting these tools in a restoration context and emphasize that regulatory and ethical frameworks are required to guide their use.
    6. 6
      Port, J. A.; O’Donnell, J. L.; Romero-Maraccini, O. C.; Leary, P. R.; Litvin, S. Y.; Nickols, K. J.; Yamahara, K. M.; Kelly, R. P. Assessing Vertebrate Biodiversity in a Kelp Forest Ecosystem Using Environmental DNA. Mol. Ecol. 2016, 25, 527– 541,  DOI: 10.1111/mec.13481
      6
      Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA
      Port, Jesse A.; O'Donnell, James L.; Romero-Maraccini, Ofelia C.; Leary, Paul R.; Litvin, Steven Y.; Nickols, Kerry J.; Yamahara, Kevan M.; Kelly, Ryan P.
      Molecular Ecology (2016), 25 (2), 527-541CODEN: MOECEO; ISSN:0962-1083. (Wiley-Blackwell)
      Preserving biodiversity is a global challenge requiring data on species' distribution and abundance over large geog. and temporal scales. However, traditional methods to survey mobile species' distribution and abundance in marine environments are often inefficient, environmentally destructive, or resource-intensive. Metabarcoding of environmental DNA (eDNA) offers a new means to assess biodiversity and on much larger scales, but adoption of this approach for surveying whole animal communities in large, dynamic aquatic systems has been slowed by significant unknowns surrounding error rates of detection and relevant spatial resoln. of eDNA surveys. Here, we report the results of a 2.5 km eDNA transect surveying the vertebrate fauna present along a gradation of diverse marine habitats assocd. with a kelp forest ecosystem. Using PCR primers that target the mitochondrial 12S rRNA gene of marine fishes and mammals, we generated eDNA sequence data and compared it to simultaneous visual dive surveys. We find spatial concordance between individual species' eDNA and visual survey trends, and that eDNA is able to distinguish vertebrate community assemblages from habitats sepd. by as little as ∼60 m. eDNA reliably detected vertebrates with low false-neg. error rates (1/12 taxa) when compared to the surveys, and revealed cryptic species known to occupy the habitats but overlooked by visual methods. This study also presents an explicit accounting of false negatives and positives in metabarcoding data, which illustrate the influence of gene marker selection, replication, contamination, biases impacting eDNA count data and ecol. of target species on eDNA detection rates in an open ecosystem.
    7. 7
      Everett, M. V.; Park, L. K. Exploring Deep-Water Coral Communities Using Environmental DNA. Deep Sea Res., Part II 2018, 150, 229– 241,  DOI: 10.1016/j.dsr2.2017.09.008
      7
      Exploring deep-water coral communities using environmental DNA
      Everett, Meredith V.; Park, Linda K.
      Deep Sea Research, Part II: Topical Studies in Oceanography (2018), 150 (), 229-241CODEN: DSROEK; ISSN:0967-0645. (Elsevier Ltd.)
      Environmental DNA (eDNA) sequencing has emerged as a valuable tool for biodiversity surveys, allowing identification of taxa that may be missed by more traditional methods. Deep-sea corals, while increasingly recognized as a valuable source of habitat in the deep-ocean, have traditionally been challenging to survey. Obstacles to traditional visual surveys of these animals include the expense and complexity inherent to working in the deep marine environment, as well as the existing taxonomic uncertainty and morphol. variation which can make deep-sea octocorals difficult to identify visually to the species level. This study tests an eDNA protocol for identification of deep-sea octocorals from water samples collected during the E/V Nautilus 2016 cruise season. Using this protocol, we were able to sequence eDNA from octocorals, and use these data along with image data collected during the cruise to identify taxa to the species level in a variety of habitats. eDNA sampling has the potential to complement traditional deep-sea coral surveys by overcoming the difficulty in visually identifying deep-sea octocorals and characterizing their diversity.
    8. 8
      Kutti, T.; Johnsen, I. A.; Skaar, K. S.; Ray, J. L.; Husa, V.; Dahlgren, T. G. Quantification of eDNA to Map the Distribution of Cold-Water Coral Reefs. Front. Mar. Sci. 2020, 7, 1– 12,  DOI: 10.3389/fmars.2020.00446
      There is no corresponding record for this reference.
    9. 9
      Whitaker, J. M.; Brower, A. L.; Janosik, A. M. Invasive Lionfish Detected in Estuaries in the Northern Gulf of Mexico Using Environmental DNA. Environ. Biol. Fishes 2021, 104, 1475– 1485,  DOI: 10.1007/s10641-021-01177-6
      There is no corresponding record for this reference.
    10. 10
      Keller, A. G.; Grason, E. W.; McDonald, P. S.; Ramón-Laca, A.; Kelly, R. P. Tracking an Invasion Front with Environmental DNA. Ecol. Appl. 2022, e2561  DOI: 10.1002/eap.2561
      There is no corresponding record for this reference.
    11. 11
      Barnes, M. A.; Turner, C. R. The Ecology of Environmental DNA and Implications for Conservation Genetics. Conserv. Genet. 2016, 17, 1– 17,  DOI: 10.1007/s10592-015-0775-4
      11
      The ecology of environmental DNA and implications for conservation genetics
      Barnes, Matthew A.; Turner, Cameron R.
      Conservation Genetics (2016), 17 (1), 1-17CODEN: CGOEAC; ISSN:1566-0621. (Springer)
      A review. Environmental DNA (eDNA) refers to the genetic material that can be extd. from bulk environmental samples such as soil, water, and even air. The rapidly expanding study of eDNA has generated unprecedented ability to detect species and conduct genetic analyses for conservation, management, and research, particularly in scenarios where collection of whole organisms is impractical or impossible. While the no. of studies demonstrating successful eDNA detection has increased rapidly in recent years, less research has explored the "ecol." of eDNA-myriad interactions between extraorganismal genetic material and its environment-and its influence on eDNA detection, quantification, anal., and application to conservation and research. Here, we outline a framework for understanding the ecol. of eDNA, including the origin, state, transport, and fate of extraorganismal genetic material. Using this framework, we review and synthesize the findings of eDNA studies from diverse environments, taxa, and fields of study to highlight important concepts and knowledge gaps in eDNA study and application. Addnl., we identify frontiers of conservation-focused eDNA application where we see the most potential for growth, including the use of eDNA for estg. population size, population genetic and genomic analyses via eDNA, inclusion of other indicator biomols. such as environmental RNA or proteins, automated sample collection and anal., and consideration of an expanded array of creative environmental samples. We discuss how a more complete understanding of the ecol. of eDNA is integral to advancing these frontiers and maximizing the potential of future eDNA applications in conservation and research.
    12. 12
      Mauvisseau, Q.; Harper, L. R.; Sander, M.; Hanner, R. H.; Kleyer, H.; Deiner, K. The Multiple States of Environmental DNA and What Is Known about Their Persistence in Aquatic Environments. Environ. Sci. Technol. 2022, 5322,  DOI: 10.1021/acs.est.1c07638
      12
      Multiple states of environmental DNA and what is known about their persistence in aquatic environments
      Mauvisseau, Quentin; Harper, Lynsey R.; Sander, Michael; Hanner, Robert H.; Kleyer, Hannah; Deiner, Kristy
      Environmental Science & Technology (2022), 56 (9), 5322-5333CODEN: ESTHAG; ISSN:1520-5851. (American Chemical Society)
      A review. Increased use of environmental DNA (eDNA) anal. for indirect species detection has spurred the need to understand eDNA persistence in the environment. Understanding the persistence of eDNA is complex because it exists in a mixt. of different states (e.g., dissolved, particle adsorbed, intracellular, and intraorganellar), and each state is expected to have a specific decay rate that depends on environmental parameters. Thus, improving knowledge about eDNA conversion rates between states and the reactions that degrade eDNA in different states is needed. Here, we focus on eukaryotic extraorganismal eDNA, outline how water chem. and suspended mineral particles likely affect conversion among each eDNA state, and indicate how environmental parameters affect persistence of states in the water column. On the basis of deducing these controlling parameters, we synthesized the eDNA literature to assess whether we could already derive a general understanding of eDNA states persisting in the environment. However, we found that these parameters are often not being measured or reported when measured, and in many cases very few exptl. data exist from which to draw conclusions. Therefore, further study of how environmental parameters affect eDNA state conversion and eDNA decay in aquatic environments is needed. We recommend analytic controls that can be used during the processing of water to assess potential losses of different eDNA states if all were present in a water sample, and we outline future exptl. work that would help det. the dominant eDNA states in water.
    13. 13
      Maruyama, A.; Nakamura, K.; Yamanaka, H.; Kondoh, M.; Minamoto, T. The Release Rate of Environmental DNA from Juvenile and Adult Fish. PLoS One 2014, 9, e114639  DOI: 10.1371/journal.pone.0114639
      13
      The release rate of environmental DNA from juvenile and adult fish
      Maruyama, Atsushi; Nakamura, Keisuke; Yamanaka, Hiroki; Kondoh, Michio; Minamoto, Toshifumi
      PLoS One (2014), 9 (12), e114639/1-e114639/13, 13 pp.CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      The environmental DNA (eDNA) technique is expected to become a powerful, non-invasive tool for estg. the distribution and biomass of organisms. This technique was recently shown to be applicable to aquatic vertebrates by collecting extraorganismal DNA floating in the water or absorbed onto suspended particles. However, basic information on eDNA release rate is lacking, despite it being essential for practical applications. In this series of expts. with bluegill sunfish (Lepomis macrochirus), we examd. the effect of fish developmental stage on eDNA release rate. EDNA concn. reached equil. 3 days after the individual fish were introduced into the sep. containers, enabling calcn. of the eDNA release rate (copies h-1) from individual fish on the assumption that the no. of eDNA released from the fish per unit time equals total degrdn. in the container (copies h -1). The eDNA release rate was 3-4 times higher in the adult (body wt.: 30-75 g) than in the juvenile group (0.5-2.0 g). Such pos. relationship between fish size and eDNA release rate support the possibility of biomass rather than d. estn. using eDNA techniques. However, the eDNA release rate per fish body wt. (copies h -1 g -1) was slightly higher in the juvenile than the adult group, which is likely because of the ontogenetic redn. in metabolic activity. Therefore, quant. eDNA data should be carefully interpreted to avoid overestimating biomass when the population is dominated by juveniles, because the age structure of the focal population is often variable and unseen in the field. EDNA degrdn. rates (copies l-1 h -1), calcd. by curve fitting of time-dependent changes in eDNA concns. after fish removal, were 5.1-15.9% per h (half-life: 6.7 h). This suggests that quant. eDNA data should be cor. using a degrdn. curve attained in the target field.
    14. 14
      Klymus, K. E.; Richter, C. A.; Chapman, D. C.; Paukert, C. Quantification of eDNA Shedding Rates from Invasive Bighead Carp Hypophthalmichthys nobilis and Silver Carp Hypophthalmichthys molitrix. Biol. Conserv. 2015, 183, 77– 84,  DOI: 10.1016/j.biocon.2014.11.020
      There is no corresponding record for this reference.
    15. 15
      Lacoursière-Roussel, A.; Côté, G.; Leclerc, V.; Bernatchez, L. Quantifying Relative Fish Abundance with EDNA: A Promising Tool for Fisheries Management. J. Appl. Ecol. 2016, 53, 1148– 1157,  DOI: 10.1111/1365-2664.12598
      15
      Quantifying relative fish abundance with eDNA: a promising tool for fisheries management
      Lacoursiere-Roussel, Anais; Cote, Guillaume; Leclerc, Veronique; Bernatchez, Louis
      Journal of Applied Ecology (2016), 53 (4), 1148-1157CODEN: JAPEAI; ISSN:0021-8901. (Wiley-Blackwell)
      Summary : Assessment and monitoring of exploited fish populations are challenged by costs, logistics and neg. impacts on target populations. These factors therefore limit large-scale effective management strategies. Evidence is growing that the quantity of eDNA may be related not only to species presence/absence, but also to species abundance. In this study, the concns. of environmental DNA (eDNA) from a highly prized sport fish species, Lake Trout Salvelinus namaycush (Walbaum 1792), were estd. in water samples from 12 natural lakes and compared to abundance and biomass data obtained from standardized gillnet catches as performed routinely for fisheries management purposes. To reduce environmental variability among lakes, all lakes were sampled in spring, between ice melt and water stratification. The eDNA concn. did not vary significantly with water temp., dissolved oxygen, pH and turbidity, but was significantly pos. correlated with relative fish abundance estd. as catch per unit effort (CPUE), whereas the relationship with biomass per unit effort (BPUE) was less pronounced. The value of eDNA to inform about local aquatic species distribution was further supported by the similarity between the spatial heterogeneity of eDNA distribution and spatial variation in CPUE measured by the gillnet method. Synthesis and applications. Large-scale empirical evidence of the relationship between the eDNA concn. and species abundance allows for the assessment of the potential to integrate eDNA within fisheries management plans. As such, the eDNA quant. method represents a promising population abundance assessment tool that could significantly reduce the costs assocd. with sampling and increase the power of detection, the spatial coverage and the frequency of sampling, without any neg. impacts on fish populations.
    16. 16
      Sansom, B. J.; Sassoubre, L. M. Environmental DNA (eDNA) Shedding and Decay Rates to Model Freshwater Mussel EDNA Transport in a River. Environ. Sci. Technol. 2017, 51, 14244– 14253,  DOI: 10.1021/acs.est.7b05199
      16
      Environmental DNA (eDNA) Shedding and Decay Rates to Model Freshwater Mussel eDNA Transport in a River
      Sansom, Brandon J.; Sassoubre, Lauren M.
      Environmental Science & Technology (2017), 51 (24), 14244-14253CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)
      Freshwater mussels are vital components of stream ecosystems, yet remain threatened. Thus, timely and accurate species counts are crit. for proper conservation and management. Mussels live in stream sediments and can be challenging to survey given constraints related to water depth, flow, and time of year. The use of environmental DNA (eDNA) to monitor mussel distributions and diversity is a promising tool. Before it can be used as a monitoring tool, however, we need to know how much eDNA mussels shed into their environment and how long the eDNA persists. We present a novel application of eDNA to est. both the presence/absence and abundance of a freshwater mussel species, Lampsilis siliquoidea. The eDNA shedding and decay rates reported within are the 1st for freshwater mussels. We detd. that eDNA shedding was statistically similar across mussel densities, but that 1st-order decay consts. varied between exptl. treatments. We effectively modeled downstream transport of eDNA and present a model that can be used as a complementary tool to est. mussel d. Our results suggest that eDNA has the potential to be a complementary tool to survey mussels and enhance current efforts to monitor and protect freshwater mussel biodiversity.
    17. 17
      Jo, T.; Arimoto, M.; Murakami, H.; Masuda, R.; Minamoto, T. Estimating Shedding and Decay Rates of Environmental Nuclear DNA with Relation to Water Temperature and Biomass. Environ. DNA 2020, 2, 140– 151,  DOI: 10.1002/edn3.51
      There is no corresponding record for this reference.
    18. 18
      Ostberg, C. O.; Chase, D. M. Ontogeny of eDNA Shedding during Early Development in Chinook Salmon (Oncorhynchus tshawytscha). Environ. DNA 2022, 339,  DOI: 10.1002/edn3.258
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      Ontogeny of eDNA shedding during early development in Chinook Salmon (Oncorhynchus tshawytscha)
      Ostberg, Carl O.; Chase, Dorothy M.
      Environmental DNA (2022), 4 (2), 339-348CODEN: EDNNAZ; ISSN:2637-4943. (John Wiley & Sons, Inc.)
      Knowledge of the timing of major life history events in aquatic species is important for informing conservation and resource management planning. Accordingly, surveys of environmental DNA (eDNA) have been performed to det. the efficacy of eDNA for providing information on life history events, primarily focusing on the timing of events assocd. with spawning, and these studies have proved successful. However, spawning represents only one part of the life history, and therefore, information on eDNA shedding during other life history stages is needed to fill gaps in knowledge. Here, we explored eDNA shedding during early life history (from fertilized eggs until near yolk sac absorption) in Chinook Salmon (Oncorhynchus tshawytscha) at three biomasses in a lab. environment. We found that fertilized eggs shed little eDNA prior to hatching. Hatching coincided with a spike in eDNA, and we obsd. a significant and pos. relationship between eDNA concn. and the no. of hatched eggs. The concn. of eDNA shed by larvae after hatching was not consistent across post-hatch sampling days, suggesting developmental and behavioral changes assocd. with larval ontogeny may affect eDNA shedding rate. These results indicate that eDNA data may be used to identify hatch timing and verify successful reprodn. in oviparous aquatic fishes. The application of eDNA to early life history broadens the capacity of eDNA-based methods for assessing population status and trends.
    19. 19
      Thalinger, B.; Rieder, A.; Teuffenbach, A.; Pütz, Y.; Schwerte, T.; Wanzenböck, J.; Traugott, M. The Effect of Activity, Energy Use, and Species Identity on Environmental DNA Shedding of Freshwater Fish. Front. Ecol. Evol. 2021, 9, 1– 13,  DOI: 10.3389/fevo.2021.623718
      There is no corresponding record for this reference.
    20. 20
      Jo, T.; Arimoto, M.; Murakami, H.; Masuda, R.; Minamoto, T. Particle Size Distribution of Environmental DNA from the Nuclei of Marine Fish. Environ. Sci. Technol. 2019, 53, 9947– 9956,  DOI: 10.1021/acs.est.9b02833
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      Particle Size Distribution of Environmental DNA from the Nuclei of Marine Fish
      Jo, Toshiaki; Arimoto, Mio; Murakami, Hiroaki; Masuda, Reiji; Minamoto, Toshifumi
      Environmental Science & Technology (2019), 53 (16), 9947-9956CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)
      Environmental DNA (eDNA) analyses have enabled a more efficient surveillance of species distribution and compn. than conventional methods. However, the characteristics and dynamics of eDNA (e.g., origin, state, transport, and fate) remain unknown. This is esp. limited for the eDNA derived from nuclei (nu-eDNA), which has recently been used in eDNA analyses. Here, we compared the particle size distribution (PSD) of nu-eDNA from Japanese Jack Mackerel (Trachurus japonicus) with that of mt-eDNA (eDNA derived from mitochondria) reported in previous studies. We repeatedly sampled rearing water from the tanks under multiple temps. and fish biomass levels, and quantified the copy nos. of size-fractioned nu-eDNA. We found that the concn. of nu-eDNA was higher than that of mt-eDNA at 3-10 μm size fraction. Moreover, at the 0.8-3 μm and 0.4-0.8 μm size fractions, eDNA concns. of both types increased with higher temp. and their degrdn. tended to be suppressed. These results imply that the prodn. of eDNA from large to small size fractions could buffer the degrdn. of small-sized eDNA, which could improve its persistence in water. Our findings will contribute to refine the difference between nu- and mt-eDNA properties, and assist eDNA analyses as an efficient tool for the conservation of aquatic species.
    21. 21
      Jo, T.; Murakami, H.; Yamamoto, S.; Masuda, R.; Minamoto, T. Effect of Water Temperature and Fish Biomass on Environmental DNA Shedding, Degradation, and Size Distribution. Ecol. Evol. 2019, 9, 1135– 1146,  DOI: 10.1002/ece3.4802
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      Effect of water temperature and fish biomass on environmental DNA shedding, degradation, and size distribution
      Jo Toshiaki; Yamamoto Satoshi; Minamoto Toshifumi; Murakami Hiroaki; Masuda Reiji; Yamamoto Satoshi
      Ecology and evolution (2019), 9 (3), 1135-1146 ISSN:2045-7758.
      Environmental DNA (eDNA) analysis has successfully detected organisms in various aquatic environments. However, there is little basic information on eDNA, including the eDNA shedding and degradation processes. This study focused on water temperature and fish biomass and showed that eDNA shedding, degradation, and size distribution varied depending on water temperature and fish biomass. The tank experiments consisted of four temperature levels and three fish biomass levels. The total eDNA and size-fractioned eDNA from Japanese Jack Mackerels (Trachurus japonicus) were quantified before and after removing the fish. The results showed that the eDNA shedding rate increased at higher water temperature and larger fish biomass, and the eDNA decay rate also increased at higher temperature and fish biomass. In addition, the small-sized eDNA fractions were proportionally larger at higher temperatures, and these proportions varied among fish biomass. After removing the fish from the tanks, the percentage of eDNA temporally decreased when the eDNA size fraction was >10 μm, while the smaller size fractions increased. These results have the potential to make the use of eDNA analysis more widespread in the future.
    22. 22
      Jo, T.; Minamoto, T. Complex Interactions between Environmental DNA (eDNA) State and Water Chemistries on eDNA Persistence Suggested by Meta-analyses. Mol. Ecol. Resour. 2021, 21, 1490– 1503,  DOI: 10.1111/1755-0998.13354
      22
      Complex interactions between environmental DNA (eDNA) state and water chemistries on eDNA persistence suggested by meta-analyses
      Jo, Toshiaki; Minamoto, Toshifumi
      Molecular Ecology Resources (2021), 21 (5), 1490-1503CODEN: MEROCJ; ISSN:1755-098X. (Wiley-Blackwell)
      Understanding the processes of environmental DNA (eDNA) persistence and degrdn. is essential to det. the spatiotemporal scale of eDNA signals and accurately est. species distribution. The effects of environmental factors on eDNA persistence have previously been examd.; however, the influence of the physiochem. and mol. states of eDNA on its persistence is not completely understood. Here, we performed meta-anal. including 26 previously published papers on the estn. of first-order eDNA decay rate consts., and assessed the effects of filter pore size, DNA fragment size, target gene, and environmental conditions on eDNA decay rates. Almost all supported models included the interactions between the filter pore size and water temp., between the target gene and water temp., and between the target gene and water source, implying the influence of complex interactions between the eDNA state and environmental conditions on eDNA persistence. These findings were generally consistent with the results of a reanal. of a previous tank expt. which measured the time-series changes in marine fish eDNA concns. in multiple size fractions after fish removal. Our results suggest that the mechanism of eDNA persistence and degrdn. cannot be fully understood without knowing not only environmental factors but also cellular and mol. states of eDNA in water. Further verification of the relationship between eDNA state and persistence is required by obtaining more information on eDNA persistence in various exptl. and environmental conditions, which will enhance our knowledge on eDNA persistence and support our findings.
    23. 23
      Weltz, K.; Lyle, J. M.; Ovenden, J.; Morgan, J. A. T.; Moreno, D. A.; Semmens, J. M. Application of Environmental DNA to Detect an Endangered Marine Skate Species in the Wild. PLoS One 2017, 12, e0178124  DOI: 10.1371/journal.pone.0178124
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      Application of environmental DNA to detect an endangered marine skate species in the wild
      Weltz, Kay; Lyle, Jeremy M.; Ovenden, Jennifer; Morgan, Jessica A. T.; Moreno, David A.; Semmens, Jayson M.
      PLoS One (2017), 12 (6), e0178124/1-e0178124/16CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Environmental DNA (eDNA) techniques have only recently been applied in the marine environment to detect the presence of marine species. Species-specific primers and probes were designed to detect the eDNA of the endangered Maugean skate (Zearaja maugeana) from as little as 1 L of water collected at depth (10-15 m) in Macquarie Harbor (MH), Tasmania. The identity of the eDNA was confirmed as Z. maugeana by sequencing the qPCR products and aligning these with the target sequence for a 100% match. This result has validated the use of this eDNA technique for detecting a rare species, Z. maugeana, in the wild. Being able to investigate the presence, and possibly the abundance, of Z. maugeana in MH and Bathurst harbor (BH), would be addressing a conservation imperative for the endangered Z. maugeana. For future application of this technique in the field, the rate of decay was detd. for Z. maugeana eDNA under ambient dissolved oxygen (DO) levels (55% satn.) and lower DO (20% satn.) levels, revealing that the eDNA can be detected for 4 and 16 h resp., after which eDNA concn. drops below the detection threshold of the assay. With the rate of decay being influenced by starting eDNA concns., it is recommended that samples be filtered as soon as possible after collection to minimize further loss of eDNA prior to and during sample processing.
    24. 24
      Collins, R. A.; Wangensteen, O. S.; O’Gorman, E. J.; Mariani, S.; Sims, D. W.; Genner, M. J. Persistence of Environmental DNA in Marine Systems. Commun. Biol. 2018, 1, 1– 11,  DOI: 10.1038/s42003-018-0192-6
      24
      Persistence of environmental DNA in marine systems
      Collins, Rupert A.; Wangensteen, Owen S.; O'Gorman, Eoin J.; Mariani, Stefano; Sims, David W.; Genner, Martin J.
      Communications Biology (2018), 1 (1), 1-11CODEN: CBOIDQ; ISSN:2399-3642. (Nature Research)
      As environmental DNA (eDNA) becomes an increasingly valuable resource for marine ecosystem monitoring, understanding variation in its persistence across contrasting environments is crit. Here, we quantify the breakdown of macrobial eDNA over a spatio-temporal axis of locally extreme conditions, varying from ocean-influenced offshore to urban-inshore, and between winter and summer. We report that eDNA degrades 1.6 times faster in the inshore environment than the offshore environment, but contrary to expectation we find no difference over season. Anal. of environmental covariables show a spatial gradient of salinity and a temporal gradient of pH, with salinity-or the biotic correlates thereof-most important. Based on our estd. inshore eDNA half-life and naturally occurring eDNA concns., we est. that eDNA may be detected for around 48 h, offering potential to collect ecol. community data of high local fidelity. We conclude by placing these results in the context of previously published eDNA decay rates.
    25. 25
      Harrison, J. B.; Sunday, J. M.; Rogers, S. M. Predicting the Fate of eDNA in the Environment and Implications for Studying Biodiversity. Proc. R. Soc. B 2019, 286, 20191409,  DOI: 10.1098/rspb.2019.1409
      25
      Predicting the fate of eDNA in the environment and implications for studying biodiversity
      Harrison, Jori B.; Sunday, Jennifer M.; Rogers, Sean M.
      Proceedings of the Royal Society B: Biological Sciences (2019), 286 (1915), 20191409CODEN: PRSBC7 ISSN:. (Royal Society)
      A review. Environmental DNA (eDNA) applications are transforming the std. of characterizing aquatic biodiversity via the presence, location and abundance of DNA collected from environmental samples. As eDNA studies use DNA fragments as a proxy for the presence of organisms, the ecol. properties of the complex and dynamic environments from which eDNA is sampled need to be considered for accurate biol. interpretation. In this review, we discuss the role that differing environments play on the major processes that eDNA undergoes between organism and collection, including shedding, decay and transport. We focus on a mechanistic understanding of these processes and highlight how decay and transport models are being developed towards more accurate and robust predictions of the fate of eDNA. We conclude with five recommendations for eDNA researchers and practitioners, to advance current best practices, as well as to support a future model of eDNA spatio-temporal persistence.
    26. 26
      Andruszkiewicz, E. A.; Koseff, J. R.; Fringer, O. B.; Ouellette, N. T.; Lowe, A. B.; Edwards, C. A.; Boehm, A. B. Modeling Environmental DNA Transport in the Coastal Ocean Using Lagrangian Particle Tracking. Front. Mar. Sci. 2019, 6, 1– 14,  DOI: 10.3389/fmars.2019.00477
      There is no corresponding record for this reference.
    27. 27
      Strickler, K. M.; Fremier, A. K.; Goldberg, C. S. Quantifying Effects of UV-B, Temperature, and pH on eDNA Degradation in Aquatic Microcosms. Biol. Conserv. 2015, 183, 85– 92,  DOI: 10.1016/j.biocon.2014.11.038
      There is no corresponding record for this reference.
    28. 28
      Eichmiller, J. J.; Best, S. E.; Sorensen, P. W. Effects of Temperature and Trophic State on Degradation of Environmental DNA in Lake Water. Environ. Sci. Technol. 2016, 50, 1859– 1867,  DOI: 10.1021/acs.est.5b05672
      28
      Effects of Temperature and Trophic State on Degradation of Environmental DNA in Lake Water
      Eichmiller, Jessica J.; Best, Sendrea E.; Sorensen, Peter W.
      Environmental Science & Technology (2016), 50 (4), 1859-1867CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)
      Degrdn. of environmental DNA (eDNA) in aquatic habitats can affect the interpretation of eDNA data and the ability to detect aquatic organisms. The effect of temp. and trophic state on the decay of Common Carp (Cyprinus carpio) eDNA was evaluated using lake water microcosms and quant. PCR for a Common Carp-specific genetic marker in 2 expts. The 1st expt. tested the effect of temp. on Common Carp eDNA decay. Common Carp eDNA exhibited exponential decay that increased with temp. The slowest decay rate was obsd. at 5°, with a T90 value (time to 90% redn. from initial concn.) of 6.6 days, as opposed to ∼1 day at higher temps. In a 2nd expt., decay was compared across waters from lakes of different trophic states. In this expt., Common Carp eDNA exhibited biphasic exponential decay, characterized by rapid decay for 3-8 days followed by slow decay. Decay rate was slowest in dystrophic water and fastest in oligotrophic water, and decay rate was neg. correlated to dissolved org. C concn. The overall rapid decay of eDNA and the effects of temp. and water quality should be considered in protocols for water sample storage and field sampling design.
    29. 29
      Lance, R.; Klymus, K.; Richter, C.; Guan, X.; Farrington, H.; Carr, M.; Thompson, N.; Chapman, D.; Baerwaldt, K. Experimental Observations on the Decay of Environmental DNA from Bighead and Silver Carps. MBI 2017, 8, 343– 359,  DOI: 10.3391/mbi.2017.8.3.08
      There is no corresponding record for this reference.
    30. 30
      Tsuji, S.; Ushio, M.; Sakurai, S.; Minamoto, T.; Yamanaka, H. Water Temperature-Dependent Degradation of Environmental DNA and Its Relation to Bacterial Abundance. PLoS One 2017, 12, 1– 13,  DOI: 10.1371/journal.pone.0176608
      There is no corresponding record for this reference.
    31. 31
      Kasai, A.; Takada, S.; Yamazaki, A.; Masuda, R.; Yamanaka, H. The Effect of Temperature on Environmental DNA Degradation of Japanese Eel. Fish. Sci. 2020, 86, 465– 471,  DOI: 10.1007/s12562-020-01409-1
      31
      The effect of temperature on environmental DNA degradation of Japanese eel
      Kasai, Akihide; Takada, Shingo; Yamazaki, Aya; Masuda, Reiji; Yamanaka, Hiroki
      Fisheries Science (Tokyo, Japan) (2020), 86 (3), 465-471CODEN: FSCIEH; ISSN:0919-9268. (Springer Japan)
      Abstr.: The environmental DNA (eDNA) technique is a convenient and powerful tool to detect rare species. Knowledge of the degrdn. rate of eDNA in water is important for understanding how degrdn. influences the presence and/or est. biomass of aquatic animals. We developed a new set of species-specific primers and probe to detect eDNA of Japanese eel Anguilla japonica, which is a com. important and endangered species, and then conducted a lab. expt. to quantify the temp.-dependent degrdn. of emitted eDNA. Eels were held in tanks at five different temp. levels from 10 to 30°C and water from each tank was sampled and kept in bottles at each temp. over 6 days. The concn. of eDNA was measured every day and the results showed that temp. (T) had a significant and pos. effect on the degrdn. rate (k) as k = 0.02T + 0.18. Improved understanding of the effect of temp. on degrdn. rates would help data interpretations and adjustments would increase the reliability of eDNA anal. in future studies.
    32. 32
      Allan, E. A.; Zhang, W. G.; Lavery, A. C.; Govindarajan, A. F. Environmental DNA Shedding and Decay Rates from Diverse Animal Forms and Thermal Regimes. Environ. DNA 2021, 3, 492– 514,  DOI: 10.1002/edn3.141
      There is no corresponding record for this reference.
    33. 33
      Caza-Allard, I.; Laporte, M.; Côté, G.; April, J.; Bernatchez, L. Effect of Biotic and Abiotic Factors on the Production and Degradation of Fish Environmental DNA: An Experimental Evaluation. Environ. DNA 2022, 4, 453– 468,  DOI: 10.1002/edn3.266
      33
      Effect of biotic and abiotic factors on the production and degradation of fish environmental DNA: An experimental evaluation
      Caza-Allard, Isabeau; Laporte, Martin; Cote, Guillaume; April, Julien; Bernatchez, Louis
      Environmental DNA (2022), 4 (2), 453-468CODEN: EDNNAZ; ISSN:2637-4943. (John Wiley & Sons, Inc.)
      Environmental DNA (eDNA) is a very promising approach to facilitate and improve the aquatic species monitoring, which is crucial for their management and conservation. In comparison with the plethora of monitoring studies in the fields, relatively few studies have focused on exptl. investigating the "ecol." of eDNA, in particular pertaining to processes influencing the detection of eDNA. The paucity of knowledge about its ecol. hampers the use of eDNA anal. to its full potential. In this study, we exptl. evaluated the impact of several biotic and abiotic factors on the rate of prodn. and degrdn. of eDNA. Individuals of three freshwater fish species (brown bullhead, tench, and yellow perch) with distinct ecol. were placed in two types of water from the St. Lawrence River (Quebec, Canada) with very distinct physicochem. characteristics and at three different temps. Water samples were then filtered at predetd. time intervals, and quant. PCR was used to quantify the eDNA in each sample. We found that temp., species, water types, and some interactions between these factors had a strong effect on the prodn. and degrdn. of eDNA. The results of this study enhance our knowledge about the ecol. of eDNA, thus improving eDNA data interpretation.
    34. 34
      Lindahl, T.; Nyberg, B. Rate of Depurination of Native Deoxyribonucleic Acid. Biochemistry 1972, 11, 3610– 3618,  DOI: 10.1021/bi00769a018
      34
      Rate of depurination of native deoxyribonucleic acid
      Lindahl, Tomas; Nyberg, Barbro
      Biochemistry (1972), 11 (19), 3610-18CODEN: BICHAW; ISSN:0006-2960.
      The rate of depurination of double-stranded Bacillus subtilis DNA, radioactively labeled in the purine residues, has been followed as a function of temp., pH, and ionic strength. In a Mg2+-contg. buffer of physiol. ionic strength, the rate const. for depurination of DNA is 4 × 10-9 sec-1 at 70° and pH 7.4. The activation energy of the reaction is 31 ± 2 kcal/mole. These data strongly indicate that depurination of DNA occurs at a physiol. significant rate under in vivo conditions and consequently that the lesions introduced in this fashion must be repaired.
    35. 35
      Cheng, Y.-K.; Pettitt, B. M. Stabilities of Double- and Triple-Strand Helical Nucleic Acids. Prog. Biophys. Mol. Biol. 1992, 58, 225– 257,  DOI: 10.1016/0079-6107(92)90007-s
      35
      Stabilities of double- and triple-strand helical nucleic acids
      Cheng, Yuen Kit; Pettitt, B. Montgomery
      Progress in Biophysics & Molecular Biology (1992), 58 (3), 225-57CODEN: PBIMAC; ISSN:0079-6107.
      A review, with ∼200 refs., centered around the authors' interest in DNA double- and triple-helix formation and stability.
    36. 36
      Lindahl, T. Instability and Decay of the Primary Structure of DNA. Nature 1993, 362, 709– 715,  DOI: 10.1038/362709a0
      36
      Instability and decay of the primary structure of DNA
      Lindahl, Tomas
      Nature (London, United Kingdom) (1993), 362 (6422), 709-15CODEN: NATUAS; ISSN:0028-0836.
      A review with 84 refs. Although DNA is the carrier of genetic information, it has limited chem. stability. Hydrolysis oxidn. and nonenzymic methylation of DNA occur at significant rates in vivo, and are counteracted by specific DNA repair processes. The spontaneous decay of DNA is likely to be a major factor in mutagenesis, carcinogenesis and aging, and also sets limits for the recovery of DNA fragments from fossils.
    37. 37
      Seymour, M.; Durance, I.; Cosby, B. J.; Ransom-Jones, E.; Deiner, K.; Ormerod, S. J.; Colbourne, J. K.; Wilgar, G.; Carvalho, G. R.; de Bruyn, M.; Edwards, F.; Emmett, B. A.; Bik, H. M.; Creer, S. Acidity Promotes Degradation of Multi-Species Environmental DNA in Lotic Mesocosms. Commun. Biol. 2018, 1, 1– 8,  DOI: 10.1038/s42003-017-0005-3
      37
      Acidity promotes degradation of multi-species environmental DNA in lotic mesocosms
      Seymour, Mathew; Durance, Isabelle; Cosby, Bernard J.; Ransom-Jones, Emma; Deiner, Kristy; Ormerod, Steve J.; Colbourne, John K.; Wilgar, Gregory; Carvalho, Gary R.; de Bruyn, Mark; Edwards, Francois; Emmett, Bridget A.; Bik, Holly M.; Creer, Simon
      Communications Biology (2018), 1 (1), 1-8CODEN: CBOIDQ; ISSN:2399-3642. (Nature Research)
      Accurate quantification of biodiversity is fundamental to understanding ecosystem function and for environmental assessment. Mol. methods using environmental DNA (eDNA) offer a non-invasive, rapid, and cost-effective alternative to traditional biodiversity assessments, which require high levels of expertise. While eDNA analyses are increasingly being utilized, there remains considerable uncertainty regarding the dynamics of multispecies eDNA, esp. in variable systems such as rivers. Here, we utilize four sets of upland stream mesocosms, across an acid-base gradient, to assess the temporal and environmental degrdn. of multispecies eDNA. Sampling included water column and biofilm sampling over time with eDNA quantified using qPCR. Our findings show that the persistence of lotic multispecies eDNA, sampled from water and biofilm, decays to non-detectable levels within 2 days and that acidic environments accelerate the degrdn. process. Collectively, the results provide the basis for a predictive framework for the relationship between lotic eDNA degrdn. dynamics in spatio-temporally dynamic river ecosystems.
    38. 38
      van Bochove, K.; Bakker, F. T.; Beentjes, K. K.; Hemerik, L.; Vos, R. A.; Gravendeel, B. Organic Matter Reduces the Amount of Detectable Environmental DNA in Freshwater. Ecol. Evol. 2020, 10, 3647– 3654,  DOI: 10.1002/ece3.6123
      38
      Organic matter reduces the amount of detectable environmental DNA in freshwater
      van Bochove Kees; Bakker Freek T; van Bochove Kees; Beentjes Kevin K; Vos Rutger A; Gravendeel Barbara; Beentjes Kevin K; Gravendeel Barbara; Hemerik Lia; Gravendeel Barbara
      Ecology and evolution (2020), 10 (8), 3647-3654 ISSN:2045-7758.
      Environmental DNA (eDNA) is used for monitoring the occurrence of freshwater organisms. Various studies show a relation between the amount of eDNA detected and target organism abundance, thus providing a potential proxy for reconstructing population densities. However, environmental factors such as water temperature and microbial activity are known to affect the amount of eDNA present as well. In this study, we use controlled aquarium experiments using Gammarus pulex L. (Amphipoda) to investigate the relationship between the amount of detectable eDNA through time, pH, and levels of organic material. We found eDNA to degrade faster when organic material was added to the aquarium water, but that pH had no significant effect. We infer that eDNA contained inside cells and mitochondria is extra resilient against degradation, though this may not reflect actual presence of target species. These results indicate that, although estimation of population density might be possible using eDNA, measured eDNA concentration could, in the future, be corrected for local environmental conditions in order to ensure accurate comparisons.
    39. 39
      Lalli, C. M.; Parsons, T. R. Biological Oceanography: An Introduction; Elsevier: (Second Edition). 1997.
      There is no corresponding record for this reference.
    40. 40
      Paulmier, A.; Ruiz-Pino, D. Oxygen Minimum Zones (OMZs) in the Modern Ocean. Prog. Oceanogr. 2009, 80, 113– 128,  DOI: 10.1016/j.pocean.2008.08.001
      There is no corresponding record for this reference.
    41. 41
      Addamo, A. M.; Vertino, A.; Stolarski, J.; García-Jiménez, R.; Taviani, M.; Machordom, A. Merging Scleractinian Genera: The Overwhelming Genetic Similarity between Solitary Desmophyllum and Colonial Lophelia. BMC Evol. Biol. 2016, 16, 108,  DOI: 10.1186/s12862-016-0654-8
      41
      Merging scleractinian genera: the overwhelming genetic similarity between solitary Desmophyllum and colonial Lophelia
      Addamo, Anna Maria; Vertino, Agostina; Stolarski, Jaroslaw; Garcia-Jimenez, Ricardo; Taviani, Marco; Machordom, Annie
      BMC Evolutionary Biology (2016), 16 (), 108/1-108/17CODEN: BEBMCG; ISSN:1471-2148. (BioMed Central Ltd.)
      Background: In recent years, several types of mol. markers and new microscale skeletal characters have shown potential as powerful tools for phylogenetic reconstructions and higher-level taxonomy of scleractinian corals. Nonetheless, discrimination of closely related taxa is still highly controversial in scleractinian coral research. Here we used newly sequenced complete mitochondrial genomes and 30 microsatellites to define the genetic divergence between two closely related azooxanthellate taxa of the family Caryophylliidae: solitary Desmophyllum dianthus and colonial Lophelia pertusa. Results: In the mitochondrial control region, an astonishing 99.8 % of nucleotides between L. pertusa and D. dianthus were identical. Variability of the mitochondrial genomes of the two species is represented by only 12 non-synonymous out of 19 total nucleotide substitutions. Microsatellite sequence (37 loci) anal. of L. pertusa and D. dianthus showed genetic similarity is about 97 %. Our results also indicated that L. pertusa and D. dianthus show high skeletal plasticity in corallum shape and similarity in skeletal ontogeny, micromorphol. (septal and wall granulations) and microstructural characters (arrangement of rapid accretion deposits, thickening deposits). Conclusions: Molecularly and morphol., the solitary Desmophyllum and the dendroid Lophelia appear to be significantly more similar to each other than other unambiguous coral genera analyzed to date. This consequently leads to ascribe both taxa under the generic name Desmophyllum (priority by date of publication). Findings of this study demonstrate that coloniality may not be a robust taxonomic character in scleractinian corals.
    42. 42
      Cordes, E. E.; McGinley, M. P.; Podowski, E. L.; Becker, E. L.; Lessard-Pilon, S.; Viada, S. T.; Fisher, C. R. Coral Communities of the Deep Gulf of Mexico. Deep Sea Res., Part I 2008, 55, 777– 787,  DOI: 10.1016/j.dsr.2008.03.005
      There is no corresponding record for this reference.
    43. 43
      Lessard-Pilon, S. A.; Podowski, E. L.; Cordes, E. E.; Fisher, C. R. Megafauna Community Composition Associated with Lophelia pertusa Colonies in the Gulf of Mexico. Deep Sea Res., Part II 2010, 57, 1882– 1890,  DOI: 10.1016/j.dsr2.2010.05.013
      There is no corresponding record for this reference.
    44. 44
      Andruszkiewicz, E. A.; Sassoubre, L. M.; Boehm, A. B. Persistence of Marine Fish Environmental DNA and the Influence of Sunlight. PLoS One 2017, 12, e0185043  DOI: 10.1371/journal.pone.0185043
      44
      Persistence of marine fish environmental DNA and the influence of sunlight
      Andruszkiewicz, Elizabeth A.; Sassoubre, Lauren M.; Boehm, Alexandria B.
      PLoS One (2017), 12 (9), e0185043/1-e0185043/18CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Harnessing information encoded in environmental DNA (eDNA) in marine waters has the potential to revolutionize marine biomonitoring. Whether using organism-specific quant. PCR assays or metabarcoding in conjunction with amplicon sequencing, scientists have illustrated that realistic organism censuses can be inferred from eDNA. The next step is establishing ways to link information obtained from eDNA analyses to actual organism abundance. This is only possible by understanding the processes that control eDNA concns. The present study uses mesocosm expts. to study the persistence of eDNA in marine waters and explore the role of sunlight in modulating eDNA persistence. We seeded solute-permeable dialysis bags with water contg. indigenous eDNA and suspended them in a large tank contg. seawater. Bags were subjected to two treatments: half the bags were suspended near the water surface where they received high doses of sunlight, and half at depth where they received lower doses of sunlight. Bags were destructively sampled over the course of 87 h. eDNA was extd. from water samples and used as template for a Scomberjaponicus qPCR assay and a marine fish-specific 12S rRNA PCR assay. The latter was subsequently sequenced using a metabarcoding approach. S. japonicus eDNA, as measured by qPCR, exhibited first order decay with a rate const. ∼0.01 h-1 with no difference in decay rate consts. between the two exptl. treatments. eDNA metabarcoding identified 190 organizational taxonomic units (OTUs) assigned to varying taxonomic ranks. There was no difference in marine fish communities as measured by eDNA metabarcoding between the two exptl. treatments, but there was an effect of time. Given the differences in UVA and UVB fluence received by the two exptl. treatments, we conclude that sunlight is not the main driver of fish eDNA decay in the expts. However, there are clearly temporal effects that need to be considered when interpreting information obtained using eDNA approaches.
    45. 45
      Tedetti, M.; Sempéré, R. Penetration of Ultraviolet Radiation in the Marine Environment. A Review. Photochem. Photobiol. 2006, 82, 389– 397,  DOI: 10.1562/2005-11-09-ir-733
      45
      Penetration of ultraviolet radiation in the marine environment. A review
      Tedetti, Marc; Sempere, Richard
      Photochemistry and Photobiology (2006), 82 (Mar./Apr.), 389-397CODEN: PHCBAP; ISSN:0031-8655. (American Society for Photobiology)
      A review concerning underwater instruments used to measure UVR (UV radiation), presenting data dealing with UVR depth penetration in different oceanic areas, is given. Topics discussed include: introduction; materials and methods (radiometers, dosimeters); and results and discussion (UVR penetration in different ocean areas [open ocean, Antarctic water, coastal water], relationship between UV-B penetration and DNA damage ED).
    46. 46
      Spens, J.; Evans, A. R.; Halfmaerten, D.; Knudsen, S. W.; Sengupta, M. E.; Mak, S. S. T.; Sigsgaard, E. E.; Hellström, M. Comparison of capture and storage methods for aqueous macrobial eDNA using an optimized extraction protocol: advantage of enclosed filter. Methods Ecol. Evol. 2017, 8, 635– 645,  DOI: 10.1111/2041-210x.12683
      There is no corresponding record for this reference.
    47. 47
      Govindarajan, A. F.; Francolini, R. D.; Jech, J. M.; Lavery, A. C.; Llopiz, J. K.; Wiebe, P. H.; Zhang, W. G. Exploring the Use of Environmental DNA (eDNA) to Detect Animal Taxa in the Mesopelagic Zone. Front. Ecol. Evol. 2021, 9, 1– 17,  DOI: 10.3389/fevo.2021.574877
      There is no corresponding record for this reference.
    48. 48
      Klymus, K. E.; Merkes, C. M.; Allison, M. J.; Goldberg, C. S.; Helbing, C. C.; Hunter, M. E.; Jackson, C. A.; Lance, R. F.; Mangan, A. M.; Monroe, E. M.; Piaggio, A. J.; Stokdyk, J. P.; Wilson, C. C.; Richter, C. A. Reporting the Limits of Detection and Quantification for Environmental DNA Assays. Environ. DNA 2020, 2, 271– 282,  DOI: 10.1002/edn3.29
      There is no corresponding record for this reference.
    49. 49
      Wickham, H. Ggplot2: Elegant Graphics for Data Analysis; Springer-Verlag: New York, 2016.
      There is no corresponding record for this reference.
    50. 50
      Shogren, A. J.; Tank, J. L.; Egan, S. P.; August, O.; Rosi, E. J.; Hanrahan, B. R.; Renshaw, M. A.; Gantz, C. A.; Bolster, D. Water Flow and Biofilm Cover Influence Environmental DNA Detection in Recirculating Streams. Environ. Sci. Technol. 2018, 52, 8530– 8537,  DOI: 10.1021/acs.est.8b01822
      50
      Water Flow and Biofilm Cover Influence Environmental DNA Detection in Recirculating Streams
      Shogren, Arial J.; Tank, Jennifer L.; Egan, Scott P.; August, Olivia; Rosi, Emma J.; Hanrahan, Brittany R.; Renshaw, Mark A.; Gantz, Crysta A.; Bolster, Diogo
      Environmental Science & Technology (2018), 52 (15), 8530-8537CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)
      The increasing use of environmental DNA (eDNA) for detn. of species presence in aquatic ecosystems is an invaluable technique for both ecol. as a field and for the management of aquatic ecosystems. We examd. the degrdn. dynamics of fish eDNA using an exptl. array of recirculating streams, also using a "nested" primer assay to est. degrdn. among eDNA fragment sizes. We introduced eDNA into streams with a range of water velocities (0.1-0.8 m s-1) and substrate biofilm coverage (0-100%) and monitored eDNA concns. over time (∼10 d) to assess how biophys. conditions influence eDNA persistence. We found that the presence of biofilm significantly increased initial decay rates relative to previous studies conducted in nonflowing microcosms, suggesting important differences in detection and persistence in lentic vs lotic systems. Lastly, by using a nested primer assay that targeted different size eDNA fragments, we found that fragment size altered both the estd. rate const. coeffs., as well as eDNA detectability over time. Larger fragments (>600 bp) were quickly degraded, while shorter fragments (<100 bp) remained detectable for the entirety of the expt. When using eDNA as a stream monitoring tool, understanding environmental factors controlling eDNA degrdn. will be crit. for optimizing eDNA sampling strategies.
    51. 51
      Bylemans, J.; Furlan, E. M.; Gleeson, D. M.; Hardy, C. M.; Duncan, R. P. Does Size Matter? An Experimental Evaluation of the Relative Abundance and Decay Rates of Aquatic Environmental DNA. Environ. Sci. Technol. 2018, 52, 6408– 6416,  DOI: 10.1021/acs.est.8b01071
      51
      Does Size Matter? An Experimental Evaluation of the Relative Abundance and Decay Rates of Aquatic Environmental DNA
      Bylemans, Jonas; Furlan, Elise M.; Gleeson, Dianne M.; Hardy, Christopher M.; Duncan, Richard P.
      Environmental Science & Technology (2018), 52 (11), 6408-6416CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)
      Environmental DNA (eDNA) is increasingly used to monitor aquatic macrofauna. Typically, short mitochondrial DNA fragments are targeted because these should be relatively more abundant in the environment as longer fragments will break into smaller fragments over time. However, longer fragments may permit more flexible primer design and increase taxonomic resoln. for eDNA metabarcoding analyses, and recent studies have shown that long mitochondrial eDNA fragments can be extd. from environmental water samples. Nuclear eDNA fragments have also been proposed as targets, but little is known about their persistence in the aquatic environment. Here we measure the abundance of mitochondrial eDNA fragments of different lengths and of short nuclear eDNA fragments, originating from captive fish in exptl. tanks, and we test whether longer mitochondrial and short nuclear fragments decay faster than short mitochondrial fragments following fish removal. We show that when fish are present, shorter mitochondrial fragments are more abundant in water samples than both longer mitochondrial fragments and short nuclear eDNA fragments. However, the rate of decay following fish removal was similar for all fragment types, suggesting that the differences in abundance resulted from differences in the rates at which different fragment types were produced rather than differences in their decay rates.
    52. 52
      Muggeo, V. M. R. Estimating Regression Models with Unknown Break-points. Stat. Med. 2003, 22, 3055– 3071,  DOI: 10.1002/sim.1545
      52
      Estimating regression models with unknown break-points
      Muggeo Vito M R
      Statistics in medicine (2003), 22 (19), 3055-71 ISSN:0277-6715.
      This paper deals with fitting piecewise terms in regression models where one or more break-points are true parameters of the model. For estimation, a simple linearization technique is called for, taking advantage of the linear formulation of the problem. As a result, the method is suitable for any regression model with linear predictor and so current software can be used; threshold modelling as function of explanatory variables is also allowed. Differences between the other procedures available are shown and relative merits discussed. Simulations and two examples are presented to illustrate the method.
    53. 53
      Bates, D.; Mächler, M.; Bolker, B.; Walker, S. Fitting Linear Mixed-Effects Models Using Lme4. J. Stat. Softw. 2015, 67, 1– 48,  DOI: 10.18637/jss.v067.i01
      There is no corresponding record for this reference.
    54. 54
      Kuznetsova, A.; Brockhoff, P. B.; Christensen, R. H. B. LmerTest Package: Tests in Linear Mixed Effects Models. J. Stat. Softw. 2017, 82, 1– 26,  DOI: 10.18637/jss.v082.i13
      There is no corresponding record for this reference.
    55. 55
      Ben-Shachar, M.; Lüdecke, D.; Makowski, D. Effectsize: Estimation of Effect Size Indices and Standardized Parameters. J. Open Source Softw. 2020, 5, 2815,  DOI: 10.21105/joss.02815
      There is no corresponding record for this reference.
    56. 56
      Harrison, X. A.; Donaldson, L.; Correa-Cano, M. E.; Evans, J.; Fisher, D. N.; Goodwin, C. E. D.; Robinson, B. S.; Hodgson, D. J.; Inger, R. A Brief Introduction to Mixed Effects Modelling and Multi-Model Inference in Ecology. Peerj 2018, 6, e4794  DOI: 10.7717/peerj.4794
      There is no corresponding record for this reference.
    57. 57
      Thomsen, P. F.; Kielgast, J.; Iversen, L. L.; Møller, P. R.; Rasmussen, M.; Willerslev, E. Detection of a Diverse Marine Fish Fauna Using Environmental DNA from Seawater Samples. PLoS One 2012, 7, e41732  DOI: 10.1371/journal.pone.0041732
      57
      Detection of a diverse marine fish fauna using environmental DNA from seawater samples
      Thomsen, Philip Francis; Kielgast, Jos; Iversen, Lars Loensmann; Moller, Peter Rask; Rasmussen, Morten; Willerslev, Eske
      PLoS One (2012), 7 (8), e41732CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      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 com. species, and restricted to particular areas with favorable 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 1/2-L 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). Addnl., 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 expt. addressing eDNA degrdn. 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.
    58. 58
      Sassoubre, L. M.; Yamahara, K. M.; Gardner, L. D.; Block, B. A.; Boehm, A. B. Quantification of Environmental DNA (eDNA) Shedding and Decay Rates for Three Marine Fish. Environ. Sci. Technol. 2016, 50, 10456– 10464,  DOI: 10.1021/acs.est.6b03114
      58
      Quantification of Environmental DNA (eDNA) Shedding and Decay Rates for Three Marine Fish
      Sassoubre, Lauren M.; Yamahara, Kevan M.; Gardner, Luke D.; Block, Barbara A.; Boehm, Alexandria B.
      Environmental Science & Technology (2016), 50 (19), 10456-10464CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)
      Anal. of environmental DNA (eDNA) to identify macroorganisms and biodiversity has the potential to significantly augment spatial and temporal biol. monitoring in aquatic ecosystems. Current monitoring methods relying on the phys. identification of organisms can be time consuming, expensive, and invasive. Measuring eDNA shed from organisms provides detailed information on the presence and abundance of communities of organisms. However, little is known about eDNA shedding and decay in aquatic environments. In the present study, we designed novel Taqman qPCR assays for three ecol. and economically important marine fish-Engraulis mordax (Northern Anchovy), Sardinops sagax (Pacific Sardine), and Scomber japonicas (Pacific Chub Mackerel). We subsequently measured fish eDNA shedding and decay rates in seawater mesocosms. eDNA shedding rates ranged from 165 to 3368 pg of DNA per h per g of biomass. First-order decay rate consts. ranged from 0.055 to 0.101 per h. We also examd. the size fractionation of eDNA and concluded eDNA is both intra- and extracellular. Finally, we derived a simple mass-balance model to est. fish abundance from eDNA concn. The mesocosm-derived shedding and decay rates inform the interpretation of eDNA concns. measured in environmental samples and future use of eDNA as a monitoring tool.
    59. 59
      Minamoto, T.; Fukuda, M.; Katsuhara, K. R.; Fujiwara, A.; Hidaka, S.; Yamamoto, S.; Takahashi, K.; Masuda, R. Environmental DNA Reflects Spatial and Temporal Jellyfish Distribution. PLoS One 2017, 12, e0173073  DOI: 10.1371/journal.pone.0173073
      59
      Environmental DNA reflects spatial and temporal jellyfish distribution
      Minamoto, Toshifumi; Fukuda, Miho; Katsuhara, Koki R.; Fujiwara, Ayaka; Hidaka, Shunsuke; Yamamoto, Satoshi; Takahashi, Kohji; Masuda, Reiji
      PLoS One (2017), 12 (2), e0173073/1-e0173073/15CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Recent development of environmental DNA (eDNA) anal. allows us to survey underwater macro-organisms easily and cost effectively; however, there have been no reports on eDNA detection or quantification for jellyfish. Here we present the first report on an eDNA anal. of marine jellyfish using Japanese sea nettle (Chrysaora pacifica) as a model species by combining a tank expt. with spatial and temporal distribution surveys. We performed a tank expt. monitoring eDNA concns. over a range of time intervals after the introduction of jellyfish, and quantified the eDNA concns. by quant. real-time PCR. The eDNA concns. peaked twice, at 1 and 8 h after the beginning of the expt., and became stable within 48 h. The estd. release rates of the eDNA in jellyfish were higher than the rates previously reported in fishes. A spatial survey was conducted in June 2014 in Maizuru Bay, Kyoto, in which eDNA was collected from surface water and sea floor water samples at 47 sites while jellyfish near surface water were counted on board by eye. The distribution of eDNA in the bay corresponded with the distribution of jellyfish inferred by visual observation, and the eDNA concn. in the bay was ∼13 times higher on the sea floor than on the surface. The temporal survey was conducted from March to Nov. 2014, in which jellyfish were counted by eye every morning while eDNA was collected from surface and sea floor water at three sampling points along a pier once a month. The temporal fluctuation pattern of the eDNA concns. and the nos. of obsd. individuals were well correlated. We conclude that an eDNA approach is applicable for jellyfish species in the ocean.
    60. 60
      Wood, S. A.; Biessy, L.; Latchford, J. L.; Zaiko, A.; von Ammon, U.; Audrezet, F.; Cristescu, M. E.; Pochon, X. Release and Degradation of Environmental DNA and RNA in a Marine System. Sci. Total Environ. 2020, 704, 135314  DOI: 10.1016/j.scitotenv.2019.135314
      60
      Release and degradation of environmental DNA and RNA in a marine system
      Wood, Susanna A.; Biessy, Laura; Latchford, Janie L.; Zaiko, Anastasija; von Ammon, Ulla; Audrezet, Francois; Cristescu, Melania E.; Pochon, Xavier
      Science of the Total Environment (2020), 704 (), 135314CODEN: STENDL; ISSN:0048-9697. (Elsevier B.V.)
      Over the last decade, there has been growing interest in the anal. of environmental DNA (eDNA) to infer the presence of organisms in aquatic environments. The efficacy of eDNA/eRNA based tools are highly depend on the turnover rate of the mol. (their release and degrdn.). Environmental DNA has been shown to persist for days, weeks or years in environmental samples. Environmental RNA (eRNA) is thought to degrade faster than eDNA, however to our knowledge, no exptl. studies have explored this. Here we present an aquarium study to investigate eDNA and eRNA shedding rates and degrdn. for two sessile marine invertebrates. The copy nos. for eDNA and eRNA were assessed using droplet digital PCR targeting the mitochondrial Cytochrome c Oxidase subunit 1 (COI) gene. Environmental RNA persisted after organism removal for much longer than expected with detections for up to 13 h. In contrast, eDNA was detected is samples collected up to 94 h after organism removal. There was no evidence that the decay rates consts. for eDNA and eRNA were different (p = 0.6, Kruskal-Wallis tests). Both eDNA and eRNA was detected in biofilms collected at the end of the expt. (day 21). This suggests binding with org. or inorg. compds. or stabilization of these mols. in the biofilm matrix. The finding of the prolonged persistence of eRNA may provide new opportunities for improved biodiversity surveys through reducing false positives caused by legacy DNA and could also facilitate new research on environmental transcriptomics.
    61. 61
      Kwong, S. L. T.; Villacorta-Rath, C.; Doyle, J.; Uthicke, S. Quantifying Shedding and Degradation Rates of Environmental DNA (eDNA) from Pacific Crown-of-Thorns Seastar (Acanthaster cf. solaris). Mar. Biol. 2021, 168, 1– 10,  DOI: 10.1007/s00227-021-03896-x
      There is no corresponding record for this reference.
    62. 62
      Kirtane, A.; Wieczorek, D.; Noji, T.; Baskin, L.; Ober, C.; Plosica, R.; Chenoweth, A.; Lynch, K.; Sassoubre, L. Quantification of Environmental DNA (eDNA) Shedding and Decay Rates for Three Commercially Harvested Fish Species and Comparison between EDNA Detection and Trawl Catches. Environ. DNA 2021, 3, 1142– 1155,  DOI: 10.1002/edn3.236
      There is no corresponding record for this reference.
    63. 63
      Turner, C. R.; Barnes, M. A.; Xu, C. C. Y.; Jones, S. E.; Jerde, C. L.; Lodge, D. M. Particle size distribution and optimal capture of aqueous macrobial eDNA. Methods Ecol. Evol. 2014, 5, 676– 684,  DOI: 10.1111/2041-210x.12206
      There is no corresponding record for this reference.
    64. 64
      Wilcox, T. M.; McKelvey, K. S.; Young, M. K.; Lowe, W. H.; Schwartz, M. K. Environmental DNA Particle Size Distribution from Brook Trout (Salvelinus fontinalis). Conserv. Genet. Resour. 2015, 7, 639– 641,  DOI: 10.1007/s12686-015-0465-z
      There is no corresponding record for this reference.
    65. 65
      Zaiko, A.; von Ammon, U.; Stuart, J.; Smith, K. F.; Yao, R.; Welsh, M.; Pochon, X.; Bowers, H. A. Assessing the Performance and Efficiency of Environmental DNA/RNA Capture Methodologies under Controlled Experimental Conditions. Methods Ecol. Evol. 2022,  DOI: 10.1111/2041-210x.13879
      There is no corresponding record for this reference.
    66. 66
      Bythell, J. C.; Wild, C. Biology and Ecology of Coral Mucus Release. J. Exp. Mar. Biol. Ecol. 2011, 408, 88– 93,  DOI: 10.1016/j.jembe.2011.07.028
      There is no corresponding record for this reference.
    67. 67
      Murray, F.; De Clippele, L. H.; Hiley, A.; Wicks, L.; Roberts, J. M.; Hennige, S. Multiple Feeding Strategies Observed in the Cold-Water Coral Lophelia pertusa. J. Mar. Biol. Assoc. U. K. 2019, 99, 1281– 1283,  DOI: 10.1017/s0025315419000298
      There is no corresponding record for this reference.
    68. 68
      Canals, O.; Mendibil, I.; Santos, M.; Irigoien, X.; Rodríguez-Ezpeleta, N. Vertical Stratification of Environmental DNA in the Open Ocean Captures Ecological Patterns and Behavior of Deep-sea Fishes. Limnol. Oceanogr. Lett. 2021, 6, 339– 347,  DOI: 10.1002/lol2.10213
      68
      Vertical stratification of environmental DNA in the open ocean captures ecological patterns and behavior of deep-sea fishes
      Canals, Oriol; Mendibil, Inaki; Santos, Maria; Irigoien, Xabier; Rodriguez-Ezpeleta, Naiara
      Limnology & Oceanography Letters (2021), 6 (6), 339-347CODEN: LOLIBT; ISSN:2378-2242. (John Wiley & Sons Ltd.)
      Establishing the foundations for a sustainable use of deep-sea resources relies on increasing knowledge on this inaccessible ecosystem, which is challenging with traditional methods. The anal. of environmental DNA (eDNA) emerges as an alternative, but it has been scarcely applied to deep-sea fish. Here, we have analyzed the fish eDNA contained in oceanic vertical profile samples (up to 2000 m depth) collected throughout the continental slope of the Bay of Biscay. We detected 52 different fish species, of which 25 were classified as deep-sea fish. We found an increase of deep-sea fish richness and abundance with depth, and that eDNA reflects day-night community patterns and species-specific vertical distributions that are consistent with the known diel migratory behavior of many mesopelagic fishes. These findings highlight the potential of eDNA to improve knowledge on the fish species inhabiting the dark ocean before this still pristine ecosystem is further exploited.
    69. 69
      Monuki, K.; Barber, P. H.; Gold, Z. eDNA Captures Depth Partitioning in a Kelp Forest Ecosystem. PLoS One 2021, 16, e0253104  DOI: 10.1371/journal.pone.0253104
      69
      eDNA captures depth partitioning in a kelp forest ecosystem
      Monuki, Keira; Barber, Paul H.; Gold, Zachary
      PLoS One (2021), 16 (11), e0253104CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Environmental DNA (eDNA) metabarcoding is an increasingly important tool for surveying biodiversity in marine ecosystems. However, the scale of temporal and spatial variability in eDNA signatures, and how this variation may impact eDNA-based marine biodiversity assessments, remains uncertain. To address this question, we systematically examd. variation in vertebrate eDNA signatures across depth (0 m to 10 m) and horizontal space (nearshore kelp forest and surf zone) over three successive days in Southern California. Across a broad range of teleost fish and elasmobranchs, results showed significant variation in species richness and community assemblages between surface and depth, reflecting microhabitat depth preferences of common Southern California nearshore rocky reef taxa. Community assemblages between nearshore and surf zone sampling stations at the same depth also differed significantly, consistent with known habitat preferences. Addnl., assemblages also varied across three sampling days, but 69% of habitat preferences remained consistent. Results highlight the sensitivity of eDNA in capturing fine-scale vertical, horizontal, and temporal variation in marine vertebrate communities, demonstrating the ability of eDNA to capture a highly localized snapshot of marine biodiversity in dynamic coastal environments.
    70. 70
      Allan, E. A.; DiBenedetto, M. H.; Lavery, A. C.; Govindarajan, A. F.; Zhang, W. G. Modeling Characterization of the Vertical and Temporal Variability of Environmental DNA in the Mesopelagic Ocean. Sci. Rep. 2021, 11, 21273,  DOI: 10.1038/s41598-021-00288-5
      70
      Modeling characterization of the vertical and temporal variability of environmental DNA in the mesopelagic ocean
      Allan, Elizabeth Andruszkiewicz; DiBenedetto, Michelle H.; Lavery, Andone C.; Govindarajan, Annette F.; Zhang, Weifeng G.
      Scientific Reports (2021), 11 (1), 21273CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
      Increasingly, researchers are using innovative methods to census marine life, including identification of environmental DNA (eDNA) left behind by organisms in the water column. However, little is understood about how eDNA is distributed in the ocean, given that organisms are mobile and that phys. and biol. processes can transport eDNA after release from a host. Particularly in the vast mesopelagic ocean where many species vertically migrate hundreds of meters diurnally, it is important to link the location at which eDNA was shed by a host organism to the location at which eDNA was collected in a water sample. Here, we present a one-dimensional mechanistic model to simulate the eDNA vertical distribution after its release and to compare the impact of key biol. and phys. parameters on the eDNA vertical and temporal distribution. The modeled vertical eDNA profiles allow us to quantify spatial and temporal variability in eDNA concn. and to identify the most important parameters to consider when interpreting eDNA signals. We find that the vertical displacement by advection, dispersion, and settling has limited influence on the eDNA distribution, and the depth at which eDNA is found is generally within tens of meters of the depth at which the eDNA was originally shed from the organism. Thus, using information about representative vertical migration patterns, eDNA concn. variability can be used to answer ecol. questions about migrating organisms such as what depths species can be found in the daytime and nighttime and what percentage of individuals within a species diurnally migrate. These findings are crit. both to advance the understanding of the vertical distribution of eDNA in the water column and to link eDNA detection to organism presence in the mesopelagic ocean as well as other aquatic environments.
    71. 71
      West, K. M.; Stat, M.; Harvey, E. S.; Skepper, C. L.; DiBattista, J. D.; Richards, Z. T.; Travers, M. J.; Newman, S. J.; Bunce, M. eDNA Metabarcoding Survey Reveals Fine-Scale Coral Reef Community Variation across a Remote, Tropical Island Ecosystem. Mol. Ecol. 2020, 29, 1069– 1086,  DOI: 10.1111/mec.15382
      71
      eDNA metabarcoding survey reveals fine-scale coral reef community variation across a remote, tropical island ecosystem
      West, Katrina M.; Stat, Michael; Harvey, Euan S.; Skepper, Craig L.; Di Battista, Joseph D.; Richards, Zoe T.; Travers, Michael J.; Newman, Stephen J.; Bunce, Michael
      Molecular Ecology (2020), 29 (6), 1069-1086CODEN: MOECEO; ISSN:0962-1083. (Wiley-Blackwell)
      Environmental DNA metabarcoding, a technique for retrieving multispecies DNA from environmental samples, can detect a diverse array of marine species from filtered seawater samples. There is a growing potential to integrate eDNA alongside existing monitoring methods in order to establish or improve the assessment of species diversity. We investigated the utility of eDNA metabarcoding as a high-resoln., multitrophic biomonitoring tool at the Cocos Islands, Australia (CKI)-a remote tropical coral reef atoll situated within the eastern Indian Ocean. Metabarcoding assays targeting the mitochondrial 16S rRNA and CO1 genes, as well as the 18S rRNA nuclear gene, were applied to 252 surface seawater samples collected from 42 sites within a 140 km2 area. Our assays successfully detected a wide range of bony fish and elasmobranchs (244 taxa), crustaceans (88), molluscs (37) and echinoderms (7). Assemblage compn. varied significantly between sites, reflecting habitat partitioning across the island ecosystem and demonstrating the localisation of eDNA signals, despite extensive tidal and oceanic movements. In addn., we document putative new occurrence records for 46 taxa and compare the efficiency of our eDNA approach to visual survey techniques at CKI. Our study demonstrates the utility of a multimarker metabarcoding approach in capturing multitrophic biodiversity across an entire coral reef atoll and sets an important baseline for ongoing monitoring and management.
    72. 72
      Lamy, T.; Pitz, K. J.; Chavez, F. P.; Yorke, C. E.; Miller, R. J. Environmental DNA Reveals the Fine-Grained and Hierarchical Spatial Structure of Kelp Forest Fish Communities. Sci. Rep. 2021, 11, 1– 13,  DOI: 10.1038/s41598-021-93859-5
      There is no corresponding record for this reference.
    73. 73
      Laroche, O.; Kersten, O.; Smith, C. R.; Goetze, E. Environmental DNA Surveys Detect Distinct Metazoan Communities across Abyssal Plains and Seamounts in the Western Clarion Clipperton Zone. Mol. Ecol. 2020, 29, 4588– 4604,  DOI: 10.1111/mec.15484
      73
      Environmental DNA surveys detect distinct metazoan communities across abyssal plains and seamounts in the western Clarion Clipperton Zone
      Laroche, Olivier; Kersten, Oliver; Smith, Craig R.; Goetze, Erica
      Molecular Ecology (2020), 29 (23), 4588-4604CODEN: MOECEO; ISSN:0962-1083. (Wiley-Blackwell)
      The deep seafloor serves as a reservoir of biodiversity in the global ocean, with >80% of invertebrates at abyssal depths still undescribed. These diverse and remote deep-sea communities are critically under-sampled and increasingly threatened by anthropogenic impacts, including future polymetallic nodule mining. Using a multigene environmental DNA (eDNA) metabarcoding approach, we characterized metazoan communities sampled from sediments, polymetallic nodules and seawater in the western Clarion Clipperton Zone (CCZ) to test the hypotheses that deep seamounts (a) are species richness hotspots in the abyss, (b) have structurally distinct communities in comparison to other deep-sea habitats, and (c) that seafloor particulate org. carbon (POC) flux and polymetallic nodule d. are pos. correlated with metazoan diversity. eDNA metabarcoding was effective at characterizing distinct biotas known to occur in assocn. with different abyssal substrate types (e.g., nodule- and sediment-specific fauna), with distinct community compn. and few taxa shared across substrates. Seamount faunas had higher overall taxonomic richness, and different community compn. and biogeog. than adjacent abyssal plains, with seamount communities displaying less connectivity between regions than comparable assemblages on the abyssal plains. Across an estd. gradient of low to moderate POC flux, we find lowest taxon richness at the lowest POC flux, as well as an effect of nodule size on community compn. Our results suggest that while abyssal seamounts are important reservoirs of metazoan diversity in the CCZ, given limited taxonomic overlap between seamount and plains fauna, conservation of seamount assemblages will be insufficient to protect biodiversity and ecosystem function in regions targeted for mining.

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    • Supplementary methods; supplementary figures and tables referenced in the main text; supplementary table supporting qPCR inhibition testing as described in the Supplementary Methods (PDF)


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