Emile Zuckerkandl, Linus Pauling, and the Molecular ...

Journal of the History of Biology 31: 155–178, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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Emile Zuckerkandl, Linus Pauling, and the Molecular Evolutionary Clock, 1959–1965 GREGORY J. MORGAN Department of Philosophy The Johns Hopkins University Baltimore, MD 21218, U.S.A.

Introduction In 1965, in an often-cited paper, Emile Zuckerkandl and Linus Pauling coined a term to describe a hypothesis that they had been promoting for the previous three years. They called the hypothesis the “molecular evolutionary clock.”1 The molecular clock hypothesis, as it came to be known, proposed that the rate of evolution in a given protein (or later, DNA) molecule is approximately constant over time and among evolutionary lineages. More specifically, it proposed that there exists a statistical proportionality between the time elapsed since the last common ancestor of two contemporary homologous protein chains and the number of amino acid differences between their sequences. In practice, it allows biologists to give a temporal dimension to phylogenetic trees constructed from molecular data. The clock hypothesis was one of the key concepts that defined the thenemerging field of molecular evolution. Supporters of the hypothesis later wrote that “the discovery of the molecular clock stands out as the most significant result of research in molecular evolution.”2 Roger Lewin, in a recent book, describes the evolutionary molecular clock as “one of the simplest and most powerful concepts in the field of evolution.”3 Francis Crick called the molecular clock “a very important idea” that has “turned out to be much truer 1 Emile Zuckerkandl and Linus Pauling, “Evolutionary Divergence and Convergence in

Proteins,” in Vernon Bryson and Henry Vogel, eds., Evolving Genes and Proteins (New York: Academic Press, 1965), pp. 97–166. The molecular evolutionary clock is not a “metronomic” clock; rather, it “ticks” are stochastic events. 2 A. Wilson, S. Carlson, and T. White, “Biochemical Evolution,” Ann. Rev. Biochem. 46 (1977), 573–639. 3 Roger Lewin, Patterns in Evolution: The New Molecular View (New York: Freeman, 1997), p. 107.

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than people thought at the time.”4 Simon Easteal and colleagues, in a book devoted entirely to the molecular evolutionary clock, claim that it is “one of the most elegantly simple concepts in biology, but it is also one of the most contentious.”5 Dietrich (1994), and Suárez and Barahona (1996) have forcefully argued that Pauling and Zuckerkandl’s research was necessary for the development of the Neutral Theory of Molecular Evolution.6 The molecular evolutionary clock became intertwined with the selectionism/neutralism debate that ensued over the next thirty years and that continues today.7 A clock-like molecular evolution was seen to stand in stark opposition to the received view that selection pressures and therefore the rate of evolution, measured in terms of amino acid substitutions, varied over time.8 However, even before the neutralist consequences of the clock idea were developed more fully by Kimura, biologists of the stature of Ernst Mayr and George Gaylord Simpson objected to the idea on the grounds that for evolutionary studies, biologists should not privilege the molecular level over the traditional morphological level. The goal of this paper is to characterize Pauling and Zuckerkandl’s role in the early development of the molecular evolutionary clock. I will characterize the respective contributions of Linus Pauling and his collaborator Emile Zuckerkandl. I will also sample the reaction of two of the most prominent members of the traditional biological community – Ernst Mayr and George Gaylord Simpson – to Pauling and Zuckerkandl’s research. I will explain why Pauling, a physical chemist by training and research reputation, was actively investigating the “biological” evolution of hemoglobin. I will show 4 Francis Crick, “The Impact of Linus Pauling on Molecular Biology,” in The Pauling Symposium: A Discourse in the Art of Biography, ed. Ramesh Krishnamurthy (Corvallis: Special Collections, Oregon State University, 1996), p. 17. 5 Simon Easteal, Chris Collet, and David Betty, The Mammalian Molecular Clock (Austin: R. G. Lands, 1995), p. 1. 6 Michael R. Dietrich, “The Origins of the Neutral Theory of Molecular Evolution,” J. Hist. Biol., 20 (1994), 21–59; Edna Su´arez and Ana Barahona, “The Experimental Roots of the Neutral Theory of Molecular Evolution,” Hist. Phil. Life Sci., 18 (1996), 55–81. See also Motoo Kimura, The Neutral Theory of Molecular Evolution (Cambridge: Cambridge University Press, 1983), p. 23; Motoo Kimura, “The Rate of Molecular Evolution Considered from the Standpoint of Population Genetics,” Proc. Nat. Acad. Sci., 63 (1969), 1181–1188; and Motoo Kimura, “The Molecular Evolutionary Clock and the Neutral Theory,” J. Mol. Evol., 26 (1987), 24–33. 7 According to the bibliographic search engine Medline more than fifty articles over the past four years discuss the evolutionary molecular clock. See also John Gillespie, The Causes of Molecular Evolution (Oxford: Oxford University Press, 1991). 8 Emile Zuckerkandl, “Around the Molecular Clock: Aspects of Molecular Evolution” (ca. 1986, unpublished), p. 2; Easteal et al., The Mammalian Molecular Clock (above, n. 5) devotes a chapter to the relationship between the clock and the neutral theory.

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that his interest in genetic damage due to radioactive fallout was a principal determinant of his interest in molecular evolution.9 Furthermore, Pauling and Zuckerkandl’s molecular evolution research was compatible with Pauling’s larger ethical framework based upon minimization of human suffering since, for Pauling and Zuckerkandl, molecular evolution and molecular disease were inextricably linked. The historical analysis also uncovers a surprising result for the supporters of neutralism: when the clock hypothesis was first proposed, it was intended to be consistent with natural selection at the molecular level, and only later became associated with neutralism.

Zuckerkandl and Pauling’s Comparative Hemoglobin Research Emile Zuckerkandl was born into an eminent Viennese family on July 4, 1922. His family provided an academically stimulating environment – it included a biochemist-turned-philosopher, a famous anatomist, an artist, a surgeon, and a psychoanalyst. As the Second World War drew closer to Austria, Zuckerkandl and his family fled, first to Paris, and then to Algiers. Fearing for his family’s and his own safety, Zuckerkandl, with the help of family friend Walther Mayer’s connection with Albert Einstein, sought out a scholarship to study in the United States. Although he had originally intended to study medicine, he discovered that pure biology captured his interests. His plans, however, were delayed due to the landing of allied troops in Algeria. After the war and a year’s biological study at the Sorbonne in Paris, Zuckerkandl began a master’s degree in physiology at the University of Illinois under the direction of C. Ladd Prosser, a well-known physiologist. On completing his degree, Zuckerkandl returned to France, earned a doctoral degree at the Sorbonne, and secured a job at a marine laboratory in Roscoff, Brittany. He, however, would not have characterized himself as a marine biologist but as a biologist with an inclination for molecular problems. His early work on the molting cycle of crabs developed into an interest in the roles of copper oxidases and hemocyanin in the molting cycle. Although the position at the marine laboratory was pleasant and secure, and allowed Zuckerkandl to meet internationally respected biologists such as Ernst Mayr, Zuckerkandl and his wife, Jane, considered returning to America. He wrote to Linus Pauling, who was planning a trip to France, and arranged a meeting with him in Paris in the summer of 1957.10 9 This point is made in Hager’s biography but not developed at any length. Thomas Hager,

Force of Nature: The Life of Linus Pauling (New York: Simon & Schuster, 1995), p. 541. 10 See Ave Helen and Linus Pauling Papers, Oregon State University (hereinafter OSU), box 160, folder 1, for Pauling’s travel plans.

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As described by Kay (1993), Pauling had by then become immersed in studies of the molecular basis of life.11 Pauling’s research in molecular biology had its roots in hemoglobin research of late 1930s. Pauling and Charles Coryell, a postdoctoral fellow, both motivated by purely chemical questions, examined the magnetic properties of oxyhemoglobin and deoxyhemoglobin.12 In 1949, using Tiselius moving band electrophoresis, Harvey Itano and Linus Pauling, with help from S. J. Singer and I. C. Wells, had shown that sickle cell hemoglobin had a significantly different electrophoretic mobility. Accordingly, they coined the term “molecular disease” to describe sickle cell anemia.13 Pauling’s work with hemoglobin continued into the 1950s.14 For example, he and his collaborators found that HbA contains two types of polypeptide chains (the α- and β-chains).15 Pauling, therefore, had a history of research with hemoglobin and contact with other hemoglobin researchers. In the late 1950s, Pauling had begun to think about evolution at the molecular level. This interest had both a scientific and political dimension. Pauling was aware of early evolutionary work with hemoglobin through reading Karl Landsteiner’s landmark book, The Specificity of Serological Reactions in 1936.16 In the fall of 1937, while Pauling was the George Fisher Baker Lecturer in Chemistry at Cornell University, Landsteiner visited Cornell and spoke at length with Pauling about serology. Landsteiner and his work made a lasting impression on Pauling: “This was a great experience for me – to have

11 Lily Kay, The Molecular Vision of Life: Cal Tech, the Rockefeller Foundation and the Rise of the New Biology (New York: Oxford University Press, 1993). 12 See Linus Pauling and D. Coryell, “The Magnetic Properties Hemoglobin, Oxyhemoglobin and Carbonmonoxyhemoglobin,” Proc. Nat. Acad. Sci. USA, 22 (1936), 159–163; and Linus Pauling, “The Magnetic Properties and Structure of Hemoglobin and Related Substances,” Science, 83 (1936), 488. For their motivation, see Robert Olby, “ ‘The Mad Pursuit’: X-Ray Crystallographers’ Search for the Structure of Haemoglobin,” Hist. Phil. Life Sci., 7 (1985), 171–193, especially p. 181. 13 Linus Pauling et al., “Sickle Cell Anemia, a Molecular Disease,” Science, 109 (1949), 443. But see also Linus Pauling et al., “Sickle Cell Anemia Hemoglobin,” Science, 111 (1950), 459. To Pauling’s knowledge, the 1949 paper was the first use of the term “molecular disease.” Letter from Linus Pauling to Richard Sasuly, 16 October 1961, OSU, box 128, folder 1. 14 See Pauling’s Research Book 17, OSU, pp. 110–111. 15 H. Rhinesmith, Walter Schroeder, and Linus Pauling, “A Quantitative Study of the Hydrolysis of Human Dinitrophenyl (DNP) Globin: The Number and Kind of Polypeptide Chains in Normal Adult Hemoglobin,” J. Amer. Chem. Soc. 79 (1957), 4682–4686. 16 Hager, Force of Nature (above, n. 9), p. 236; Pauling, “Fifty Years of Progress in Structural Chemistry and Molecular Biology,” Daedalus, 99 (1970), 998–1014, on p. 1005. I thank Tom Hager for pointing this out to me. Pauling’s personal copy of the first edition of The Specificity of Serological Reactions contains many margin notes throughout the book.


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this field of knowledge presented and clarified to me by its great master.”17 In the second chapter of his book, Landsteiner discusses work that uses chemical differences to measure differences between species. For example, he reviews the early work of Reichert and Brown (1909), who found that the shapes and angles of hemoglobin crystals are characteristic for each species, and “the differences stand in relation to the distance between species in the zoological system.”18 In the latter half of his life – post World War II – Pauling became more interested in humanistic issues. Ava Helen, his wife, who was perhaps more liberal and politically aware than he, influenced his political and ethical development. His ethical framework was made explicit – Pauling believed that the correct moral code should be based upon the minimization of human suffering.19 Accordingly, this ethical principle provided a motivational force that influenced two other elements in his life that led to his evolutionary work with hemoglobin: his spirited protest against the testing of nuclear weapons, and his interest in disease, especially molecular diseases such as sickle cell anemia. Robert Paradowski, a Pauling historian, broadly characterizes Pauling’s life from 1950 to 1963 as his “increasing involvement in world peace.”20 Pauling’s involvement in the debate over the genetic effects of radioactive fallout led him to think more deeply about mutation, molecular disease, and evolution, and provided motivation to conduct research on molecular evolution with Zuckerkandl.21 After Pauling was awarded the Nobel Prize for Chemistry in 1954, he used his heightened prominence to promote peace issues more vigorously.22 By 1955, Pauling was quoting the views of prominent geneticists such as Herman Muller, Kurt Stern, and Alfred Sturtevant in

17 Linus Pauling, “The Harrison Howe Lecture: Analogies between Antibodies and Simpler

Chemical Substances,” Chem. Eng. News, 24 (1964), p. 1064, quoted in R. J. Paradowski, “The Structural Chemistry of Linus Pauling” Ph. D. diss., University of Wisconsin, 1972, p. 87. 18 K. Landsteiner, The Specificity of Serological Reactions Springfield, Illinois: (Charles Thomas, 1936). E. T. Reichert and A. P. Brown, “The Differentiation and Specificity of Corresponding Proteins and other Vital Substances in Relation to Biological Classification and Organic Evolution” Carnegie Institute Washington (1909), 116. Incidentally, the revised edition of Landsteiner’s book included an introductory chapter by Pauling. 19 Hager interview. This was contrasted with the more typical utilitarian formulation of maximization of human happiness. Happiness, Pauling thought, was too subjective and difficult to measure, unlike suffering, which, it was hoped could be given a scientific basis. 20 Paradowski, “The Structural Chemistry of Linus Pauling” (above, n. 17), p. 107. 21 I am expanding upon a point made by Hager, Force of Nature (above, n. 9), p. 541. 22 Ibid., p. 461.

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his ongoing debate with Willard Libby over the dangers of natural radiation and man-made radiation.23 Pauling’s involvement in the nuclear testing debate intensified in 1957 and 1958. In May 1957, he initiated a petition against nuclear testing that was eventually signed by more than eleven thousand scientists from around the world. By early 1958, Linus Pauling and Edward Teller had “emerged as the leading spokesmen for the contending viewpoints.”24 Edward Teller, the Hungarian physicist known as “the father of the H-bomb,” had been involved with atomic weapons since the building of Los Alamos. He argued that the risk from fallout was very slight and much less than the risk to national security if the testing were to cease. He also argued that the testing should continue in order to develop a “clean” bomb – a hydrogen bomb based almost entirely on fusion, hence having minimal radioactive fallout. In February, Teller and Pauling debated nuclear testing on KQED, an educational television channel based in San Francisco. Teller brought up the question of genetic damage, arguing, “It is possible that there is damage. It is even possible, to my mind, that there is no damage; and there is the possibility, furthermore, that very small amounts of radioactivity are helpful.”25 Countering this type of argument required that Pauling discuss “evolution through mutation and natural selection.”26 After Life magazine refused to publish a reply to a defamatory pro-testing article by Teller and Albert Latter, Pauling began writing his book No More War!27 In this popular book, Pauling included a six-page section called “Mutation and Evolution.” To write his book and participate in the fallout debate, Pauling read, discussed, and became quite proficient in genetics and evolutionary theory.28 For example, he mentions Dobzhansky, Mayr’s theory of speciation, Muller and the steady state theory, Fisher, and Sewall Wright, and the balanced theory in his notes for the six Messenger Lectures given at Cornell in October 1959.29 He also consulted with the California 23 Letter from Linus Pauling to Willard Libby, 30 March 1955, OSU, box 64, folder 1. See

also Alfred Sturtevant, “Social Implications of the Genetics of Man,” Science, 120 (1954), 52–53. 24 Robert Divine, Blowing in the Wind: The Nuclear Test Ban Debate, 1954–1960 (New York: Oxford University Press, 1978), p. 182. 25 Edward Teller; transcript of KQED debate, p. 14, OSU, box 1958a, folder 4. A similar statement made in 1962 is quoted by S. Blumberg and G. Owens, Energy and Conflict: The Life and Times of Edward Teller (New York: G.P. Putnam’s Sons, 1976), p. 411. 26 Linus Pauling, No More War! (New York: Dodd, Mead, 1958), p. 53; See also Linus Pauling, “Genetic and Somatic Effects of Carbon-14,” Science, 128 (1958), 1183–1186. 27 Letter from Linus Pauling to Willard Libby, 5 March 1958, OSU, box 64, folder 1; Hager, Force of Nature (above, n. 9), pp. 485–486. 28 Hager, personal communication, July 1996. 29 Pauling’s small black notebook, undated, OSU safe; Interoffice memo from Bea to Joan, OSU, box 186, folder 5.


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Institute of Technology (Cal Tech) geneticist E. B. Lewis, who gave him his manuscripts on the linearity of radioactive exposure and mutational damage and then proofread the manuscript for No More War!30 By the time Zuckerkandl arrived from France in September 1959, Pauling was well versed in evolutionary theory and genetics.31 In a hotel in Paris, Zuckerkandl proposed a research project on hemocyanin and copper oxidases to a receptive Linus Pauling. The famous chemist was impressed by the young researcher and recommended him for a postdoctoral fellowship in chemistry under his direction.32 In September 1959, Emile and Jane Zuckerkandl arrived at Cal Tech. Zuckerkandl recounted his first meeting with Pauling: “He said, you know this subject of yours on hemocyanin and copper oxidases, I think the results are going to be difficult to interpret and I think you would do better to work on a protein about which more is known, . . . why don’t you work on hemoglobin?33 Pauling suggested that Zuckerkandl analyze the hemoglobin of various primates using the newly invented electrophoretic-chromatographic technique of “finger printing,” recently popularized by hemoglobin researcher Vernon Ingram at the Medical Research Council (MRC) in Cambridge.34 This technique combined the “one-dimensional” techniques of paper chromatography and paper electrophoresis to form unique two-dimensional patterns of tryptic hydrolysates of hemoglobin. By using the technique, Pauling hoped, they could draw evolutionary conclusions from the comparative study.35 Pauling arranged for Zuckerkandl to work with his graduate student Richard T. Jones 30 Letter from Linus Pauling to Willam Libby, 10 May 1957; Letter from William Libby to Linus Pauling, 17 May 1957; Comments on book, from E. B. Lewis to Linus Pauling, undated, OSU, box 411, folder 15. 31 It is perhaps significant that Pauling ordered George Gaylord Simpson’s The Meaning of Evolution and his Behavior and Evolution, as well as Charles Darwin’s On the Origin of Species in February 1959. Book Orders, OSU, box 187, folder 11. It is also significant that when asked to write an article for Scientific American on the role of mutation in evolution, Pauling replied that Dobzhansky, L. C. Dunn, or J. B. S. Haldane would be better because he was not a “competent geneticist.” Letter from R. H. Kent to Linus Pauling, 7 February 1959, OSU box 59, folder 3; Letter from Linus Pauling to R. H. Kent, 11 February 1959, OSU, box 59, folder 3. 32 Memo from Linus Pauling to Prof. Swift, 2 March 1959, OSU, box 153, folder 5. 33 Interview with Emile Zuckerkandl, July 24, 1996 (hereafter Zuckerkandl interview). 34 Vernon M. Ingram, “Abnormal Human Haemoglobins: The Comparison of Normal Human and Sickle Cell Haemoglobins by Finger Printing,” Biochim. et Biophys. Acta, 28 (1958), 539. 35 Interview with Richard T. Jones, July 26, 1996, (hereafter Jones interview); Chemistry and Chemical Engineering Division, “Chemical Structure Studies of Animal Hemoglobins,” Chemistry and Chemical Engineering Divisional Report (Pasadena: California Institute of Technology, 1959–1960), p. 82, stored at the Cal Tech Archives.

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in Professor Walter Schroeder’s laboratory, since at that time Pauling did not have a laboratory of his own. Richard T. Jones, like Pauling, was a native of Oregon. As an undergraduate, he had gone to Cal Tech because of its strength in chemistry, a strength due, in large part, to Linus Pauling’s presence.36 Jones then completed a M.D. degree at the University of Oregon. Pauling recognized that Jones’s undergraduate research in chemistry was “first rate” and gladly accepted him back to conduct experimental research at Cal Tech.37 As in the case of Zuckerkandl, Pauling “suggested” that Jones work on hemoglobin, and his doctoral dissertation concerned the minor components of hemoglobin.38 For the first two or three months of Zuckerkandl’s appointment, Jones taught Zuckerkandl the technique of “fingerprinting.”39 After Zuckerkandl had perfected the technique, he widened the number of species in the analysis from primates to include cow, pig, shark (Cephaloscyllium), bony fish (Pimelometopon), lungfish (Lepidosiren), and Echiurid “worm” (Urechis).40 Using this technique, Zuckerkandl, Pauling, and Jones drew qualitative conclusion from their comparative study, which they completed in the summer of 1960. “The gorilla, chimpanzee and human patterns are almost identical in appearance . . . the difference from human patterns is somewhat greater for orangutan peptide. . . . As one gets further away from the group of Primates, the amount of primary structure that is shared with human hemoglobin decreases.” 41 Due to the pictorial nature of fingerprinting, the technique did not allow for quantitative comparisons. Further progress would require a more detailed description of the amino acid sequences. At this time, three rival laboratories wre working on the complete amino acid sequences of the α- and β-chains of human hemoglobin: Walter Schroeder’s laboratory at Cal Tech; Gerhard Braunitzer’s laboratory at the Max Planck Institute in Munich; and Lyman Craig’s laboratory at the Rockefeller Institute in New York City.42 Max Delbrück, returning to Cal Tech from a visit to Braunitzer’s laboratory in Germany, brought back the sequence of 36 Jones interview. 37 Recommendation for Richard Jones, 3 January 1958, OSU, box 56, folder 23. 38 Biochemists now believe that the minor components are glycosylated forms of

hemoglobin. 39 Jones interview. 40 Zuckerkandl interview. 41 Emile Zuckerkandl, Richard T. Hones, and Linus Pauling, “A Comparison Animal Hemoglobins by Tryptic Peptide Pattern Analysis,” Proc. Nat. Acad. Sci. 46 (1960), 1349– 1360. Italics mine. 42 Jones interview. Incidentally, Pauling had a “high opinion” of Braunitzer’s work, although he met him only once. Letter from Linus Pauling to W. Grassman, 26 November 1963, OSU, box 42, folder 7.


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the thirty terminal residues of the human β-chain, and Zuckerkandl was able to compare it with the preliminary results of Schroeder’s group – a number of unordered but sequenced tryptic peptides. Through this comparison, Zuckerkandl inferred that the α- and β-chains are homologous, that is, they have a common ancestor and arose as distinct chains through a duplication event.43 Once Schroeder returned from sabbatical in Denmark, he and Zuckerkandl discussed whether or not the similarity in sequence was evidence for common ancestry. Zuckerkandl remained convinced that it was, but Schroeder refused to publish a collaborative paper, perhaps due in part to his creationist religious beliefs, and Zuckerkandl could not use Schroeder’s data independently.44 The realization that the hemoglobin chains were homologous legitimated further evolutionary analysis of the different chains within a single species as well as chains from different species. Zuckerkandl began working with Schroeder in determining the amino acid composition of gorilla hemoglobin.45 Their collaboration was partially arranged by Pauling in January 1961.46 The results of the compositional analysis were published in Nature: “The α-chains of gorilla and human hemoglobin may well differ by only two residues and the β-chains by one residue.”47 Zuckerkandl and Pauling used these quantitative results in their next paper to calculate the time of divergence between gorilla and human, using the evolutionary molecular clock. In late November 1960, Pauling accepted an invitation to submit a paper to be published in a volume dedicated to Albert Szent-Györgyi, the Nobel prize-winning discoverer of vitamin C.48 On June 22, 1961, Pauling wrote to 43 Zuckerkandl, “On the Molecular Evolutionary Clock,” J. Mol. Evol., 26 (1987), 34–46,

see p. 35. This homology was independently hypothesized first by Itano in 1957 and later by Ingram in 1961: Harvey A. Itano, “The Human Hemoglobins: Their Properties and Genetic Control,” Adv. Protein Chem., 12 (1957), 215–268; Vernon Ingram, “Gene Evolution and the Haemoglobins,” Nature 139 (1961), 704–708. 44 Emile Zuckerkandl, personal communication, 30 October 1996. 45 Chemistry and Chemical Engineering Division, “Investigation of the Amino Acid Sequences of Gorilla Hemoglobin,” Chemistry and Chemical Engineering Divisional Report (Pasadena: California Institute of Technology, 1961–1962), p. 94, stored at the Cal Tech Archives. 46 Interoffice memo from Linus Pauling to Dr. Walter Schroeder, 20 January 1961, OSU, box 153, folder 3. 47 Emile Zuckerkandl and Walter A. Schroeder, “Amino-Acid Composition of the Polypeptide Chains of Gorilla Hemoglobin,” Nature, 192 (1961), 984–985, see p. 985. It was later found that the gorilla and human α-chain actually differed by only one residue. 48 Letter from Linus Pauling to Dr. Bernard Pullman, 22 November 1960. OSU, box 1962a, folder 2. Szent-Györgyi was an active promoter of Pauling for the Nobel Prize in Chemistry in 1953. See the correspondence beginning with the letter from Szent-Györgyi to Linus Pauling, 8 November 1952, OSU, box 118, folder 1.

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inform Dr. Bernard Pullman, an editor of the volume, that the paper would concern “The Molecular Basis for Disease.”49 Zuckerkandl recounts how Pauling came down from his office to Schroeder’s lab, a floor below, to ask him to collaborate on the paper: “I said, I would with pleasure, and he said, ‘you know it is for Szent-Györgyi, so we should say something outrageous!”’50 The collaboration set the tone for much of the future collaboration between Pauling and Zuckerkandl – Pauling would be invited to submit to a Festschrift volume without peer review, and together they would publish pioneering papers on molecular evolution. The Szent-Györgyi paper was written by Zuckerkandl,51 and many people did find parts of it outrageous, especially those traditional biologists and anthropologists who though that the idea of the molecular clock was “crazy.”52 After Zuckerkandl traveled to Seattle and Berkeley to check some final details with geneticists there, the paper was finally completed and sent to the publishers on November 1, 1961.53 The most novel feature of the historic article titled “Molecular Disease, Evolution, and Genic Heterogeneity” is the first application of the thenunnamed molecular evolutionary clock. The idea of using the number of amino acid substitutions to make temporal divergence estimates “evolved” as Zuckerkandl wrote the paper.54 Zuckerkandl and Pauling explicitly assume the homology of the globin genes: “In the course of time the hemoglobin chain genes duplicate, . . . the descendants of the duplicate genes ‘mutate away’ from each other, and the duplicates eventually become distributed through translocations over different parts of the genome.”55 In a somewhat cautious manner, Zuckerkandl and Pauling compare horse and human α-chains to calibrate the clock: It is possible to evaluate very roughly and tentatively the time that has elapsed since any of the hemoglobin chains present in a given species and controlled by non-allelic genes diverged from a common chain an49 Letter from Linus Pauling to Dr. Bernard Pullman, 22 June 1961. OSU, box 1962a, folder 2. 50 Zuckerkandl interview. 51 Letter from Linus Pauling to Dr. Herbert York, 15 September 1962: “. . . I enclose a paper by [Zuckerkandl] and me – mainly written by him; Zuckerkandl interview: “I essentially wrote it [“Molecular Disease, Evolution, and Genic Heterogeneity”].” Note also that Zuckerkandl has first authorship. 52 Zuckerkandl interview. I pursue this further below. 53 Interoffice memo from Pauling to Zuckerkandl, 24 October 1961, OSU box 153, folder 3; letter, Pauling to Academic Publishers, 1 November 1961, OSU box, 1962a, folder 2. 54 Zuckerkandl interview. 55 Emile Zuckerkandl and Linus Pauling, “Molecular Disease, Evolution and Genic Heterogeneity,” in Horizons in Biochemistry: Albert Szent-Györgyi Dedicatory Volume ed. Michael Kasha and Bernard Pullman (New York: Academic Press, 1962), pp. 189–225, see p. 198.


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cestor. . . . From paleontological evidence it may be estimated that the common ancestor of man and horse lived in the Cretaceous or possibly the Jurassic period, say between 100 and 160 million years ago. . . . the presence of 18 differences between human and horse α-chains would indicate that each chain had 9 evolutionary effective mutations in 100 to 160 millions of years. This yields a figure of 11 to 18 million years per amino acid substitution in a chain of about 150 amino acids, with a medium [sic] figure of 14.5 million years.56 Using the figure of 14.5 million years per amino acid substitution per 150 residue polypeptide, Zuckerkandl and Pauling calculate the time of derivation from the common chain ancestor of the β and δ, β and γ , α and β, α and γ , gorilla α and human α, and gorilla β and human β-chains, as 44, 260, 565, 600, 14.5, and 7.3 million years respectively.57 They note that these figures agree with the observation that γ -chains are present only in primates, which “furnishes evidence” that their evaluation is “not unreasonable.”58 Furthermore, the inferred divergence of gorilla and human of 11 million years is consistent with the range estimated by traditional paleontological methods.59 The section of the paper dealing with the molecular clock ends with a discussion of possible sources of error in the estimates.60 Easteal and colleagues claim that “[Zuckerkandl and Pauling’s 1962] analysis did not provide evidence of a molecular clock; it assumed one.”61 From this claim, they argue that this paper did not mark the “discovery” of the molecular clock.62 However, Zuckerkandl and Pauling did provide confirming evidence, albeit slight. They compared the estimates derived from a clock postulate with the available paleontological evidence. Pauling wrote, “In 1962, . . . [Emile and I] found that the amino acid sequences of hemoglobin molecules manufactured by animals of different species provided strong 56 Ibid., p. 201. 57 Ibid. Note that when the correction value of one substitution difference between gorilla α-

chain and human α-chain is taken into account, the divergence time from the common ancestor of gorilla-α and human-α is 7.3 million years. 58 Ibid. 59 Ibid., p. 202. Eleven million years as an estimate of the human-gorilla divergence is on the lower side of the range estimated by anthropologists at the time. 60 Zuckerkandl and Pauling deal with the problems of accounting for back mutations, “minor” components of hemoglobin, change from an aquatic to a terrestrial environment, and changing population sizes. They conclude that their estimates are probably underestimates. Zuckerkandl and Pauling, “Molecular Disease, Evolution and Genic Heterogeneity” (above, n. 55), pp. 203–204. 61 Easteal et al., The Mammalian Molecular Clock (above, n. 5), p. 9. 62 As Lindley Darden pointed out to me, Easteal et al., The Mammalian Molecular Clock (above, n. 5), appear to conflate discovery and justification.

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evidence about the evolution of species supporting the evolutionary history as formulated by paleontologists on the basis of macroscopic characters.”63 If, in this case, there is any historical value in attributing “discovery” to any paper, then the 1962 paper deserves more recognition than Easteal and colleagues give. The analysis Zuckerkandl and Pauling provided to estimate divergence times was revolutionary, but the remainder of the paper including their justification of the analysis was more conservative. They wrote, justifying their analysis, “Our best excuse for making this present evaluation is that it affords us the opportunity to point out why it is probably wrong [i.e., an idealization].”64 One might think that, in line with the later neutral theory, Zuckerkandl and Pauling would argue that random drift explains the molecular clock. In the same vein, one might think that they would explicitly deny a linkage between rates of evolution at the morphological and molecular levels, since this independence would reconcile a constant rate of molecular evolution with a varying rate of morphological evolution. However, Zuckerkandl and Pauling do not argue along these lines. In fact, they anticipate an approximate long-term linkage between the rate of morphological and molecular evolution. “We may expect that this generalization [in the course of time, evolution has accelerated] based upon morphological characteristics has its counterpart in the speed of evolution of deoxyribonucleic acid (DNA) and of the proteins.”65 Furthermore, nowhere in the paper do Zuckerkandl and Pauling trivialize the role of natural selection at the molecular level. In fact, they argue natural selection is necessary to keep functional genes from mutating into functionless “dormant” genes.66 In a recent interview, Zuckerkandl expressed how he saw the connections between the molecular clock, natural selection, and neutralism: “The me, natural selection was never contradictory to a molecular clock, but for Kimura the clock was one of the foundations for neutral drift.”67 The vague idea of differential selection pressures causing differential resistances to change along the sequence was pivotal in the gen63 Linus Pauling, “Fifty Years of Progress” (above, n. 16), p. 1014. More specifically, regarding the paternity of the molecular clock Pauling writes, “I think it was my idea, but I am not sure. We were just collaborating on these studies. Perhaps it was Emile’s idea.” Linus Pauling, “An Extraordinary Life: An Autobiographical Ramble.” in Creativity: Paradoxes and Reflections. ed. Harry A. Wilmer (Wilmette, Ill.: Chiron Publications, 1991), pp. 69–85, p. 74. 64 Zuckerkandl and Pauling, “Molecular Disease, Evolution and Genic Heterogeneity” (above, n. 55), p. 203. 65 Ibid., p. 204, italics mine. 66 Ibid., p. 209. Other than the first application of the clock concept, the paper is novel in introducing the notion of a dormant gene, the conceptual precursor to what are now called pseudogenes. 67 Zuckerkandl interview.


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esis of the clock idea. Zuckerkandl recounts his earliest thoughts about the molecular clock: . . . after a few chains had been partially sequenced it was possible to compare them and it became immediately clear that there are places along the sequence that are more variable than others. . . . That [differential variability] gives one a feeling – because feelings about things play a role in the origination of ideas – that there are certain resistances to change that are set [by natural selection]. One has particular sites where the resistances are very great and one has sites where it is less great and you observe the most changes. There is nothing extraordinary about thinking that the degree of resistance to change at an amino acid site could be fairly constant during long periods of evolution. Then if you consider the most changeable sites – they will make the clock tick if there is a clock – there could be a certain regularity in this ticking because the resistance to change at these changeable sites is more or less constant and perhaps averages out over time.68 It must be kept in mind that, as Zuckerkandl himself notes, it is “dangerous” to reconstruct from memory the detailed trails of discovery that took place more than thirty years ago.69 It is clear, however, that the molecular clock, as it was originally conceived in 1961, was not intended to be a “neutral” clock. Pauling liked the idea of the molecular clock and wove it into a number of lectures he gave. For example, at the First Inter-American Conference on Congenital Defects on January 22, 1962, he spoke of the molecular evidence for the time of separation of human and gorilla lineages.70 On November 15, 1962, while in Norway, Pauling delivered talk entitled “Molecular Disease and Evolution” at Oslo University.71 Pauling’s William Lloyd Williams lectures at Ohio State were entitled ‘Molecules, Evolution and Disease.”72 He gave a similar talk at Modesto Junior College to the science educators of 68 Ibid. 69 Ibid. 70 Linus Pauling, “The Molecular Basis of Genetic Defects” (typescript, 1962), p. 14, OSU, box 1962a, folder 1. This speech was published in International Medical Congress, First InterAmerican Conference on Congenital Defects (Philadelphia: Lippincott, 1963), pp. 15–21. For Pauling’s correspondence regarding this conference, see OSU, box 164, folder 3. 71 Date book 1962, OSU. 72 Itineraries: July 1961–July 1963, OSU, box 164, folder 1. Incidentally, these lectures were performed under “rather tense” conditions partly due to Pauling’s disapproval of Kennedy’s actions during “the Cuban Situation.” Letter from William White to Linus Pauling, 7 November 1962; letter from A. B Garrett to Linus Pauling, 8 November 1962, OSU, box 164, folder 12.

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central California in January 196373 and again in June to the Orange Country Medical Society.74 His date book indicates that he gave the same talk August 24, December 12, and December 17, 1963.75 By 1964, the title was occasionally changed to “Abnormal Molecules and Molecular Disease,” but the content was, for the most part, unchanged.76 Often, as Pauling preferred, he would combine a lecture on molecular disease and evolution and a lecture on science and peace.77 Late in 1966, disease and evolution.78 Perhaps Pauling’s most famous lecture pertaining to molecular evolution was given to the Rudolf Virchow Medical Society in New York on November 5, 1962. Using the clock, he calculated the figure of one substitution every 14.5 million years per hemoglobin chain in the same manner as before, adding, “We may use this value to discuss other evolutionary epochs.” Pauling then speculated upon the future value of the idea of an evolutionary molecular clock and molecular evolutionary studies in general: “I believe that it will be possible, through the detailed determination of amino acid sequences of hemoglobin and other molecules, to obtain much information about the course of the evolutionary process. . . . ”79 The future collaboration between Pauling and Zuckerkandl in part actualized Pauling’s optimism over extracting evolutionary information from amino acid sequences. The next day, Pauling traveled to Arden House, Columbia University, to attend a small informal conference on hemoglobin organized by Vernon Ingram.80 In his keynote speech, he discussed the molecular clock. Alexander Rich, a former student of Pauling’s, attended the conference. He recalled 73 Itineraries: July 1961–July 1963, OSU, box 164, folder 1; letter from William Grum to

Linus Pauling, 12 March 1963, box 78, folder 5. 74 Date book entry for 6 June 1963, OSU. Pauling notes that there were fifty people picketing against him outside. See also OSU, box 164, folder 1. 75 Date book 1963, OSU. 76 OSU, box 164, folder 16. Pauling literally takes the speech “Molecular Disease and Evolution,” makes some minor stylistic changes, and retitles it “Abnormal Hemoglobin and Molecular Disease.” This “new” speech was given at Congreso del Centenario de la Academia Nacional de Medicina, in Mexico on May 3, 1964. See also letter from Edward Pols to Linus Pauling 15 October 1963, OSU, computer search. 77 Letter from Linus Pauling to Tanya Menci, 29 November 1966, OSU, computer search. 78 Letter from Linus Pauling to R. S. Ramakrishna; letter, Linus Pauling to V. K. Gokak, OSU, box 164, folder 18. 79 OSU, box 1962s; this speech was printed as Linus Pauling, “Molecular Disease and Evolution,” Proc. Rudolf Med. Soc. N.Y., 21 (1963), 131–140. It was reprinted in Karger Gazette, Basel/New York 7/8, 3–4, and Bulletin N. Y. Acad. Med., 40 (1964) 334–342. 80 Letter from Vernon Ingram to Linus Pauling, 16 February 1962. OSU, box 164, folder 12; Pauling’s date book entry for 6 November 1962: “to Arden House (50 mins out) V. Ingram, I participate – opening address Hb conf.” The proceedings of this conference were not published.


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Pauling’s presentation of Zuckerkandl and Pauling’s research: “In one stroke he [and Zuckerkandl] united the fields of paleontology, evolutionary biology and molecular biology.”81 Emile Zuckerkandl also attended the Arden House workshop. Pauling suggested to him that one should be able to calculate an ancestral sequence, given the contemporary descendent sequences.82 Although, at first Zuckerkandl was skeptical about the possibility of achieving this reconstruction, he set about writing the paper that would bring Pauling’s idea to fruition.83 The article, “Chemical Paleogenetics, Molecular ‘Restoration Studies’ of Extinct Forms of Life,” was published in a Festschrift volume of Acta Chemica Scandinavica for the Swedish Nobel Prize-winning biochemist Hugo Theorell. 84 The paper reconstructs the probable common ancestral sequences of the human α-, β-, γ -, and δ-chains.85 In the final paragraph, conceived and written by Zuckerkandl,86 it was suggested that these paleobiochemical studies could conceivably be extended to proteins that differ in function (i.e., nonhemoglobin proteins) and hence give a more complete molecular phylogeny. The idea that molecules furnish evidence for evolutionary history was developed further in Zuckerkandl and Pauling’s next collaborative paper. On June 26, 1963, Pauling accepted an invitation to write an article for a volume celebrating the seventieth birthday of Alexandr Oparin, the famous Russian origin-of-life researcher.87 The same day he asked Zuckerkandl to collaborate with him and “think of some aspect of evolutionary biochemistry that [they] might discuss.”88 The resulting paper, finished in November, has the suggestive title “Molecules as Documents of Evolutionary History” and was written principally by Zuckerkandl.89 In this paper, Zuckerkandl and Pauling argue that “semantides,” that is, DNA, RNA, and polypeptides, contain the most 81 Alexander Rich, quoted in Hager, Force of Nature (above, n. 9), p. 541; Tom Hager,

personal communication, July 1996. 82 Zuckerkandl interview. 83 Ibid. 84 Linus Pauling and Emile Zuckerkandl, “Chemical Paleogenetics Molecular ‘Restoration Studies’ of Extinct Forms of Life,” Acta Chem. Scand., 17 (1963), S9–S16. 85 With current synthesizing technology, researchers are beginning to synthesize postualed ancestral sequences to determine their biochemical properties. This possibility was first suggested by Pauling and Zuckerkandl in this article (p. S15). 86 Letter from Emile Zuckerkandl to Linus Pauling, 25 March 1963, OSU, box 1963a, folder 1; Pauling and Zuckerkandl, “Chemical Paleogenetics” (above, n. 84), p. S16; Zuckerkandl interview. 87 Letter from Linus Pauling to W. L. Kretovich, OSU, box 1963a, folder 3. 88 Interoffice memo from Linus Pauling to Emile Zuckerkandl, OSU, box 1963a, folder 3. 89 The article was published in English as Emile Zuckerkandl and Linus Pauling, “Molecules as Documents of Evolutionary History,” J. Theoret. Biol., 8 (1965), 357–366.

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information and preserved history of any biological molecule and, hence, are to be preferred for providing a basis for molecular phylogeny.90 Zuckerkandl’s choice of terminology reflects the climate of early-1960s molecular biology, where much effort was aimed at understanding the nature of the genetic code and the transfer of information from DNA to protein. Not surprisingly, Pauling and Zuckerkandl briefly discuss the use of the degenerate genetic code for phylogenetic inferences. They show that some amino acid substitutions should be more probable, given the nature of the relationship between the codons for different amino acids. Zuckerkandl had spent the summer at Stanford by invitation of Joshua Lederberg, and they discussed the implications of the genetic code.91 The section on the genetic code was intended to be longer, but Thomas Jukes, another pioneer in the study of molecular evolution, was thinking along similar lines and published first.92 Late 1963 was a busy time for Pauling and the Zuckerkandls. Pauling was awarded the Nobel Peace Prize on the day a partial nuclear weapon test ban treaty went into effect. Within two months of the award, Pauling resigned from Cal Tech, partially because of the university’s lack of recognition of the prize.93 Emile and Jane Zuckerkandl visited Richard T. Jones, now a researcher at the University of Oregon Medical School in Portland, to separate and identify orangutan tryptic peptides using Jones’s newly developed automatic column chromatograph.94 Even though the Zuckerkandls extended their time in Portland, many experiments remained unfinished as they left on the day of President Kennedy’s assassination.95 90 The article is a very explicit use of the “metaphor” that semantides contain information

and hence have some form of meaning. 91 Zuckerkandl interview. The original reason for Lederberg’s inviting Zuckerkandl to Stanford was to discuss possible potential forms of life on Mars. For the particular genetic code Zuckerkandl considered, see R. V. Eck, “Genetic Code: Emergence of a Symmetrical Pattern,” Science, 140 (1963), 477–481. Joshua Lederberg does not recall the details of their discussions (Lederberg, personal communication, April 1997). 92 Thomas Jukes, “Some Recent Advances in Studies of the Transcription of the Genetic Message,” Adv. Biol. Med. Phys., 9 (1963), 1–42; see also Thomas Jukes “Relations between Mutations and Base Sequences in the Amino Acid Code,” Proc. Nat. Acad. Sci., 48 (1962), 1809–1815; letter from Emile Zuckerkandl to Linus Pauling, 21 July 1963, OSU, box 153, folder 5; letter from Linus Pauling to Emile Zuckerkandl, 29 July 1963, OSU, box 153, folder 5; letter from Thomas Jukes to Linus Pauling, 13 June 1963, OSU, box 56, folder 28; letter from Zuckerkandl to Linus Pauling, 26 October 1963, OSU, box 153, folder 5. 93 Hager, Force of Nature (above, n. 9), p. 549. 94 Pauling oversaw this trip. Interoffice memo from Linus Pauling to Emile and Jane Zuckerkandl, 10 September 1963, OSU, box 153, folder 5; letter from Richard Jones to Linus Pauling, 26 November 1962, OSU, box 54, folder 10; Jones interview; for a review of automatic peptide chromatography, see Richard T. Jones, “Automatic Peptide Chromatography,” Methods Biochem. Analysis, 18 (1970), 206–251. 95 Emile and Jane Zuckerkandl, Zuckerkandl interview.


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In 1964, Zuckerkandl attended two important conferences that discussed molecular evolution. The first was held in Bruges, Belgium, from April 29 to May 4. The second was held at Rutgers in September. In his presentation in Bruges, Zuckerkandl compared the rates of evolution of hemoglobin and cytochrome c, the other molecule that had been the subject of early evolutionary studies.96 He noted that the cytochromes seem to be evolving nearly 1.5 times faster than hemoglobin, and that in both molecules “the difference between chains may reach and pass 50 percent without any gross deviation from linearity in the number of differences in sequence and the time of the common ancestral chain.”97 The still nameless molecular clock had been refined somewhat: in 1962 it was a simple linear function, in 1964 it was approximately linear until around 50 percent of the sites were changed and then asymptotic to 100 percent.98 The refinement results, in part, from a change of emphasis from the actual number of amino acid substitutions to the number of observed amino acid substitutions.99 (Multiple substitutions at the same site are unobserved.) Zuckerkandl also considered a proposal that seemed to oppose the clock hypothesis: “Contemporary organisms shaped much like ancestral forms [i.e., “living fossils”] probably contain polypeptide chains that, in turn, mostly resemble quite closely the polypeptide chains of the ancestral forms.”100 Two things are noteworthy concerning this apparent about-face. First, Zuckerkandl did not believe the clock was contrary to natural selection; he was not interested in defending the independence of molecular evolution and organismal evolution. Second, the presentation considered “living fossils” as an exception rather than the rule – the second “slide” of his presentation recalculated, with more recent data, the divergence times of the four human hemoglobin chains using a molecular clock.101 In any 96 Emanual Margoliash, “Primary Structure and Evolution of Cytochrome c,” Proc. Nat. Acad. Sci., 50 (1963), 672–679. 97 Manuscript, “The Chemical Paleogenetics of Hemoglobin,” dated April 64, box 153, folder 5, p. 8. 98 The difference is in part due to a difference in perspective: if one considers the actual number of substitution events, then the function is linear; if one considers the number of observed substitutions, then the function is asymptotic. Although the terminology is a little loose, the 1962 paper implicitly assumes the latter as an estimate of the former. 99 If one focuses on observable substitutions, then this number will always be equal to or less than the number of actual changes. 100 Emile Zuckerkandl, “Further Principles of Chemical Paleogenetics as Applied to the Evolution of Hemoglobin,” in Protides of the Biological Fluids, ed. Hubert Peeters (Amsterdam: Elsevier, 1964), pp. 102–109. See also the earlier manuscript in OSU, box 153, folder 5. The paper is also significant in that it is Zuckerkandl’s first use of the “parsimony principle” to construct the topology of a phylogenetic tree. 101 Manuscript, “The Chemical Paleogenetics of Hemoglobin” (above, n. 97). The table on slide #2 was not included in the published paper.

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case, Zuckerkandl had rejected the proposal by the time he wrote the paper for the important September 1964 conference.102 On September 17 and 18, the Institute of Microbiology at Rutgers University held the seminal symposium “Evolving Genes and Proteins.”103 Many eminent biologists were present to hear Zuckerkandl deliver a collaborative paper that Robert Paradowski described as the “most influential of Pauling’s later career.”104 This lengthy paper, principally written by Zuckerkandl, named the molecular evolutionary clock and incorporated much of the material discussed in earlier papers: construction of a globin chain phylogeny, a system of nomenclature for describing ancestral nodes in a phylogenetic tree, implications of a genetic code for molecular evolution, and so on.105 It also responded to objections to the expression of “evolutionary transformations” as a simple function of time.106 The mathematical function that characterizes the clock was also derived. At the request of Zuckerkandl, Pauling derived this function and sent it to Zuckerkandl on September 12.107 The derivation provides an algebraic description of the curve that Zuckerkandl presented in Bruges. Let the chance of a mutation affecting one amino acid site of a polypeptide chain in time t be αt. For N sites, the total number of mutations is about Nαt. Let us consider the time, t at which Nαt mutations have occurred. We assume equal probability for all sites. The probability that the first site is unchanged after the first mutation is P1 =

N −1 1 =1− N N

102 Emile Zuckerkandl, personal communication, 20 July 1996. He, however, saw “living

fossils” as a test of the molecular clock hypothesis. 103 The full proceedings were published; see Bryson and Vogel, Evolving Genes and Proteins (above, n. 1). A short summary by Bryson and Vogel was published in Science, 147 (1965), 68–72. 104 Robert Paradowski, “Chronology,” in Linus Pauling: A Man of Intellect and Action, ed. Fumikazu Miyazaki (Tokyo, Japan: Cosmos Japan International, 1991), pp. 186–211, see p. 206. 105 It is common for people to mistakenly cite this paper as the origin of the molecular clock concept. For example, Wen-Hsiung Li, “So, What about the Molecular Clock Hypothesis,” Curr. Opin. Genet. Devel., 3 (1993), 896–901; Naoyuki Takahata, “Molecular Clock,” in Population Genetics, Molecular Evolution and the Neutral Theory: Selected Papers of Motoo Kimura, ed. Naoyuki Takahata (Chicago: University of Chicago Press, 1994), pp. 535–536. 106 I discuss this further in the next section. 107 Manuscript dated September 12, OSU, unsorted; letter from Zuckerkandl to Pauling, 3 [sic] September 1964. The date of this letter is obviously incorrect – the letter reports on the meeting that occurred on September 17 and 18. A similar equation was used by Margoliash and Smith at the conference. Emanual Margoliash and Emil Smith, in Evolving Genes and Proteins (above, n. 103), p. 233.


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and after Nαt mutations is

1 Nαt PNαt = P1Nαt = 1 − N At the limit, as N increase to infinity, 1 1 N = lim 1 − N→∞ N e Hence αt 1 ∼ = e−αt = At PNαt = e where A = e−α . Let us use the expression P (t) as the average probability for any given site to be unchanged at time t. Let τ be a unit lapse of time. Then P (nτ ) = P n Notice that the above derivation does not mention any selective processes. However, the remainder of the lengthy text gives ample evidence to suggest that natural selection leads to different probabilities of substitution at each site, and is consistent with Zuckerkandl’s claim that “natural selection is perfectly compatible with the clock.”108 On the other hand, Zuckerkandl and Pauling’s selectionism was not “cut and dried,”109 and they toyed with apparently protoneutralist explanations: “Along lines of descent marked by high evolutionary stability, the shuttle motion between functionally similar amino acid residues will . . . occur. The changes in amino acid sequences will, however, be limited almost exclusively to functionally nearly neutral changes.”110 These seemingly neutralist sentiments should be taken with a grain of salt, since Zuckerkandl and Pauling did not reject selectionist explanations and in the same paragraph argue that natural selection is needed to spread the “nearly neutral” substitutions through the population. It is noteworthy that the above treatment does not treat back mutations. In a sense, it assumes that there is an infinite number of amino acids rather than twenty, such that the probability of a back mutation is zero. This simplification worried Zuckerkandl, and he brought it to Pauling’s attention.111 Pauling was not interested in complicating his derivation because then it would no longer be aesthetically pleasing.112 Nonetheless, the 1965 article 108 Zuckerkandl interview. 109 Zuckerkandl, personal communication, October 30, 1996. 110 Zuckerkandl and Pauling, “Evolutionary Divergence” (above, n. 1), p. 149. 111 Letter from Zuckerkandl to Pauling, 3 [sic] September 1964 (above, n. 107). 112 Zuckerkandl interview.

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represented the pinnacle and culmination of the previous five fruitful years of collaboration between Linus Pauling and Emile Zuckerkandl. Early Reaction to the Molecular Evolutionary Clock The biological and anthropological communities were at first unreceptive to the idea that evolution at the molecular level might proceed at a constant rate. An important confrontation between the champions of the new molecular approach and the heirs of the organismal orthodoxy occurred at a milestone conference, “Classification and Human Evolution,” at Burg Wartenstein, Austria, in the summer of 1962. Zuckerkandl presented a paper whose title introduced the new controversial term “molecular anthropology.” A “restricted committee” meeting consisting of B. Campbell, T. Dobzhansky, M. Goodman, G. A. Harrison, H. P. Klinger, E. Mayr, G. G. Simpson, and Zuckerkandl considered the potentialities of the molecular approach to anthropology and the study of evolution.113 Only Morris Goodman, who had used immunological properties of proteins to reconstruct phylogenies, shared Zuckerkandl’s optimism about the utility of the molecular approach.114 Simpson and Mayr were skeptical of the clock hypothesis and molecular evolution in general. Concerning the use of amino acid sequences as discontinuous characters, Simpson argued that they had no important advantage over morphological characters.115 For example, Simpson pointed out that the clock ignores variation in the rates of evolution and would be highly inaccurate when applied to short lapses of time.116 Furthermore, they were skeptical of the use of single characters, as they took a molecule to be, to measure accurately evolutionary rates and similarity between species. “Seemingly contradictory evidence (e.g., that of the hemoglobins as reported by Zuckerkandl in this book) indicates merely that in certain characters Homo and its allies [e.g., gorilla] retain ancestral resemblances and that these are not the characters involved in their radical divergence. . . . ”117 Simpson later reiterated his “well received”118 comments even more strongly: 113 Emile Zuckerkandl, “Perspectives in Molecular Anthropology,” in Classification and

Evolution ed. Sherwood Washburn (Chicago: Aldine, 1963), pp. 243–272, see p. 254. 114 Zuckerkandl interview. Incidentally, Goodman sent Pauling some papers on his work in 1961. Letter from Morris Goodman to Linus Pauling, 12 May 1961, OSU. box 42, folder 5. 115 Zuckerkandl, “Perspectives in Molecular Anthropology” (above, n. 113), p. 259. 116 Ibid., p. 268. 117 George Gaylord Simpson, “The Meaning of Taxonomic Statements,” in Classification and Evolution, ed. Sherwood Washburn (Chicago: Aldine, 1963), pp. 1–31, see p. 25, italics in original. 118 George Gaylord Simpson, Concession to the Improbable: An Unconventional Autobiography (New Haven: Yale University Press, 1978), p. 217.


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Zuckerkandl has shown that “From the point of view of hemoglobin structure, it appears that gorilla is just an abnormal gorilla, and the two species form actually one continuous population.” From any other point of view other than that properly specified, that is, of course, nonsense. What the comparison seems really to indicate is that in this case, at least, hemoglobin is a bad choice and has nothing to tell us about affinities, or indeed tells us a lie. . . . Of course, as Zuckerkandl points out, we should not use just one kind of molecule but many, preferably proteins. However, if one can be misleading, so can many!119 Mayr reconciles morphological and molecular evidence in a similar manner: Man’s shift into the niche of the bipedal, tool-making, speech-using hominid necessitated a drastic reconstruction of his morphology, but his morphology did not, in turn, require a revamping of his biochemical system. Different characters and character complexes thus diverged at different rates.120 Hence the implication is that one should not arbitrarily pick one character to calculate divergence times. Interestingly, Mayr and Simpson appear to be little concerned with defending any strong dependencies between evolution at the molecular and organismal levels. The 1964 meeting at Rutgers also proved to be a battleground between the molecularly inclined and organismally inclined biologists. In the brief report of the meeting published in Science, the organizers commented that “much of the discussion during the meeting revolved around views of the relation between macromolecular and organismal evolution.”121 The atmosphere was described as “lively,” and there was “an unusually fruitful exchange among biochemists, molecular biologists, evolutionists, geneticists, taxonomists, exobiologists, and microbiologists.”122 Zuckerkandl wrote to Pauling informing him that a long paper was needed, given the mostly negative reaction of people to what he now called “chemical paleogenetics.”123 Ernst 119 George Gaylord Simpson, “Organisms and Molecules in Evolution,” in Protides of the

Biological Fluids, ed. Hubert Peeters (Amsterdam: Elsevier, 1965), pp. 29–35, see p. 31. 120 E. Mayr. “The Taxonomic Evaluation of Early Hominids,” Classification and Evolution, ed. Sherwood Washburn (Chicago: Aldine, 1963), pp. 332–346, see p. 344. However, in his book Animal Species and Evolution, Mayr acknowledges the evolutionary work done by Goodman and Pauling; Ernst Mayr, Animal Species and Evolution (Cambridge, Mass.: Harvard University Press, Belknap Press, 1963), p. 628. Pauling annotates this in his copy of the book. 121 Bryson and Vogel, summary (above, n. 103), p. 68. 122 Ibid. 123 Letter from Zuckerkandl to Pauling, September 3 [sic], 1964 (see above, n. 107).

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Mayr attended the meeting. In discussion, he affirmed his 1962 comments about the dangers of relying on only one character but seemed more receptive to the value of molecular evidence for evolution. I think that we actually are much closer together [on the value of molecular characters] than we appear to be. Everybody agrees that when you get down to the gene level and deal with components of the genotype, you are obviously better off than with purely phenotypical characters, where you go by inference. . . . The danger of basing a classification on single cistrons, no matter how important they are is nicely illustrated by hemoglobin of the primates. The baboon, which is a ceropithecoid very close to the macaques, has a remarkably different hemoglobin pattern, while the South American cebiods, which – according to the paleontologists – did not evolve out of the cercopithecoid group, but independently from the prosimians nevertheless have a hemoglobin similar to that of the ceropithecoid monkeys. Here is clearly a case where we would come up with a rather misleading classification, if we relied on these molecular characters.124 Mayr then highlighted the fact that the rate at which characters change depends heavily on the lineage that one considers: “ . . . the Homo Sapiens line came under an enormous selection pressure. The resulting change in certain characters is almost unprecedented. . . . ” Mayr repeatedly emphasized the role of selection even at the molecular level, for example, “most likely the replacement of residues [of cytochrome c] have been controlled by natural selection, even though we do not yet know the function of most of the residues” and “the few [mutations] that are incorporated are those which selection let go through.” Mayr weakened his position but responded to the threat that by focusing on the molecular level, natural selection would be wrongly devalued. His responses, as before, rested on thinking of a molecule as a single character (instead of, for example, thinking of each site as a character) and placing importance on what happens after splitting events, that is, anagenic change, as it is now called.

Conclusion The 1960s saw the development of the new field of molecular evolution. The series of largely theoretical papers that arose from the collaboration between Zuckerkandl and Pauling helped to found the new discipline. In the early years, there was no disciplinary journal. In fact, Zuckerkandl would become 124 E. Mayr, in “Discussion of Part III,” in Evolving Genes and Proteins (above, n. 1), p. 198.


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the founding editor of the Journal of Molecular Evolution in 1971. Instead, Zuckerkandl and Pauling published in Festschrift volumes. As an eminent scientist, Pauling was often invited to submit papers to these volumes. He then co-opted Zuckerkandl’s coauthorship and they published papers principally written and conceived by Zuckerkandl. Because they did not have the constraints of peer review, this medium allowed them much freedom in articulating their position. These volumes then functioned to disseminate pioneering ideas such as the molecular evolutionary clock. From the beginning, Zuckerkandl and Pauling sought to use the tools of molecular biology to enlighten studies of evolution. In this light, the use of the technique of fingerprinting proved to be a degenerating research practice. It did not revolutionize the study of evolution to the same extent that a detailed study of amino acid (and later DNA) sequences did. Zuckerkandl and Pauling’s later focus of the primary structure (i.e., the amino acid sequence) of hemoglobin centered the emerging field of molecular evolution on a particular class of molecular phenomena. Compare their approach with the immunological approach, popularized by Morris Goodman and Curtis Williams, among others, which emphasizes the three-dimensional structure of molecules.125 Zuckerkandl and Pauling’s use of the terms “informational macromolecule” and “semantide” echo their bias to study a certain class of molecules and privilege the one-dimensional sequences of these molecules. Although this terminology arose out of the general climate of molecular biology at the time, especially the work on the genetic code, one may trace their preference, at least in part, to Christian Anfinsen’s book The Molecular Basis of Evolution, reviewed by Pauling one month after Zuckerkandl’s arrival at Cal Tech.126 The recent papers by Dietrich (1994) and Suárez and Barahona (1996) have emphasized the role that Zuckerkandl and Pauling’s research on rates of molecular evolution played in the development of the Neutral Theory of Molecular Evolution.127 They have been largely concerned with revealing the experimental roots of the Neutral Theory of Molecular Evolution. In contrast, but not in contradiction, I have argued that when the clock was proposed in 1962 it was not intended to be a neutral clock. Instead, Zuckerkandl and Pauling were closer to their protagonists Mayr and Simpson in emphasizing the importance of selection at the molecular level. As a chemist and a biochemist/molecular biologist respectively, Pauling and Zuckerkandl viewed the protein molecule as a series of functionally constrained amino acid sites 125 Morris Goodman, “On the Emergence of Intraspecific Differences of the Protein Anti-

gens of Human Beings,” Amer. Nat., 94 (1960), 77–92; C. A. Williams and C. T. Wemyss, “Experimental and Evolutionary Significance of Similarities among Serum Protein Antigens of Man and the Lower Primates,” Ann. N. Y. Acad. Sci., 94 (1961), 77–92. 126 Letter from Linus Pauling to Robert Gould, 19 October 1959, OSU, box, 1959a, folder 2. 127 See above, n. 6.

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with the functional constraints enforced by natural selection. For Zuckerkandl and Pauling promotion of the molecular clock hypothesis amounted to the commitment that functional constraints approximately constant over time. Acknowledgments This paper was completed with the support of an internship at the Ava Helen and Linus Pauling Papers at Oregon State University Libraries. Ramesh Krishnamurthy, Sean Goodlet, and Cliff Mead deserve thanks for their archival assistance. I would also like to thank Robert Olby, Lindley Darden, and an anonymous referee for their comments on an earlier draft. Related papers were delivered at the University of Pittsburgh Center for the Philosophy of Science in May 1997 and the Seattle meeting of the International Society for the History Philosophy and Social Studies of Biology in July 1997. I am indebted to the respective audiences for their questions and comments. Last, but not least, I would like to thank Emile Zuckerkandl, Richard T. Jones, Ernst Mayr, and Joshua Lederberg for sharing their knowledge of the period.