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In Pacific Fishing September 2008 (issue available on newstands, not yet on web) two New York City teenagers, Kate Stoeckle (my daughter!), 19, and classmate Louisa Strauss, 18, apply DNA-based identification to fish sold in their Manhattan neighborhood. The girls purchased 60 items from 14 establishments and sent samples to University of Guelph where graduate student Eugene Wong performed DNA barcode analysis. 14 (25%) of 56 samples with recoverable DNA were mislabeled, in all cases as more expensive or more desirable fish. Mislabeled items were sold at 2 of 4 restaurants and 6 of 10 grocery stores/fish markets. 

The frequency of mislabeling and the ease of high-schoolers obtaining DNA-based species identifications captured public interest. Their study was featured on page 1 in New York Times on August 22, one of the “quotes of the day” on CNN/Time website, a live segment on CBS TV Early Show, an interview on national public radio, and has appeared in over 350 print and news items and blogs from 34 countries in 10 languages so far, with particularly heavy coverage in China, Korea, and Japan, presumably related to dietary importance of fish in general and sushi in particular. 

This response demonstrates how powerful the FishBOL database is already (30,665 barcodes from 5,463 species so far, which represents about 20% of world fish), and hints at enormous uses that DNA barcoding will have as technology gets smaller and cheaper.

I will be away from Blog until mid-August.

COI barcodes aim to enable identification of species, assigning unknown specimens to known species, and helping flag genetically divergent organisms that may represent new species. Might barcodes also help understand relationships among species?

Here I look at one example from birds, comparing differences among COI barcodes to a recently revised phylogeny of terns (subfamily Sternini). According to American Ornithologists’ Union Check-list of North American Birds Supplement 47 (2006), “the data show that the genus Sterna as currently defined…is paraphyletic.”…”[W]e follow the recommendation of Bridge et al 2005 to resurrect four generic names currently placed in synonomy with Sterna.” The figure at left, taken from the 2005 paper by Bridge, Jones, and Baker, shows phylogenetic relationships based on 2800 bp of mtDNA from 33 species of terns (Bayesian tree with ML distances and ML boostrap support indices), and is juxtaposed to an NJ tree of COI barcodes from 29 of the same 33 taxa. The figure is colored according to the revised generic assignments (AOU 2006).

The topology of the COI NJ tree is similar to the larger data set tree, including that all currently recognized genera are reciprocally monophyletic, and most show similarly high boostrap values as in the Bayesian/ML analysis based on the larger data set. 

Of course mitochondrial DNA is widely used in analyzing relationships among animal species, including birds. Most of these studies are focused on relatively small groups of species, such as the tern study cited here. With growing DNA barcode libraries it will be increasingly possible to get at least a preliminary look at genetic relationships for large numbers of species (so far 2,393 avian species (24% of world birds) have barcode records in BOLD). This could be exciting! 

The DNA barcode for animals is a 648 base pair (bp) fragment from the 5′ end of mitochondrial gene cytochrome c oxidase subunit I (COI).  Does this relatively short mitochondrial sequence contain enough information to make evolutionary inferences about species limits, or is it a more of a rough survey method that needs to be confirmed by more data including from nuclear genes? 

In April 2008 Mol Ecol researchers from University of Minnesota and American Museum of Natural History, New York, analyze utility of mitochondrial as compared to nuclear DNA for inferring recent evolutionary history. Zink and Barrowclough first apply population genetic theory and then look at real data from bird species. 

Based on mathematical population genetics, they find “mitochondrial loci are generally a more sensitive indicator of population structure than are nuclear loci,” primarily due to much smaller effective population size (Ne) for mitochondrial as compared to nuclear markers, which leads to more rapid sorting of differences among genetically isolated populations. Analysis of real-world data in 45 studies of differences among and within avian species confirms this expectation, ie either the patterning is consistent between mitochondrial and nuclear genes, or there are shallow mtDNA trees which are not yet reflected in nuclear genes. Reanalysis of one study, which appears to show a split in nuclear but not mitochondrial markers, suggests possible misinterpretation. Regarding other factors that could potentially lead to mistaken inferences about species limits based on mitochondrial DNA (NUMTs, sex-biased gene flow, introgression), experimental data suggests these are rarely important, at least in birds. The authors conclude “mtDNA patterns will prove to be robust indicators of population history and species limits.” Nuclear markers ARE important for deep gene trees, for detecting hybrids, and for “quantitative estimates…of rates of population growth and values of gene flow.”  

Regarding length of mitochondrial sequence, this only has to be long enough to capture differences among closely-related species. Most populations that we recognize as species differ from their closest relatives by >1% in mitochondrial coding regions (corresponding to about 0.5 million years or more of reproductive isolation). At this level, even 100 bp is generally sufficient to distinguish most closely-related species, and a 648 bp COI barcode sequence should generally allow resolution of populations/species which have been reproductively isolated for much shorter periods of time. 

In July 2008 Wildlife Management researchers from Smithsonian Institution report on identifying otherwise unidentifiable remnants from bird-aircraft collisions (hereafter birdstrikes). Authors Dove et al point out “birdstrikes are a serious safety hazard and a major expense for the industry”. The US Federal Aviation Agency Wildlife Mitigation site shows about 600 incidents a month over the past year, peaking in late summer and early fall, presumably coincident with fall migration. The Smithsonian Institution has been identifying birdstrike species for military and civil aviation industries since the 1960s, analyzing specimens which range from whole carcasses to bits of feathers, tissue, or blood. Prior to availability DNA testing, identifications have relied on expert examination of detailed feather morphology with comparisons to Smithsonian’s vast bird specimen collection.

Of 1,715 birdstrike samples sent to Smithsonian Insitution during 4 months in fall 2006, 821(47.9%) contained only blood or tissue. Of these, 554 (67.5%) had amplifiable mtCOI DNA, and 535 (96.6%) with DNA led to species-level identifications based on reference sequences in Barcode of Life Database (BOLD). DNA barcoding identified 128 species representing 14 orders of birds, plus 2 bat species. 19 cases were deemed inconclusive as DNA barcode matched to a set of 2 or more closely-related species with overlapping barcodes, or the recovered sequence did not meet their 98% match criteria when compared to BOLD.

There was much better success recovering DNA from dry samples (70%) than from samples collected with a wet paper towel (about 23%), which had been the standard method, pointing the way toward improving yield of DNA-based ID. The authors conclude with a call for applying “a combination of morphological and molecular methods such as DNA barcoding for efficient, cost-effective birdstrike identifications”.

Just as in CSI television series, DNA-based identification can make possible what would otherwise be impossible; in this case, identifying birds from bits of tissue and blood and making birdstrike identifications available to those without access to Smithsonian’s experts or vast collections. In addition to helping airlines, birdstrike ID will inform our knowledge of bird migration routes. There are many exciting discoveries ahead.