Biochemists probably know more about hemoglobin than any other protein. Being bright red, abundant, and functionally important, it attracted attention early and was first purified and crystalized in the mid 1800's. Comparison then among hemoglobins isolated from different species showed that each had a species-specific crystal form which indicated subtile chemical differences. However, it was not until the 1950's when it became possible to determine the amino acid sequences of proteins that the actual differences could be characterized. Hemoglobins from various species were among the first proteins to be studied and thus, along with cytochrome c, it became an archetype of protein sequence comparisons for evolutionary studies and the source of the Molecular Clock Hypothesis (12a).

Now, the amino acid sequences of the alpha and beta chains of hemoglobin are known from hundreds of species including bats and primates. The data below show the positions in the amino acid sequence where amino acid replacements have occurred in one or more of the three lineages. The remaining 123 positions in the alpha-globin and 114 positions in the beta-globin are identical in all three species. Note, residue numbers are written vertically.

Tabulate separately the number of positions for each globin sequence that support one or another phylogeny. Do these data support one or an other of the possible phylogenies? What is the most parsimonious solution (13)? Based on your analysis of the hemoglobin sequences, would you conclude that flight in mammals evolved once or twice?  Which globin sequence has the greater rate of evolution?  What might this imply about the two subunits?  Does this correspond to your understanding of the structure and function of hemoglobin?

For next time (2/27/13)

After the above sequences were published, the amino acid sequences of a number of other bat and primate hemoglobins have been determined and are available from protein data bases on the Internet. You may want to check out these additional sequences to see if they support or contradict your initial phylogeny? Similarly, do your conclusion agree with the analysis of the epsilon-globin gene (14).

In addition to phylogenetic deductions based on proteins encoded by nuclear genes, e.g. hemoglobins, the bat-primate problem has been studied using mitochondrial DNA sequences (15,16). Should the phylogeny based on mitochondrial DNA be the same as that based on nuclear encoded proteins (17)? Does the high AT content of Chiropteran nuclear DNA bias phylogenetic analysis (18)? What has been reported in the past few years on this topic?

Discuss bat phylogeny issue for the first half of class on Friday, March 1. The third an final page of this case study will involve a group project with individual components and be due Friday, March 8.

References

12a. Morgan, G. J. (1998) Emile Zuckerkandl, Linus Pauling, and the molecular evolutionary clock hypothesis. J. Hist. Biol. 31(2), 155-178.
13. Stewart, C-B. (1993) The powers and pitfalls of parsimony. Nature 361, 603-607.
14. Bailey, W. J., Slightom, J. L., and Goodman, M. (1992) Rejection of the "flying primate" hypothesis by phylogenetic evidence from the epsilon-globin gene. Science 256, 86-89 and commentary on p 34.
15. Adkins, R. M. and Honeycutt, R. L. (1991) Molecular phylogeny of the superorder Archonta. Proc. Natl. Acad. Sci. USA 88, 10317-10321.
16. Mindell, D. P., Dick, C. W. and Baker, R. J. (1991) Phylogenetic relationships among megabats, microbats, and primates. Proc. Natl. Acad. Sci. USA 88, 10322-10326.
17. Naylor, G.J.P., Collin, T.M., & Brown, W.M. (1995) Hydrophobicity and phylogeny. Nature 373, 565-566.
18. Pettigrew, J. D. (1994) Flying DNA. Current Biology 4, 277-280.

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