By Steve Crookes and Fei Luo
The science behind environmental DNA (eDNA) detection and its applicability to biomonitoring was introduced to Environmental Science & Engineering’s readers in the June 2019 edition. (Gasparini, 2019). That article highlighted the sensitivity, specificity and power of eDNA and the potential of this technology to expand the scope of and fundamentally change the methodology of ecological monitoring.
However, realizing the potential of eDNA will take adoption by industry, buy-in from regulators and standardization in practice and analysis. In this article, we focus on eDNA tools that can be used over the life cycle of primary resource industry (PRI) projects for biodiversity monitoring.
Greenfield project sites will, in the absence of any destabilizing ecological forces, be home to a relatively stable ecosystem representing a baseline of biodiversity. A robust accounting of the biodiversity characteristic of that baseline can be used to frame restoration efforts. Post operations, it is imperative to know what land use is intended so that appropriate landscape features can be incorporated to support the expected biodiversity. Characterizing biodiversity over this life cycle is where eDNA technology has great potential.
There are two eDNA technology platforms. Metabarcoding is a high-throughput method that identifies many species in a single test and as such represents an untargeted approach. Quantitative PCR (qPCR) is a targeted approach, which detects only particular species of interest, but with superior sensitivity and accuracy. These two platforms are complementary, and together offer comprehensive coverage of biodiversity.
Metabarcoding is a process whereby the DNA of multiple species is identified using a standard genetic marker (a molecular barcode) that is common to all life. When analyzed using molecular methods it is diagnostic of each unique species due to variations in the sequences of DNA. Similar sequences that group together and represent the same species are called “operational taxonomic units”, or OTUs. Because each OTU has unique representative DNA sequence, they are termed “barcodes”, as they work in an analogous fashion to the UPC barcodes used in commercial transactions.
Genetic barcode markers are qualified and quantified using next-generation DNA sequencing that can analyze, by directly sequencing each molecule, millions of different DNA sequences simultaneously. Total DNA may be extracted from environmental samples (i.e., eDNA) and contain within them DNA from all the biota that had recently shed eDNA into their surroundings. This is the “meta” part of metabarcoding.
All DNA sequences are grouped into OTUs, which are then compared to a reference database of DNA barcode from known species and identified. In this way, all the species can be identified, and their relative abundances of the DNA sequences indicated (i.e., via proportion of DNA sequences sorting into each OTU).
In simple terms, the entire biological community that left traces within a sample can be approximately reconstructed whole again. Effectively we get a “snapshot” of biodiversity in time. For example, if completed in the pre-operations phase, the biodiversity assessment using eDNA is part of baseline work for a project.
These snapshots of species assemblages from DNA barcodes can be repeated across time and space. They can provide a temporal record in the change of species composition and an indication of the changes in relative abundance of species detected. If employed in this time-series fashion, eDNA metabarcoding barcode sequences may be referred to as “chronosequences” (Gastuaer et al. 2019).
The benefits to chronosequence monitoring over the lifetime of a PRI project include being able to modify operational procedures to mitigate further biodiversity loss and/or to enact habitat enhancement initiatives to restore biodiversity during the operation (i.e., progressive reclamation).
Chronosequence monitoring will allow for timely enactment of response initiatives or action plans that are part of the overall management strategy for the project.
There are a number of published field studies that have implemented chronosequence monitoring (e.g., Boschen et al., 2016; Coble et al., 2018; Fernandes et al., 2018). They showed that it can be used effectively to monitor changes in biodiversity. However, the use of metabarcoding in PRI projects is novel and is beginning to be applied in order to answer very specific questions.
As with all environmental assessment techniques, caveats are in place for proper use of eDNA metabarcoding. These include all the sampling and optimization processes associated with conducting eDNA surveys generally. eDNA metabarcoding methods, including computational advances in DNA sequence analysis and the numbers of species represented in genetic databases, will only ever increase.
Therefore, all samples collected at any point in time will be subject to less optimal analysis than samples collected in the future that will be processed using more up-to-date methods. Thus, there will be a time-directed bias to the quality/quantity of information. Rather than being a fatal flaw, all that needs to occur is that, for every survey conducted, at least half of any eDNA extract be archived in deep freeze for future analysis with the upgraded metabarcoding workflow.
Although archiving would increase costs, small amounts of eDNA representing entire ecosystems can be stored in relatively small spaces. Archiving samples is quickly being considered to be an absolute necessity in the surveillance of global biodiversity (Dysthe et al. 2018) and would likely be considered necessary by regulators in the future.
The less complex targeted (qPCR) approach also retains a prominent place in appraising lands for resource utilization and responsible environmental stewardship. Targeted eDNA surveys employ species-specific molecular probes of extreme sensitivity that can potentially detect the target species’ eDNA in an environmental sample at amounts as low as attograms—that is 10-23 g.
Such extreme sensitivities are essential in gaining more confidence that a species is not present, as well as for detecting it when it is suspected to be present. In sampling terms, by employing rigorous machine-learning based software tools for survey designs, which include increasing our statistical power to detect the species, we can be more confident in that species’ absence should it not be detected.
Put another way, the extreme sensitivities of eDNA-based molecular assays increase our power to reject false negative detection (assuming an arbitrary rate of false negative detections—usually 5%).
Being confident that a species of interest is not occupying an area earmarked for development, is often an essential first step in the pathway to initiate responsible resource-based projects.
As an example, the field team at Precision Biomonitoring Inc. recently conducted a survey for the federally protected Blanding’s turtle (Emydoidea blandingii) at a site of an historic gold mine in northern Ontario that has been slated for a resumption of operations. However, the local Ministry of Natural Resources and Forestry office had credible reports of this turtle being present in the area in the past, albeit in low numbers. This was likely as intentional release by the local human population.
Regardless of its origin, the site was intensively surveyed using an on-site eDNA sampling strategy that, in combination with a highly sensitive Blanding’s assay, constituted enough effort (assuming a 5% false negative detection rate as acceptable error) to have detected the species should it have been present.
The calculation of the sampling effort was based on previous records of eDNA-based detection of the turtle within its known range of occupancy in southern Ontario. This was further verified by a prototype machine-learning based software tool that generates sampling strategies with high statistical accuracy.
This example shows how important pilot, and antecedent studies, are to conduct eDNA surveys, particularly for areas where development poses benefits but that have important conservation value. For the full benefits of eDNA detection methods to be felt by PRIs, and, thus, by the wider public, we must work together to validate these methods and to establish the bare minimum standards required for their conduct.
A coming-together and reciprocal understanding of stakeholder concerns are necessary to unify the goals of industrial interests, economists, politicians, First Nations, regulators and conservationists to optimize environmental monitoring under a shared rubric. Luckily, egalitarian methods such as eDNA detection, which lend themselves to direct public involvement (citizen science projects), can facilitate closer ties and mutual understanding.
Steve Crookes is with Precision Biomonitoring Inc. Fei Luo is with Ecometrix Incorporated. This article appears in ES&E Magazine’s December 2019 issue.