APPENDIX

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 synthesised from eDNA research to date.

PONDS

In a pond, eDNA does not mix well due to absence of flow or wave action, so multiple water samples are key to capture the eDNA present. A pond can be sampled for eDNA in two ways: 1) independent samples, each comprised of subsamples (e.g. 2 L comprised of 5 x 400 mL subsamples), can be taken at multiple locations around the pond perimeter, or 2) subsamples can be taken at multiple locations and pooled into a single composite sample (e.g. 2 L comprised of 20 x 100 mL subsamples) representing the entire pond.

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. Composite samples will reduce cost and allow more ponds to be sampled, but DNA from rare or low-density species may not be detected. Several composite samples may be needed for adequate eDNA representation from larger ponds.

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.

  • Use the provided sampling bag 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 propped against a log, tree stump or rock to stabilise it for filtration.

  • You should use a new bag/bottle for independent samples and for each pond to avoid cross-contamination.

  • Depending on water clarity, it may be possible to pass up to 2 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.

LAKES

In a lake, eDNA can still be localised, so multiple water samples remain key to capture the eDNA present. We recommend at least 10 independent samples, each comprised of subsamples (e.g. 2 L comprised of 5 x 400 mL subsamples), are taken from multiple locations around the lake perimeter. You should consider collecting one independent sample (i.e. using 1 kit) along 400 m 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.

  • Use the provided sampling bag or a clean bottle (we recommend a 2 L mineral water bottle with the water discarded) to scoop up water every 20-50 m along a 400 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 propped against a log, tree stump or rock to stabilise it for filtration.

  • Depending on water clarity, it may be possible to pass up to 5 L of lake water through each filter used, but smaller volumes (e.g. 2 L) are more typical.

  • 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. However, detection of invertebrates and some other taxonomic groups is generally lower in colder temperatures. In some cases, it is advisable to sample by boat and at varying depths to maximise detection rates. 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, but flow means that eDNA can be transported hundreds to thousands of metres from its source. As such, multiple sampling locations along the length of the stream/river are recommended. In small streams or rivers, at least three sampling locations (i.e. upstream, mid-section, downstream) should be identified. In larger rivers, 20-60 sampling locations should be identified for comprehensive survey.

A minimum of three independent samples (e.g. 1 L), spanning the width of the stream/river section (e.g. left bank, centre, right bank) should be collected from each sampling location. More water samples at each sampling location are recommended for wider rivers, and possibly at downstream sampling locations if the river has a large catchment and/or is fast-flowing. If your budget does not allow for independent samples, subsamples (e.g. 3 x 1 L) can be taken at each sampling location, mixed, and as much of the pooled sample filtered as possible, but detection of rare or low-density species may be impeded.

When sampling rivers or streams:

  • Start at the most downstream sampling location and work your way upstream.

  • 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. If the water is too deep to enter, a boat or similar should be used for sampling.

  • Use the provided sampling bag or a clean bottle (we recommend a 2 L 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/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 propped against a log, tree stump or rock to stabilise it for filtration.

  • Depending on water clarity, it may be possible to pass up to 5 L of stream/river water through each filter used, but smaller volumes (e.g. 2 L) are more typical.

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. 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 meters. 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 characterise the marine communities at the sampling location.

eDNA in marine systems is generally much more dilute compared to that in freshwater systems. This is in part dependent on the target group or species but is particularly pertinent for larger vertebrates (extra-organismal vertebrate eDNA). Planktonic or microbial taxa usually require smaller volumes. Therefore, sample volume should be maximized to be representative of the environment and the taxa that are targeted. Each sample should be at least 2 L volume, and the volume of water filtered should be in the range of 2-5 L (more is always better). 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. 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. In order to characterize a community or compare sites, a minimum of 20 samples, even for relatively small areas, is strongly advised. This usually involves collecting independent samples (rather than subsamples) spread out across the sampling area.

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. 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.

  • Use the provided sampling bag or a Kemmerer/Niskin sampler to collect a minimum of 2 L of sea water per sample. 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.

  • You can use a manual syringe to filter the water, but vacuum pumps are more commonly used for larger volume samples.

CONTROLS

NatureMetrics recommend the inclusion of field negative controls (blanks) to detect possible contamination introduced
during sampling. NatureMetrics Standard, MAXI and Pump 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. NatureMetrics provide a free kit to be used as a field blank if 20 or more Standard, MAXI or Pump Aquatic eDNA Kits are purchased.

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

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