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Scientists Use DNA Barcoding to ID Canadian Freshwater Fish

NEW YORK (GenomeWeb News) – Scientists can accurately identify the majority of Canadian freshwater fish species using DNA “barcodes” that are just a few hundred base pairs long, new research suggests.
Using a newly developed set of barcoding standards, a team of Canadian researchers sequenced a DNA barcode — part of a mitochondrial gene — from 95 percent of the known freshwater fish species in Canada. The work, published in today’s issue of the journal PLoS ONE, demonstrates that the DNA sequence can accurately and effectively distinguish between more than 90 percent of the species tested. It also exposed sequence divergence between some individuals lumped into the same species — suggesting cryptic species may exist or be in the process of forming.
DNA barcodes are short stretches of DNA that are used as molecular tags by researchers to classify different plant and animal species. Proponents see it as a fast and efficient way to accurately identify organisms at any stage in their life cycle, uncover previously unrecognized species, and aid research on biodiversity, predator-prey relationships, and invasive species.
Even so, the concept of DNA barcoding is somewhat controversial. Some critics have claimed that the proposed benefits of DNA barcoding are overblown, arguing that barcoding is a poor alternative to other classification methods such as morphology and traditional taxonomy.
In the past, barcoding has proven itself highly accurate in marine fish, co-author Robert Hanner, associate director for the Canadian Barcode of Life and biologist at the University of Guelph, told GenomeWeb Daily News today. But, he added, some believed it would be much more difficult to distinguish between freshwater fish, which live in more changeable freshwater ecosystems or “harbors of speciation.”
“The suspicion was that there’d be more hybridization going on,” Hanner said.
As part of the international Fish Barcoding of Life, or FISH-BOL, initiative, the team set out to test the validity of DNA barcoding as a method for distinguishing between individuals from similar species, specifically freshwater fish. They amplified barcode DNA from 1,360 individual fish representing 190 of Canada’s 203 documented freshwater fish species.
To gauge variability between populations, the team attempted to sample between three and five individuals per site and at least two different sites whenever possible. For each species, they tested between one and 17 different individuals.
They then bi-directionally sequenced about 650 base pairs of “barcode” sequence from the mitochondrial cytochrome c oxidase I, or COI, gene of each individual. Based on their analysis of these barcodes, the investigators reported that the set of barcodes they had were adequate for identifying about 93 percent of the species tested.
For the most part, genetic distances were much higher between species in a genera than they were between individuals belonging to the same species. On average, the researchers found 27 times greater genetic distances between than within species. Just over a dozen species — about seven percent of those tested — had barcodes that were the same as or overlapped with those of other species.
And Hanner noted that DNA barcoding will likely be useful even for species that share sequence because it provides enough information to narrow a Canadian freshwater fish down to two species out of more than 200.
In combination with other types of data, the work may also fuel a re-assessment of freshwater fish taxonomy. The researchers detected both similarities between sister species, but also deep genetic divergence between some individuals assigned to the same species.
“This just reflects the fact that speciation is a process,” Hanner said. “We do have these sort of evolutionary bursts taking place.” But for the most part, he added, the fish species are sufficiently distinct to be distinguished from one another by DNA barcoding.
Data from the freshwater fish barcoding project will be available through the actively curated Barcode of Life Database, called BOLD, which contains raw sequence data and detailed information about specimen taxonomy.
Although there are hundreds of DNA barcoding papers in print, Hanner noted, “This is one of the first to implement BARCODE standards.” Those standards were developed by the Consortium for the Barcode of Life and denote barcoding data that meets a set of criteria for sequence quality as well as availability of additional information such as when and where specimens were collected, the primers used to amplify the barcode, and so on, Hanner explained.
Barcode sequence data that meets the standards will eventually be available through GenBank and other databases under the reserved keyword “BARCODE.”
The FISH-BOL team is currently expanding their work to include all the North American freshwater fish — work that is already underway. And, Hanner said, the consortium has gathered information about some 5,000 fish species internationally.

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