Cataloging Life

Cataloging Life Can a single barcode of DNA record biodiversity and keep us safe from poisons? By Bob Grant Related Articles 1 for soil nematodes, barcoding's genesis lies in a 2003 paper in Proceedings of the Royal Society of London2 by Paul Hebert, a Canadian researcher who some call "the father DNA barcoding," and colleagues. In that paper, Hebert's team proposed a universal animal barcode: a segment of roughly 650 base pairs of the mitochondrial gene, cytochrome c oxidase 1 (CO1). CO1 became an attractive candidate for a barcode because the primers used to amplify the gene fragment worked across many animal groups. Moreover, researchers had previously suggested that the gene evolved rapidly enough to allow for discrimination between even closely related species. CO1 appeared to work well as a barcode, at least for some animals. Using butterflies and moths Hebert collected from his own Canadian backyard and other animal sequences listed in GenBank, he found that the region was highly variable between species but almost invariable within species. "Barcoding works, and it works better than we could have hoped, because of this gap between intra- and interspecific variation," Hebert says. Success followed. Since 2003, several barcoding projects have probed the mitochondrial DNA of everything from birds3 and fish4 to leeches5 and mosquitoes,6 and they found a similar gap between intraspecific and interspecific variation. These validation studies follow a general formula: Take species that are already well described and delineated through morphology, ecology, and other characters, collect their CO1 barcodes, and see how closely the traditional classification matches with that derived from the barcode. A recent study of North American bird species7 found that 94% of the 643 bird species surveyed possessed distinct barcode clusters and could thus be identified to species based on their CO1 sequences. The idea, says Hebert, is to use DNA barcoding as a response to the dearth of information and resources, to identify living things in the face of declining traditional taxonomic expertise and vanishing species. "We're at a situation of crises, and we have no capacity to inventory biodiversity on the planet, in my view, on the scale that we need to be able to do it," he says, "so we have to change the rules." The sheer number of DNA barcoding research projects going on now illustrates the momentum behind the initiative. Barcoding milestones accumulate daily. There are now more than 300,000 barcode sequences in the central repository for barcoding data - the Barcode of Life Data Systems (BOLD) database. More than 30,000 species of mostly fish, birds, and insects have been described through barcoding. The Consortium for the Barcode of Life (CBOL), the main organizing body for barcoders, comprises more than 150 member organizations, such as museums, zoos, and botanical gardens, from more than 50 countries. International teams of researchers have established working groups within CBOL on fish, mosquitoes, fruit flies, birds, and invasive and pest species, and these groups are accumulating barcode records at a rapid pace. At least 85 barcoding papers have been published in 2007, with several more due for publication in the near future. CBOL chairman Scott Miller says CBOL hopes to have one million barcode records from 100,000 different species by 2009. In addition to CBOL, there is the Canadian Barcode of Life Network, the UK's Species 2000 (a conglomeration of databases that seeks to catalogue life on Earth), and the yet-to-launch Encyclopedia of Life, a Web-based road map envisioned to help users navigate Earth's biodiversity. Because it works with short segments of DNA, barcoding has the potential to quickly and cheaply identify processed animal or plant products that may pose a danger to public health. It may also identify and better control cryptic pest species, such as mosquitoes or fruit flies, which cause widespread disease or wreak economic havoc in many parts of the world. In the clinical setting, it also has the potential to rapidly identify biologic pathogens in ailing patients. Basic science can also benefit from barcoding, if it delivers on the promise of reliable, fast, and cheap identification of species. Currently, barcoding a single specimen can be done for about $5 per sample in about three hours. Because taxonomists have identified fewer than two million of the estimated ten million species of living things inhabiting Earth, a quick and easy genetic technique that can point out unnamed species is an obvious boon to the field. Hebert sees technology advancing so that barcodes will be produced in about 10 minutes for less than one dollar. "DNA barcoding has really demonstrated its effectiveness," says Eldridge Bermingham, a biologist at the Smithsonian Tropical Research Institute in Panama. "Now we argue the finer points." Some say it's not just the finer points that need to be argued - it's the technique itself. They note that the mitochondrial gene used as the animal barcode does not differentiate accurately between all animal species, and for some taxonomic groups it doesn't work at all. Some taxonomists remain unconvinced of the utility of barcoding every species on Earth. "Putting a lot of our energy into harvesting CO1, almost entirely to the exclusion of doing anything else, really is not an effective use of our time," says Kipling Will, a University of California-Berkeley insect systematist. "There's a pretty well-known notion that any single character system is unlikely going to be the panacea for anything." Reports of CO1's inability to differentiate particular animal species have been cropping up lately. In one genus of blowfly, for example, 60% of the species are impossible to identify using CO1, with some of the species containing identical barcodes.8 In another study9 on an order (Scleractinia) of corals, researchers found that CO1 varied so little between different species that delimiting most species using barcoding was nearly impossible. Other theoretical problems with using a mitochondrial gene to define species boundaries include confounders such as the potential for gene transfer between the mitochondria and nuclear genomes, and male-biased gene flow. Some skeptics say that the ambitious goal of barcoding the planet's life is putting the cart before the horse. "My objection is that in the early stages it was grossly oversold," says University of Alberta entomologist Felix Sperling. He says that the concept of using a mitochondrial gene as the sole marker of species boundaries was not well supported as wide DNA barcoding initiatives got underway. Some skeptics consider the concept little more than a new packaging of older ideas. "Barcoding, in and of itself, is just a marketing tool," Will says, adding that the idea of using a standardized stretch of DNA for species identification was not invented in 2003. Hebert, who has raised about $28 million in funding since 2003 for his barcoding activities and facilities (see "The Barcoding Factory," p. 40), acknowledges using strong language in the original presentation of his vision for barcoding. "I think you have to be bold," he says. "If we said, 'It's business as usual. We're not trying to change the system. We'd like a lot of money to do this, but we're not going to change anything,' then I doubt we would have gotten any money." While the bulk of US funding for barcoding is coming from private institutions, such as the Moore and Sloan foundations, public funding may soon follow. Already, in Hebert's homeland of Canada, federal funding has made up the bulk of the money he has used to collect CO1 barcodes, and the US Environmental Protection Agency has devoted $250,000 to barcoding. Sperling says that focusing the early DNA barcoding verification studies on birds or lepidopterans (butterflies and moths) was plucking the lowest-hanging fruit first. The unique biology of these species, some of the most rigorously studied and taxonomically categorized animals, meant that these groups constituted simplistic tests of the theory. "I find it's interesting that a lot of the early barcoding has used Lepidoptera and birds. Those are the groups that you would expect to have the highest probability of showing a good match between mitochondrial DNA variation and species boundaries," because those species are less likely to hybridize and back-cross, which can confound barcoding, Sperling says. "To an extent, [these problems] are being addressed, although very slowly. There's no rush on the part of the barcode advocates to really subject the barcoding to the tough tests." Hebert defends the initial proof-of-concept experiments as being "absolutely critical to begin where we began. How would you ask if barcoding is working, if you're going into a system that's taxonomic terra incognita?" Critics cite another problem with CO1 barcoding: No one's quite sure why the mitochondrial gene doesn't tend to vary within a species. Even barcoding devotees, such as University of Pennsylvania ecologist Dan Janzen, concede that the reasons why CO1 barcoding seems to work are poorly understood. He says, however, that empirically successful proofs of the technique speak for themselves. Accumulating data that validates barcoding's ability to differentiate species, he says, is "far better than any kind of logical soapboxing." Barcoding with CO1 also doesn't work in plants. Researchers say that evolution rates in plant species are much more variable than in animal species, meaning that divergence in plant mitochondrial genomes is virtually nil. Plant barcoders are lagging behind their animal-identifying counterparts and still have yet to identify a suitable and standardized alternative to CO1 for barcoding plants. "Putting a lot of our energy into harvesting CO1, almost entirely to the exclusion of doing anything else, really is not an effective use of our time" --Kipling WillAlso at issue among barcoding skeptics is the fact that the movement to barcode every species on Earth is sapping resources that could be used for traditional taxonomic studies. "Where it's costing us right now is actually in manpower," Will says. "I've seen some great [systematists], even here at Berkeley, who are now going on to postdocs and things because barcoding has money, and you can't blame them for that. But they're not going to be doing the real work of taxonomy. They're not going to be building phylogenies; they're going to be harvesting CO1." Will also says funders that pour resources into initiatives seeking to barcode all of Earth's species may abandon taxonomy once the goal is met. "When the project has run its course, and unconditional success has been declared, it's very likely that funding agencies are going to say, 'Well, we're more or less done with taxonomy now. Why should we bother to fund it? We've already barcoded everything,'" he says. "But the job will be so far from being done. We don't have endless resources to do every wild idea that somebody might think of." Hebert maintains that rather than trying to encourage a migration away from classical taxonomy, he seeks to augment it with barcoding. "I would not like to walk away from 250 years of history. It would be rather sad," he says. "I want to work with taxonomists. I want to make their job a bit easier." Deeds wasn't a barcoding skeptic to begin with, and by the end of his experience with tetrodotoxin, he was a confirmed believer. He knew to call Yancy about his unidentified fish chunks because the two had barcoded fish before. Deeds and Yancy sent the Chicago fish samples to Hebert's lab in Guelph, Ontario, and when Deeds got the results back from Canada, his suspicions were confirmed. The "monkfish" wasn't monkfish at all. It was puffer fish. "We needed that real good confirmation," of the identity of the fish product, says Deeds, before the FDA could take action against the Chinese supplier of the fish. "Barcoding allowed us to identify this fish because we didn't have any other means," he says. In this case the legal stakes were high: The only legal source of imported puffer fish in the United States is a highly regulated Japanese supplier. Barcoding helped the FDA to place the name of the Chinese supplier - Xia Wei Aquatic Products - of the illicit puffer fish on their import alert list. Now this supplier of potentially deadly fish is barred from importing anything labeled as monkfish into the United States. The FDA isn't the only US regulatory agency taking notice of barcoding. The Federal Aviation Administration is barcoding tissue from hapless birds involved with airplane strikes. Its intent is to prevent bird strikes by targeting specific bird species for control around airstrips. The US EPA is using barcoding to characterize the health of waterways through identifying complex assemblages of aquatic species that would otherwise require intense and time-consuming taxonomic study to characterize. The US Department of Agriculture is using barcoding data from CBOL's fruit fly working group to better control the agricultural pest. The continued scientific successes and practical applications of barcoding fuel Hebert's grander ambitions. "I began by arguing that we could imagine a world in which we could know the name of any species, on the spot, anywhere on our planet, in just a minute," Hebert says. "When we first talked about this, a few years ago, it seemed a bit remote. I think we can turn this vision into a reality."References 1. R. Floyd et al., "Molecular barcodes for soil nematode identification," Mol Ecol, 11:839-50, 2002. 2. P. Hebert et al., "Biological identifications through DNA barcodes," Proc R Soc Lond B, 270:313-22, 2003. 3. P. Hebert et al., "Identification of birds through DNA barcodes," PLoS Biol, 2:1657-68, 2004. 4. R.D. Ward et al., "DNA barcoding Australia's fish species," Phil Trans R Soc B, 360:1847-57, 2005. 5. M.E. Sidall et al., "DNA-barcoding evidence for widespread introductions of a leech from the South American Helobdella triserialis complex," Conserv Genet, 6:467-72, 2005. 6. P.N. Kumar et al., "DNA barcodes can distinguish species of Indian mosquitoes (Diptera: Culicidae)," J Med Entomol, 44:1-7, 2007. 7. K. Kerr et al., "Comprehensive DNA barcode coverage of North American birds," Mol Ecol Notes, 2007; doi: 10.1111/j.1471-8286.2006.01670.x 8. T.L. Whitworth et al., "DNA barcoding cannot reliably identify species of the blowfly genus Protocalliphora (Diptera: Calliphoridae)," Proc Biol Sci, 274:1731-9, 2007. 9. T.L. Shearer, M.A. Coffroth, "Barcoding corals: limited by interspecific divergence, not intraspecific variation," Mol Ecol Notes, 2007; doi:10.1111/j.1471-8286.2007.01996.x