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Project: DNA Barcoding


Background and Objectives


Assemblages of benthic invertebrate species are often used to assess sediment condition. However, traditional methods for gathering data on benthic infauna communities can be time consuming and labor intensive. Samples are traditionally processed by trained taxonomists, who must identify and count each organism by hand. This project examines a new molecular tool for rapidly identifying the species found within benthic community assemblages and other organism communities used in bioassessment, such as algae. DNA barcoding espouses the idea that all biological species can be identified using a short gene sequence from a standardized position in the genome – a “DNA barcode” – analogous to the black stripes of the Universal Product Code used to distinguish commercial products.

Building a barcode reference library of DNA sequences from known voucher specimens is the first step in being able to use DNA barcoding for sample processing. With a reference library, unknown specimens can be identified by "looking up" their sequences. Barcoding data based on species presence can then be interpreted to correspond with other benthic indices that count numbers and species of organisms. Examination of barcode data sometimes reveal instances where a reassessment of morphologically-defined species is warranted. For instance, cryptic (previously unrecognized) species may be discovered, or a merger of groups previously believed to be separate species may be indicated. Thus, one short-term benefit of DNA barcoding research will be clarification of the catalog of benthic invertebrate species taxonomy for Southern California.

The DNA Barcoding process involves analyzing the DNA signatures in a sample of benthic invertebrates.

The goal of this project is to assess the efficacy of barcoding for rapidly identifying freshwater and marine benthic invertebrate and algal species in the Southern California Bight.

Status

This project was initiated in 2009 with anticipated completion in 2015.

Methods

The project involves several steps. The first is to establish a DNA barcode reference library of voucher specimens for southern California. To sequence a specimen’s DNA barcode, DNA is first extracted from the tissue using standard and widely available tools of molecular biology. The barcode region is then isolated, replicated by PCR amplification, and sequenced. The barcode region must consistently identify all members of the species, while excluding closely related species.

Secondly, researchers must develop modified protocols for sample processing that accommodate examination by molecular methods. Current methods for preserving benthic invertebrates rely on formalin as a fixative, which renders the samples unsuitable for molecular processing.

The third task is to investigate the relationship between barcode-based taxonomy and traditional morphology-based taxonomy, and demonstrate how barcoding can expand our ability to assess species composition and identify cryptic species. Current benthic indices are based on abundance and diversity of individual species. Barcode data will elicit the diversity of species, but may not provide an accurate count of the number of individual organisms in the sample. This task focuses on determining how to convert barcode data to usable, and improved, environmental indices. 

The fourth effort will involve working toward next-generation sequencing methods to analyze composite DNA samples collected through traditional benthic sampling. In addition, researchers will evaluate the ability to determine invertebrate community composition from environmental DNA (eDNA), a composite of free-floating DNA extracted directly from the water column. 

Findings

Routine preservation methods can largely be used without adverse effects on DNA integrity. Benthic macroinvertebrates from five taxa subjected to one of six preservation treatments were processed to produce DNA barcodes. On average, successful sequences (i.e., either full or partial barcodes) were obtained for 93-99% of all specimens across all six treatments. As long as samples were initially preserved in 95% ethanol, successful barcode sequencing was not affected by a low dilution ratio of 2:1, transfer to 70% ethanol, presence of abundant organic matter, or holding times of up to six months.

The lack of quantification in species identification does not seem to be an impediment to adoption of molecular methods in biological assessments.To evaluate how well barcoding data meshes with two commonly-used environmental indices (the Southern California Benthic Response Index or BRI and the AZTI Marine Biotic Index or AMBI), species abundance information was removed from the index calculation process to simulate collection of the data through barcoding. Presence-only scores for these two indices were then compared to the scores using species abundance data. The presence and abundance scores were significantly similar, with correlation coefficients of 0.99 and 0.81 for the BRI and AMBI, respectively. To assess the importance of the size of the barcode species library, researchers also systematically removed taxa from the index calculation process. Correlation between the results remained above 0.9 with only 370 species for the BRI library. Similarly, reducing the number of species to 450 had almost no effect on correlation between the presence and abundance versions of the AMBI.

One study found that using barcoding data increased total richness at coastal stream sites by 12-40 taxa when compared to morphological identification alone. It also increased the difference between reference and impact sites in terms of lost taxa. These results suggest that macroinvertebrate biodiversity in streams has been substantially underestimated in the past, as has the biodiversity lost in response to environmental stress. In another study, 10 out of 16 metrics of community health had substantially higher statistical power when calculated with DNA barcode data as compared to morphological data. Researchers were able to discern differences between armored and unarmored stream reaches using DNA data. These differences were associated with increased taxonomic richness detected via DNA data for midges, mayflies, noninsects, caddisflies, and black flies. These results suggest identifications based on DNA barcoding have the potential to improve power to detect small changes in stream condition.

Partners

This project is being conducted in collaboration with the US Environmental Protection Agency, Canadian Centre for DNA Barcoding, Stroud Water Research Center, and SCCWRP’s member agencies.

Presentations

DNA barcoding as a tool for marine and freshwater bioassessment - January 2012 overview presentation to SCCWRP member agencies summarizing efforts in this research area.

Adaptation of the Benthic Response Index to utilize DNA barcoding data - January 2011 presentation to SCCWRP member agencies describing a method to correlate DNA barcoding data with a traditional benthic community assessment method.

Videos



Overview of DNA Barcoding


DNA Barcoding Methods

 

For more information on DNA Barcoding, contact Eric Stein at erics@sccwrp.org (714) 755-3233.
This page was last updated on: 6/30/2014