of sites that can accept amino acid replacements and the particular replacements that can occur at each site. It remains obscure why greater constraints would occur in Drosophila than in the Chymomyza or Ceratitis lineages (or, indeed, in other animals, plants, and fungi). But, in any case, the issue is not whether biologically ascertainable processes are at work, which of course they are, in GPDH, SOD, or any other enzymes. The issue rather is whether the processes are of such regularity that some sort of molecular clock may be assumed to be at work. The stark contrast between the pattern of evolution of GPDH and SOD may be an aberration rather than representative of prevailing modes of protein evolution, since protein evolution seems so often to behave in a clocklike manner. But the congruence between observations and the clock predictions are often obtained due to the fact that the data collected do not have sufficient resolution to exhibit likely discrepancies.

The operational risks of assuming that protein clocks are fairly reliable are made evident in Table 9. The rate of GPDH evolution is nearly 4 times faster between animals and plants than between Drosophila species, whereas the rate of SOD evolution is 1/5 as fast. If we were to use the observed rate of Drosophila evolution to estimate the time of divergence between plants and animals, GPDH would yield an estimate of 3,990 My, SOD an estimate of 224 My, both grossly erroneous. The practical conclusions to be drawn are that (i) protein clocks should be used cautiously and weighed against any other available evidence, rather than considered decisive; (ii) several protein clocks should be used whenever feasible, particularly

when important evolutionary events need to be determined (44); (iii) whenever possible, synonymous rather than nonsynonymous nucleotide substitutions should be used, given that substitutions that yield amino acid replacements are more constrained by natural selection. The rapid rate of synonymous nucleotide substitutions becomes, however, a problem whenever long evolutionary spans are at stake, because many superimposed substitutions will have occurred so that the differences observed have little statistical reliability for estimating the multiple hits concealed behind the observed differences. The strategy of using as many separate molecular clocks as feasible is grounded on the convergence expected from the “law of large numbers;” statistical and other biases will tend to cancel as the number of observations increases.

I am grateful to Walter M.Fitch and Richard R.Hudson for valuable comments and to the members of my laboratory who participated in the research herein reported, particularly Kevin Bailey, Eladio Barrio, Michal Jaworski, Michal Krawczyk, Jan Kwiatowski, and Douglas Skarecky. Stephen Rich’s help with computer graphics is much appreciated. This research is supported by National Institutes of Health Grant GM42397.

1. Zuckerkandl, E. & Pauling, L. (1962) in Horizons in Biochemistry, eds. Kasha, M. & Pullman, B. (Academic, New York), pp. 97–166.

2. Zuckerkandl, E. & Pauling, L. (1965) in Evolving Genes and Proteins, eds. Bryson, V. & Vogel, H.J. (Academic, New York), pp. 97–166.

3. Margoliash, E. (1963) Proc. Natl. Acad. Sci. USA 50, 672–679.

4. Kimura, M. (1968) Nature (London) 217, 624–626.

5. Kimura, M. (1969) Proc. Natl. Acad. Sci. USA 63, 1181–1188.

6. Kimura, M. (1983) The Neutral Theory of Molecular Evolution (Cambridge Univ. Press , Cambridge, U.K.).

7. Kimura, M. & Ohta, T. (1971) Nature (London) 229, 467–469.

8. O’Brien, S.J. & MacIntyre, R.J. (1978) in The Genetics and Biology of Drosophila, eds. Ashburner, M. & Wright, T.R.F. (Academic, New York), Vol. 2a, pp. 395–551.

9. O’Brien, S.J. & MacIntyre, R.J. (1972) Genetics 71, 127–138.

10. Bewley, G.C., Cook, J.L., Kusakabe, S., Mukai, T., Rigby, D.L. & Chambers, G.K. (1989) Nucleic Acids Res. 17, 8553–8567.

11. von Kalm, L., Weaver, J., DeMarco, J., MacIntyre, R.J. & Sullivan, D.T. (1989) Proc. Natl. Acad. Sci. USA 86, 5020–5024.

12. Cook, J.L., Bewley, G.C. & Shaffer, J.B. (1988) J. Biol. Chem. 263, 10858–10864.

13. Lakovaara, S., Saura, A. & Lankinen, P. (1977) Evolution 31, 319–330.

14. Kwiatowski, J., Krawezyk, M., Jaworski, M., Skarecky, D. & Ayala, F.J. (1997) J. Mol. Evol. 44, 9–22.

15. Barrio, E. & Ayala, F.J. (1997) Mol. Phylogenet. Evol. 7, 79–93.

16. Wheeler, M.R. (1981) in The Genetics and Biology of Drosophila, eds. Ashburner, M., Carson, H.L. & Thompson, J.N.J. (Academic, New York), Vol. 3a, pp. 1–97.

17. Grimaldi, D. (1990) Bull. Am. Mus. Nat. Hist. 197, 1–139.

18. Kwiatowski, J., Skarecky, D., Bailey, K. & Ayala, F.J. (1994) J. Mol. Evol. 38, 443–454.

19. Fitch, W.M. & Ayala, F.J. (1994) Proc. Natl. Acad. Sci. USA 91, 6802–6807.

20. Ayala, F.J., Barrio, E. & Kwiatowski, J. (1996) Proc. Natl. Acad. Sci. USA 93, 11729–11734.

21. Dayhoff, M.D. (1978) Atlas of Protein Sequences and Structure (Natl. Biomed. Res. Found., Washington, DC).

22. Kimura, M. (1980) J. Mol. Evol. 16, 111–120.

23. Li, W.-H. (1993) J. Mol. Evol. 36, 96–99.

24. Fridovich, I. (1986) Adv. Enzymol. 58, 61–97.

25. Steinman, H.M. (1988) Basic Life Sci. 49, 641–646.

26. Kwiatowski, J., Hudson, R.R. & Ayala, F.J. (1991) Free Radical Res. Commun. 12–13, 363–370.

27. Smith, M.W. & Doolittle, R.F. (1992) J. Mol. Evol. 34, 175–184.

28. Kwiatowski, J., Skarecky, D., Burgos, M. & Ayala, F.J. (1992) Insect Mol. Biol. 1, 3–13.

29. Kwiatowski, J., Skarecky, D. & Ayala, F.J. (1992) Mol. Phylogenet. Evol. 1, 72–82.

30. Wilson, A.C., Carlson, S.S. & White, T. (1971) Annu. Rev. Biochem. 46, 573–639.

31. Wu, C.-I. & Li, W.-H. (1985) Proc. Natl. Acad. Sci. USA 82, 1741–1745.

32. Gaut, B.S., Muse, S.V., Clark, W.D. & Clegg, M.T. (1992) J. Mol. Evol. 35, 292–303.

33. Li, W.-H., Ellsworth, D.L., Kruchkal, J.K., Chang, B.H.-J. & Hewett-Emmett, D. (1996) Mol. Phylogenet. Evol. 5, 182–187.

34. Fitch, W.M. & Markowitz, E. (1970) Biochem. Genet. 4, 579–593.

35. Fitch, W.M. & Langley, C.H. (1976) Fed. Proc. 35, 2092–2097.

36. Bousque, J., Strauss, S.H., Doerksen, A.H. & Price, R.A. (1992) Proc. Natl. Acad. Sci. USA 89, 7844–7848.

37. Gillespie, J.H. (1991) The Causes of Molecular Evolution (Oxford Univ. Press, New York).

38. Wells, R.S. (1996) Proc. R. Soc. London Ser. B 263, 393–400.

39. Kimura, M. & Ohta, T. (1972) J. Mol. Evol. 2, 87–90.

40. Li, W.-H. & Graur, D. (1991) Fundamentals of Molecular Evolution (Sinauer, Sunderland, MA).

41. Kohne, D.E. (1970) Quart. Rev. Biophys. 33, 327–375.

42. Popper, K.R. (1959) The Logic of Scientific Discovery (Hutchinson, London).

43. Ayala, F.J. (1994) Hist. Phil. Life Sci. 16, 205–240.

44. Wry, G.A., Levinton, J.L. & Shapiro, L.H. (1996) Science 274, 568–573.

Contents (R1-R2)

Genetics and the origin of species: An introduction (1-7)

Origin of Genes (8-13)

Transposable elements as sources of variation in animals and plants (14-21)

Long term trends in the evolution of H(3) HA1 human influenza type A (22-28)

Genes, people, and languages (29-34)

DNA variation at the Sod locus of Drosophila melanogaster: An unfolding story of natural selection (35-39)

Neutral behavior of shared polymorphism (40-44)

Divergent and conserved features in the spatial expression of the Drosophila pseudoobscura esterase-5B gene and the esterase-6 gene of Drosophila melanogaster (45-51)

A demographic approach to selection (52-57)

Phylogenetics and the origin of species (58-65)

Assortative fertilization in Drosophilia (66-70)

Incipient species formation in salamanders of the Ensatina complex (71-77)

Genetics and the origin of bird species (78-85)

Vagaries of the molecular clock (86-93)

Evolution of codon usage bias in Drosophila (94-100)

The evolution of plant nuclear genes (101-108)

Evolution by the birth-and-death process in multigene families of the vertebrate immune system (109-116)