DNA Barcoding Debuts for Environmental Bioassessment
By Eric D. Stein, Karen E. Setty and Peter E. Miller *
Bioassessment of marine and freshwater environments is a cornerstone of environmental monitoring throughout the world. The most common approaches infer condition or health of the aquatic habitats based on the structure and composition of the benthic macroinvertebrate community that resides in the underlying substrate (e.g., rocks, sand, sediment, or mud).
These kinds of biological health measurements add significant value to typical data on water chemistry and physical habitat characteristics by integrating fluctuations in habitat condition over time, and directly measuring the sum biological impacts of potential stressors on exposed organisms. Indexing methods have been developed in many parts of the world to translate benthic community information into meaningful and easy-to-understand scores based on the known stressor tolerance of each species.
Enter DNA Barcoding
Scientists at the Southern California Coastal Water Research Project (SCCWRP) have been researching environmental monitoring approaches since 1969. They helped develop southern California’s Benthic Response Index (BRI) for the marine environment in the 1990s, and have contributed to several Indices of Biotic Integrity (IBIs) for the freshwater environment.
In 2009, they began looking into the use of DNA barcoding as an option for identifying benthic organisms. Initial research has focused on collecting voucher specimens to build a locally-relevant barcode reference library, refining sample collection approaches, and investigating the efficacy of barcoding for use in environmental indices.
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Ultimately, the goal is to be able to batch-process entire samples using next-generation sequencing. This research, being conducted by SCCWRP and partners at the Canadian Centre for DNA Barcoding (CCDB), U.S. Environmental Protection Agency (EPA) National Exposure Research Laboratory, and Stroud Water Research Center, represents a major step toward application of DNA barcoding to routine environmental bioassessment.
Partnerships among these research centers have enabled pooling of their various strengths and resources. Because SCCWRP and its member agencies conduct regular monitoring in a variety of environments, they have made much progress in supplying organisms to the CCDB.
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The CCDB and EPA are focused on developing state-of-the-art DNA barcode methodology. The Stroud Water Research Center offers data analysis assistance as well as a point of comparison for SCCWRP’s efforts, based on experience interpreting benthic invertebrate barcoding data from other regions of the United States.
Benefits and Challenges
Benefits of DNA barcoding include reduction of sample processing time, cost, and taxonomic misidentifications, as well as increased knowledge of taxonomic diversity.
One existing barrier to benthic index application when using traditional taxonomic methods is the sometimes hefty investment to get from sample to score. A trained taxonomist must sort bulk environmental samples containing several thousand invertebrates. Identifying specimens and partial specimens by sight alone can take hours to days per sample and cost hundreds to thousands of dollars.
DNA barcoding is highly amenable to automation, so samples can be processed in days rather than months. Over the long term, next generation sequencing should result in dramatic cost and time savings.
SCCWRP has collected and processed about 500 marine invertebrate specimens and 6,000 freshwater invertebrates to date, contributing approximately 170 distinct marine species and 140 freshwater species to the reference library.
Initial analyses from Southern California freshwater streams show general agreement between traditional morphology-based taxonomy and barcoding analysis, but with some telling differences. In a handful of cases, pairs of hard-to-differentiate species like Baetis adonis and Baetis tricaudatis (mayflies) have unique barcodes, and multiple species lumped into the same category, like Simulium spp. (black flies), can also be distinguished. In addition, some newly discovered genetic similarities or differences may warrant taxonomic reclassifications (e.g., Orthocladius).
Several technical challenges to the use of barcoding are also being investigated. Since current specimen preservation methods may not be practical for application to routine field assessments, SCCWRP is researching alternative approaches that best maintain DNA integrity.
In addition, the BRI has been adapted to work with only presence/absence data. This step eliminates the need to quantify the number of each taxon in a sample, and enables comparison of new barcoding results with local historic datasets.
Next steps include completing the reference library, likely to require hundreds of additional species at a minimum, and validating the barcoding method’s ability to interpret monitoring data across a large number of samples and habitats.
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Once developed, DNA barcoding has potential for routine benthic community monitoring applications in coastal and inland waters throughout the world. Opportunities abound to integrate barcoding into existing environmental indices, and use it as a supplement for current monitoring methods.
For example, it could be extended to analyzing algal communities, another emerging bioassessment method in several U.S. states, or to bioassessment using meiofauna. It could also potentially serve as an early screening tool to detect introduced or invasive species in sensitive aquatic habitats.
SCCWRP plans to take a lead role in helping to transition DNA barcoding to routine application by developing and demonstrating tools and procedures for its use in regional and compliance monitoring programs.
* Eric Stein heads the biology department at SCCWRP, Costa Mesa, California. Karen Setty is a Science Writer with SCCWRP. Peter Miller is Director of Business Development with the Canadian Centre for DNA Barcoding in Guelph, Ontario.