SAMPLING STRATEGIES FOR AQUATIC ENVIRONMENTS

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Ideal sampling strategies will vary among habitats and monitoring contexts. We recommend that you speak to a member of our team to plan your survey. However, below is some guidance synthesized from eDNA research to date.

Note that if you are using bacterial kits, much less water is required to capture the bacterial community. You may choose to collect less water, and take fewer samples, than for other tests and kits.

PONDS

In a pond, eDNA does not mix well due to absence of flow or wave action (1-3), so multiple water samples are key to capture the eDNA present (4-6). A pond can be sampled for eDNA in two ways: 1) independent samples, each comprised of subsamples (e.g. 3 L comprised of 12 x 250 mL subsamples), can be taken at multiple locations around the pond perimeter (6-8), or 2) subsamples can be taken at multiple locations and pooled into a single composite sample (e.g. 3 L comprised of 30 x 100 mL subsamples) representing the entire pond (1,9).

Independent samples each passed through a separate filter will likely have higher detection rates as eDNA is more likely to be captured in at least one of the samples (6,10). Composite samples will reduce cost and allow more ponds to be sampled (1,10), but DNA from rare or low-density species may not be detected (7,8). Several composite samples may be needed for adequate eDNA representation from larger ponds (1).

When sampling ponds:

• Surface water (sub)samples should be collected from the shoreline at roughly equidistant intervals or targeting preferred habitat (if detection of a particular species is a priority) around the pond perimeter. (4,11)
• Use the provided sampling bag or ladle, or a clean bottle (we recommend a small mineral water bottle with the water discarded), to collect water. Deposit the collected (sub)sample in the provided sampling bag. Repeat for each subsample if applicable. Seal the bag and make sure the water is well mixed by shaking for 20-30 seconds. The bag is not self-standing, but can be supported by the surveyor or propped against a log, tree stump or rock to stabilize it for filtration.
• You should use a new bag/ladle/bottle for independent samples and for each pond to avoid cross-contamination (4,11).
• Depending on water clarity, it may be possible to pass up to 3 L of pond water through each filter used, but smaller volumes (e.g. 150-250 mL) are more typical for turbid ponds or ponds with dense vegetation (4,11).

LAKES

In a lake, eDNA can still be localized, so multiple water samples remain key to capture the eDNA present (4, 12-18). We recommend at least 10 independent samples, each comprised of subsamples (e.g. 3 L comprised of 12 x 250 mL subsamples), are taken from multiple locations around the lake perimeter (19,20). You should consider collecting one independent sample (i.e. using 1 kit) along 120 m of shoreline (maximum of shoreline), but contact NatureMetrics to discuss alternative sampling strategies to suit all budgets.

When sampling lakes:

• Surface water samples should be collected from the shoreline at roughly equidistant intervals around the lake perimeter (4).
• Use the provided sampling bag or ladle, or a clean bottle (we recommend a small mineral water bottle with the water discarded), to scoop up water every 10 m along a 120 m stretch of shoreline. Deposit the collected subsample in the provided sampling bag. Repeat for each subsample, seal the bag, and make sure the water is well mixed by shaking the bag for 20-30 seconds. The bag is not self-standing, but can be supported by the surveyor or propped against a log, tree stump or rock to stabilize it for filtration.
• Depending on water clarity, it may be possible to pass up to 3 L of lake water through each filter used, but smaller volumes (e.g. 2 L) are more typical (4).
• Sampling when the lake is not thermally stratified is ideal as more mixing of the water will occur. This means there is a higher chance of detecting eDNA from shallow and deep-water aquatic species (4,15,17,21). However, detection of invertebrates and some other taxonomic groups is generally lower in colder temperatures (22-25). In some cases, it is advisable to sample by boat and at varying depths to maximize detection rates (4,15,17,18,21). Please contact NatureMetrics to discuss your sampling plans.

RIVERS OR STREAMS

In a river or stream, eDNA can be well-mixed depending on local environmental conditions (26-30), but flow means that eDNA can be transported hundreds to thousands of meters from its source(29,31,32), and be diluted (27,33). As such, multiple sampling locations along the length of the stream/river are recommended (4). In small streams or rivers, five sampling locations should be identified, unless they are being considered as part of a broader catchment. In larger rivers, 20-60 sampling locations should be identified for a comprehensive survey (30,31,34-36).

At each sampling location, collect 3 L of water and filter through a single kit. To maximize and assess the likelihood of species detection, sampling can be split into three independent samples (kits) spanning the width of the stream/river section (e.g. separate kits for the left bank, center, and right bank) (26,34,37,38). If your budget does not allow for independent samples, subsamples at each sampling location are an alternative method to increase species detection (e.g. 1 L from the left bank, center, and right bank, pooled to give 3 L) (38-40). If sub-sampling is not feasible, alternate sampling locations between both sides of the stream/river as much as possible to account for variable eDNA distribution (27). For wide rivers, fast flowing rivers, and rivers with a large catchment, more water samples may be needed at each sampling location (26,34,37,38). We recommend speaking to our survey design team to help plan your survey in these instances or when a specific project question is being considered.

When sampling rivers or streams:

• Start at the most downstream sampling location and work your way upstream (4).
• Use of a sampling vessel/device and sampling from the shoreline is recommended. If shoreline sampling is not possible, surveyors should enter the water downstream of where they will collect the sample and be careful not to disturb sediment as they move to the collection point (4). If the water is too deep to enter, a boat or similar should be used for sampling.
• Use the provided sampling bag or ladle, or a clean bottle (we recommend a mineral water bottle with the water discarded), to collect water by holding it with the opening pointing upstream at the water surface. Stand downstream of the sampling bag/ladle/bottle to avoid collecting your own DNA. Deposit the collected sample in the provided sampling bag. Repeat for each sample, seal the bag, and make sure the water is well mixed by shaking for 20-30 seconds. The bag is not self-standing, but can be supported by the surveyor or propped against a log, tree stump or rock to stabilize it for filtration.
• Depending on water clarity, it may be possible to pass up to 3 L of stream/river water through each filter used, but smaller volumes (e.g. 2 L) are more typical.

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ESTUARIES, SEAS OR OCEANS

In marine waterbodies, less is currently known about how hydrological systems affect eDNA transportation and distribution. However, it has been shown that communities obtained from marine eDNA metabarcoding are highly representative of the immediate local habitat where the sample was collected, both on horizontal and vertical planes (41-50) and across short timescales (47,21,52). This means that when samples are collected in a transect going from shore to offshore, different communities will be detected, sometimes even within the range of tens of metres (41,44,48,53). As in lakes, vertical stratification of the water (as a result of thermoclines) restricts mixing of eDNA, meaning that water samples should be collected from each depth zone of interest to fully characterize the marine communities at the sampling location (4,44,48,53,54).

eDNA in marine systems is generally much more dilute compared to that in freshwater systems (4,55). This is in part dependent on the target group or species but is particularly pertinent for larger vertebrates (extra-organismal vertebrate eDNA) (56,57). Planktonic or microbial taxa usually require smaller volumes (4). Therefore, sample volume should be maximized to be representative of the environment and the taxa that are targeted (58). Each sample should be at least 2 L volume (59), and the volume of water filtered should be in the range of 2-5 L (more is always better) (4,60). Turbidity is usually less of a problem in marine water, although inshore areas (e.g. mangrove forests, marinas, areas with high population density) can become turbid due to coastal run-off and wave action disturbing the sea floor (4,35). In this case, filter as much water as possible from each sample until the filter completely clogs.

Sample number will depend on the spatial scale of the study or monitoring project (4). In order to characterize a community or compare sites, a minimum of 20 samples, even for relatively small areas, is strongly advised (58). This usually involves collecting independent samples (rather than subsamples) spread out across the sampling area (4).

Sampling design will equally be dependent on the size of the sampling area, and also on the type of ecosystem (coral reef, mangrove forest, pelagic, etc.) that is targeted. Season (and even time of day) may need to be taken into consideration, as many fish species move to (in)shore areas for mating and spawning and move to deeper or warmer waters in winter, while other species may prefer cold, deep water during summer (15,61). Furthermore, some fish species exhibit diel migration where they move to a different position in the water column during the day or night which is reflected by eDNA analysis (52,54,62). Thus, it is important to consider migration patterns as well as mating and spawning sites.

When sampling estuaries, seas or oceans:

• Sampling is most often done from a boat, but samples may also be collected from the shoreline (4).
• Use the provided sampling bag or a Kemmerer/Niskin sampler to collect a minimum of 2 L of sea water per sample (59). Contact NatureMetrics if you do not have access to a Kemmerer/Niskin sampler.
• When using the Kemmerer/Niskin sampler to collect water near the bottom, be careful not to disturb the sediment as sediment present in water samples can clog filters and may contain ‘old’ eDNA that can skew inferences of species’ presence (63).
• You can use a manual syringe to filter the water, but peristaltic or vacuum pumps are more commonly used for larger volume samples (64).
• We recommend filtering 5 L (60), but the actual volume filtered will depend on the clarity and turbidity of the water (35). We suggest filtering no less than 2 L.

TO REPLICATE OR NOT TO REPLICATE?

Independent versus composite samples have been discussed as sampling strategies for several aquatic environments. Independent samples are field replicates, whether of an entire waterbody (e.g. pond, lake) or of a particular location in a waterbody (e.g. streams, rivers, seas, oceans). They add spatial context to the biological data obtained using eDNA analysis as each sample has its own coordinates (4). They also increase the probability of detecting species, especially those that are rare and/or present at low density, as eDNA is more likely to be captured (4,60,65–67). As composite samples are comprised of subsamples taken from multiple, distinct points around or within a waterbody that are pooled together, spatial resolution is lost (4) and eDNA of some species may be diluted by additional water (1,14,18), but there is more budget available to sample more locations and/or waterbodies (4,50). Which strategy to use ultimately depends on project aims, questions and budget (4,11).

If the question is simply ‘what species might be present in my study area?’, rare and/or low density species are of less concern, and statistical modelling is not required, then a composite sampling strategy will suffice. For example, one composite sample could be taken at each of 20 sampling locations along a river or multiple rivers as opposed to three independent samples at each sampling location, meaning 60 sampling locations could be surveyed instead of 20 sampling locations with the same budget.

If the question is more complex, such as ‘where do detected species occur?’, ‘what is the total number of species present in my study area?’ or ‘what is the error margin around species detection?’, then replicates are required to provide statistical power for models that perform predictive mapping, interpolate and extrapolate species richness, and estimate species detection probability or likelihood of false positives (i.e. species detected that is not actually present) and false negatives (i.e. species not detected that is actually present) (4,50,60,65–69). Please contact NatureMetrics to discuss your sampling plans if you have a particular question in mind.

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CONTROLS

NatureMetrics recommend the inclusion of field negative controls (blanks) to detect possible contamination introduced during sampling. These are purified water (i.e. mineral, deionized or MilliQ water) processed in the same way as water samples (4,63,64,70).

The purified water should be poured into the sampling bag provided in the kit being used as a field blank. The bag should be sealed and shaken, then the purified water filtered as you would filter a natural water sample. Generally, filtering 500 mL of purified water is sufficient to serve as a field blank.

Once at the NatureMetrics laboratories, the field blank will be processed alongside your samples and screened with the analyses you selected to identify whether any contamination has occurred in the field. NatureMetrics Aquatic eDNA Kits contain only sterile, single-use components so no contamination is present before the kit is opened. However, contamination can be introduced by the surveyor as water samples are collected and filtered.

We recommend including at least one field blank at the end of each day that water sampling occurs, but ideally a field blank should be included for each waterbody sampled (70). NatureMetrics provide a free kit to be used as a field blank if 20 or more Aquatic eDNA Kits are purchased.

DECONTAMINATION

If you will be entering the water to collect samples, boots or waders should be decontaminated with bleach (containing sodium hypochlorite) in between waterbodies (4,11,63,64). Bleach should be disposed of responisibly. Please contact NatureMetrics for advice on appropriate decontamination procedures.

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