Current Research: DNA Barcode Results **new** 2004/11/22 13:00:43.858 US/Central 2004/12/06 12:55:46.829 US/Central Alison Fromme alisonfromme@nasw.org Alison Fromme alisonfromme@nasw.org barcode Biology DNA PLoS Biology Results species Hebert et al 2004 studied birds to determine whether or not a standard section of DNA could distinguish between species. Their results are summarized here.

Interpreting Results

Comparison of DNA differences among North American Birds Figure 1. Comparison of Nucleotide Sequence Differences in COI among 260 Species of North American birds Adapted from: Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM (2004) Identification of Birds through DNA Barcodes. PLoS Biol 2(10): e312.

The original research paper states: "Pairwise comparisons between 437 COI sequences are separated into three categories: differences between individuals in the same species, differences between individuals in the same genus (not including intraspecific differences), and differences between individuals in the same family (not including intraspecific or intrageneric differences)." What does the figure mean? Researchers used the DNA sequence from the COI gene in birds to figure out if the COI gene is useful as a DNA barcode-- in other words, a unique identifier for bird species. For the "Within Species" analysis (top panel), researchers compared one bird's 640-base pair COI sequence with another sequence from a bird of the same species. They recorded the number of differences between the two sequences (identical sequences would have zero differences, whereas completely different sequences would have 640 differences). They repeated this for all of their bird sequences. These comparisons are called "pairwise" because the data they recorded was based on each pair of sequences compared. In the graph, the x-axis shows the number of differences between sequences, and the y-axis shows the percentage of comparisons. For the "Within Genus" analysis (middle panel), researchers compared one bird COI sequence with all the others of the same genus. Again, they recorded the number of differences between the sequences, and then repeated this analysis for all the sequences. Since members of the same genus might also be members of the same species, they didn't count those comparisons. The same method was used for "Within Family" comparisons. What percentage of pairwise comparisons within species found zero differences? 0% Hint: The x-axis shows the number of differences in COI sequence comparisons. 94% 0.43% Hint: The x-axis shows the number of differences in COI sequence comparisons. Estimate the average number of sequence differences within species. About 94 Hint: Which axis shows number of sequence differences? About 8 Hint: Which axis shows number of sequence differences? Less than 1 Congratulations! The average nucleotide sequence differentiation within species was only 0.43%. Which group had the least amount of sequece variation at this gene? Family Hint: What was the average sequence variation for each group? Genus Hint: What was the average sequence variation for each group? Species Congratulations! The average nucleotide sequence differentiation within species was only 0.43%, compared with 7.93% within genus and 12.71% within family. COI sequence comparisons within families had between: 0-8 differences. Hint: Which axis shows the number of differences between COI sequences? 1-16 differences. Hint: Which axis shows the number of differences between COI sequences? 5-20 differences. Congratulations! Within family, COI sequences had at least five (and up to 20) differences. Which of the following statements best supports the use of the COI gene as a barcode to identify species? 94% of comparisons within species found zero differences. Congratulations! Because the COI sequence is nearly identical within species, it appears that COI could be used to distinguish between bird species. The average number of differences for within genus comparisons was 7.93. What is the purpose of a DNA Barcode? The average number of differences for within family comparisons was 12.71. What is the purpose of a DNA Barcode?

Your turn... How does this graph support the use of DNA barcodes to identify species? What about the 6% of individuals that did differ from others in the same species? Is there any other information would you want to know before using DNA barcodes to identify species?

Source Identification of Birds through DNA Barcodes Paul D. N. Hebert1* , Mark Y. Stoeckle2 , Tyler S. Zemlak1 , Charles M. Francis3 1 Department of Zoology, University of Guelph, Guelph, Ontario, Canada, 2 Program for the Human Environment, Rockefeller University, New York, New York, United States of America, 3 National Wildlife Research Centre, Canadian Wildlife Service, Ottawa, Ontario, Canada DOI: 10.1371/journal.pbio.0020312 Copyright: © 2004 Hebert et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Citation: Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM (2004) Identification of Birds through DNA Barcodes. PLoS Biol 2(10): e312.