PNAS CLASSIC PERSPECTIVE Phylogeny and beyond: Scientific, historical, and conceptual significance of the first tree of life Norman R. Pacea,1, Jan Sappb, and Nigel Goldenfeldc aDepartment of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309; bBiology Department, York University, Toronto, ON, Canada M3J 1P3; and cInstitute for Genomic Biology and Department of Physics, University of Illinois, Urbana, IL 61801 Edited by Edward F. DeLong, Massachusetts Institute of Technology, Cambridge, MA, and approved December 20, 2011 (received for review November 29, 2011) In 1977, Carl Woese and George Fox published a brief paper in PNAS that established, for the first time, that the overall phylogenetic structure of the living world is tripartite. We describe the way in which this monumental discovery was made, its context within the historical development of evolutionary thought, and how it has impacted our understanding of the emergence of life and the characterization of the evolutionary process in its most general form. fundamental breakthrough in Several aspects of the paper by Woese species and organism and bringing to the biological science occurred in and Fox (1) sparked skepticism. One fore the deep limitations of earlier ac- A 1977, and most biologists did was the arcane nature of the molecular counts of the evolutionary process. not notice. The paper by Woese data, which few could appreciate. The re- and Fox (1) in 1977 was 2.5 pages in length liance on a single gene to trace major Lead Up to the Paper and contained a single table of numbers trends in evolution was an equally alien The 1977 paper by Woese and Fox (1) was that compared sequence snippets derived concept. Some quibbled about the name an early example of what we would today from small subunit rRNAs of different archaebacteria; others objected to The call molecular phylogenetics—the com- organisms. The table provided the first New York Times publicity. Most impor- parison of macromolecular sequences to gene sequence-based quantitative assess- tantly, however, the conclusions of the infer genealogical and thereby, evolution- ment of phylogenetic (evolutionary) rela- paper flew into the face of the common ary relationships. The notion of comparing tionships between representatives of the wisdom of the time regarding the basic sequences to infer relationships was put major known kinds of organisms (1). The divisions of biology and the nature of early forward in 1958 by Francis Crick (3) and paper showed that all cellular life falls into evolution. It was generally believed—and more formally, by Emil Zuckerkandl and one of three large relatedness groups: still is taught in our textbooks—that life Linus Pauling in 1965 (4). This was a eukaryotes (our kind of cells, which con- is of two kinds: on one hand, eukaryotes time when determination of protein se- tain a nuclear envelope), eubacteria and on the other hand, prokaryotes, which quences had become, to some extent, [Woese and Fox (1) termed the group and lack nuclear membranes and as the name tractable with protocols developed by Fred fi this group is where classically studied implies, were supposed to have preceded Sanger (5), who received his rst Nobel bacteria fit], and archaebacteria [an un- and evolved into eukaryotes. However, Prize for that development and the de- usual group of recently described organ- eubacteria and the newly discovered group termination of the amino acid sequence of isms named by Woese and Fox (1) to of archaebacteria both lacked nuclear insulin in the 1950s (5). Protein bio- distinguish the group from eubacteria]. In membranes. Eukaryotes seemed not de- chemists began to develop phylogenetic describing the phylogenetic relationships, rived from either bacteria or arch- relatedness maps, phylogenetic trees, based on amino acid sequences derived the results also charted the first scientific aebacteria; all three kinds of organisms from various organisms, mainly animals. view of deep evolutionary history. Both seemed to represent aboriginal lines Russell Doolittle sketched out vertebrate these fundamental aspects of biology, the of descent. evolution using blood-clotting fibrinopep- phylogenetic structure of life and the In this retrospective, we view the 1977 tides in the work by Doolittle and Feng course of early evolution, previously were paper by Woese and Fox (1) from three (6); the work by Fitch and Margoliash (7) standpoints. First, we discuss the specific only realms of speculation. used the mitochondrial protein cyto- However, the methods and data used in accomplishments of this landmark paper chrome C to relate animals and some the work by Woese and Fox (1) were and how the program of research initiated fungi. However, not all organisms possess unfamiliar to most biologists, even mo- and led by Woese from the late 1960s to cytochrome C, and for that reason alone, lecular biologists. Traditional biologists, the present day has spawned a revolution fi its amino acid sequence could not be used students of plants and animals, paid little in microbiology and other elds contin- to infer the patterns of relationships attention, because the results had little gent on microbiology, including ecology among all of life. bearing on their interests. Because of and the health sciences. Second, we dis- Carl Woese came to the study of evo- a joint press release by the National cuss the place of that paper in the history lution from a background in biophysics and Aeronautics and Space Administration of evolutionary biology, where its un- and the National Science Foundation that precedented use of molecular sequences ’ fi supported Woese s research, the paper associated with rRNA provided the rst Author contributions: N.R.P., J.S., and N.G. wrote the paper. was heralded on the front page of The New window into the deep timeline of life, one The authors declare no conflict of interest. York Times for discovery of “a third form independent of theoretical prejudices that This article is a PNAS Direct Submission. of life” (2). However, the few biologists had flawed earlier efforts to classify life. See Classic Article “Phylogenetic structure of the prokary- who noticed sometimes reacted negatively, Finally, we describe how the under- otic domain: The primary kingdoms” on page 5088 in issue and articles denouncing the claims were standings sparked by the paper are bring- 11 of volume 74. published. Subsequent developments ing a new face to the study of evolution by See Classic Profile on page 1019. showed that the methods and conclusions compelling biologists to address founda- 1To whom correspondence should be addressed. E-mail: of the paper were sound. tional issues related to the very concepts of [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1109716109 PNAS | January 24, 2012 | vol. 109 | no. 4 | 1011–1018 an interest in the genetic code and its RNases, and the sequences of the resulting origins. During the early 1960s, the nature oligonucleotides were determined by di- of protein synthesis and the makeup of the gestion with other nucleases. Next, frag- genetic code were just being worked ments of incomplete digestion of the RNA out (8). Woese was a contributor to early were isolated and digested completely, thought on the genetic code and had and the digestion products were analyzed; conducted experimental studies to try to eventually, the sequence could be inferred understand the chemical basis of the ca- from the oligonucleotide contents of nonical assignments of different amino overlapping fragments. Mitchell Sogin, acids to particular codons (the DNA or then a graduate student with Woese, RNA base triplets that specify the amino learned the techniques from David H. L. acid sequence of a protein during protein Bishop, a postdoctoral student from synthesis) (9). His 1967 book The Genetic Fred Sanger’s laboratory who was then Code: The Molecular Basis for Genetic working in the Sol Spiegelman laboratory Expression focused prescient attention on at the University of Illinois. Sogin set up the RNA elements of the protein synthe- the necessary facility for Woese’s group. sizing machinery (10). Woese, along with Woese and his students determined sev- Francis Crick (11) and Leslie Orgel (12) eral bacterial 5S rRNA sequences. They after him, are considered founding cham- showed that the rRNA sequences could be pions of the idea that nucleic acids played used as phylogenetic markers for bacteria more than template roles in the origin of (15). They also showed that evolutionary biological systems, thus giving rise to the variation in sequences could be used to notion of a hypothetical prebiotic RNA determine how the RNAs fold into sec- world in which nucleic acids served as both ondary structure (so-called phylogenetic catalytic entities and genetic templates comparative RNA structure analysis) (16). (13). Woese was concerned that the However, it soon became clear that 5S Fig. 1. Ribonuclease T1 oligonucleotide finger- emerging paradigm for the mechanism of rRNA, at only 120 nt in length, was too print. As outlined in the text, data reported in the protein synthesis was too static and had small in size and hence, information con- paper by Woese and Fox (1) in 1977 consisted of no evolutionary dimension. As early as tent to provide for accurate phylogenetic catalogs of oligonucleotide sequences derived 1969, as articulated in a letter to Francis assessments. from RNase T1 digestion of small subunit (SSU) Crick, he understood that the only way to The SSU rRNA, at 1,500–2,000 nt, was rRNAs. The first step of the analysis involved res- reveal the essence of the process was to information-rich, but because of its rela- olution of RNase T1 digests of 32P-labeled rRNA study its conservation and variation in tively large size, it was practically impos- by 2D electrophoresis and locating labeled oligo- different organisms—its evolution—in sible to determine the entire sequence nucleotides on the 80 × 100-cm sheet of electro- a phylogenetic framework.