- Email: [email protected]
Searching for the common ancestor
Res. Microbiol. 151 (2000) 85–89 © 2000 Éditions médicales et scientifiques Elsevier SAS. All rights reserved S092325080000124-8/REV
Searching for the common ancestor Russell F. Doolittle* Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0634, USA
Abstract — In this brief mini-review I address the current controversy surrounding the nature of the last common ancestor of all life on Earth. The pros and cons of the various positions have been hotly debated of late with no sign of the tumult subsiding. As such, I could not possibly do justice to all the varied and opposing views, nor even cite them. Let me just say at the outset that my own views on the subject at hand have been greatly influenced by the writings of T. Cavalier-Smith, W.F. Doolittle, P. Gogarten, R. Gupta, H. Hartman, O. Kandler, E. Koonin, J. Lake, W. Martin, M. Sogin, and C. Woese, inter alia. I hope they will forgive me if my depiction of events does not do full justice to their contributions. © 2000 Éditions médicales et scientifiques Elsevier SAS anomalous phylogenies / horizontal gene transfers / endosymbiont imports
1. The sequence revolution Forty years ago, the prospect of being able to determine amino acid sequences lent hope to the notion that the history of life on Earth could in large part be reconstructed. Twenty years ago, the advent of DNA sequencing momentarily made that hope border on certainty. The problem is that the more we have learned, the more the details have mattered. Dealing with ancient events of necessity involves conjecture. Controversy has dogged us every step of the way, whether it be the origin of spliceasomal introns, horizontal gene transfers, the technical limits of phylogenetic reconstruction, or, now the most sacred of Darwinian constructs, the notion of the last common ancestor. Certainly no thinking biologist doubts that all living organisms on the Earth today are genetically related. It is the nature of that relationship that is under fire. For the first three-quarters of the 20th century, all cellular life was categorized
* Tel.: +1 858 534 4417; fax: +1 858 534 4985; [email protected]
as being either prokaryotic or eukaryotic [17]. A membrane-enshrouded nucleus was either present or not. Then, in 1977, two lines of evidence suggested that the prokaryotes came in two kinds. The distinction was made on the basis of ribosomal RNA sequences [19] on the one hand, and the nature of cell walls on the other [11]. The now three groups of organisms were initially designated Archaebacteria, Eubacteria, and Eukaryotes, but subsequently renamed Archaea, Bacteria, and Eucarya [20]. Given the limited data at hand, it was not possible to root a tree of life for the three domains. Did the bacterial nature of the Archaea and Bacteria imply recent common ancestry, as many still believe, or, as many others propose, have the Archaea shared a more recent common ancestry with Eucarya than they have with Bacteria? There is also the third possibility that Bacteria are nearer kin to Eucarya. Evidence favoring each of these ‘trees of life’ has been posed by Kandler [9] in the form of a sphynxian triangle (figure 1). Thus, Archaea and Bacteria both have circular genomes but Eucarya do not. Bacteria and Eucarya have ester-based lipids, while Archaea use ethers; but Archaea and Eucarya are clearly
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Doolittle
Figure 1. Symbolic triangle posed by Kandler [9] showing paradoxical affinities of the three domains of life.
united by the kind of elongation factors they possess. Which of these properties is telling the truth? It was assumed that amino acid sequences would eventually be able to resolve the matter, especially when several groups were able to use the device of rooting the phylogeny by paralogous gene products. The robust tree that emerged from those studies had the eukaryotes appearing as an offshoot of archaebacteria. By 1990, this tree of life was the generally accepted model and served as the basis for the nomenclature change mentioned above [20]. As the last decade of the millenium progressed, however, the model came under increasing fire because of the large number of discrepancies that have arisen, many of which have come to light as the result of completely sequenced microbial genomes. Some of these have struck at the very heart of the three-domain concept. The use of ‘signature sequences’, for example, has led to the almost heretical proposal that the Archaea are really kin to Gram-positive eubacteria [6]. (I say almost only because I recall that CavalierSmith had at one point suggested that Archaea and posibacteria shared a common ancestor which had lost the outer membrane common to more primitive bacteria.)
2. Was there a common ancestor? Serious doubt about the existence of a specific last universal common ancestor was first raised
by Kandler, who suggested that the three different lineages began at a time when life was still colonial and undifferentiated [10]. As more modern traits were added and exchanged, the tree of life became more bush-like. The idea was extended by Woese [18], mostly in an effort to explain the large numbers of lateral gene transfers that were being uncovered by the large-scale genome comparisons. W. Ford Doolittle [4] pushed the idea even further, raising the prospect that we will never be able to trace organismal lineages as such, an idea that has provoked a storm of comment from others. If the rate of lateral gene transfer exceeds the rate of species diversification, he suggested, then it is possible we will never be able to reconstruct a true tree of life.
3. How rampant is rampant? Although it is not a simple matter to assess the frequency of horizontal gene transfer, there are some approaches to consider. First, we should categorize the different kinds of transfer. To that end, let us put aside for a moment all the transfers that might have occurred as a consequence of organellar import. These will be considered in the next section. The remainder can be divided into two major kinds: a) those that confer some significant new advantage on the recipient; and b) those that merely displace an equivalent gene in the recipient. The latter kind typically involve stand-alone enzymes [8]. In a kind of neutral evolution mode, the frequency of occurrence for these equivalent displacements may be relatively high, but the fraction surviving may be small. In contrast, transfers offering new opportunities may be rare, but the chances of survival will be great. One way to get a handle on the frequency of transfer is from simple measures of sequence identity. For example, if a gene were transferred very recently, say within the last million years, its protein sequence in the new setting (recipient) ought to be virtually identical with what it was in the donor organism. We can ask, then, how similar are the most similar proteins that occur in Archaea and Bacteria? As it happens,
Searching for the common ancestor
87
Table I. Strongest sequence resemblances between Archaea and Bacteria. Protein Nitrogenase HS70 Enolase HMG-CoA reductase Nucleotide diP kinase Gapdh B Serine RS Dihydrolip. Dh Glu-NH3 synthase
Archaea
Bacteria
Percent ID
Methanosarcina barkeri Methanococcus mazeii Methanococcus jannaschii Archaeoglobus fulgidus Methanococcus jannaschii Haloarcula vallismortis Haloarcula vallismortis Halobacterium volcanii Methanococcus voltae
Clostridium pasteurianum Clostridium acetobutyl. Zymomonas mobilis Pseudomonas sp. Staphylococcus aureus Bacillus subtilis Bacillus subtilis Bacillus subtilis Bacillus cereus
74.0 67.7 59.7 57.0 56.1 51.8 50.6 49.9 48.0
the most similar pair of sequences that I have been able to find, where one member is from the Archaea and the other from Bacteria, is 74% identical (table I). In contrast, in an earlier survey of 34 enzymes common to Archaea and Bacteria, the average resemblance was found to be 38% identical [5]. In fact, the most similar sequences between any two species of Archaea and Bacteria (table I) are all candidates for horizontal transfers, as shown by phylogenetic tree construction. Some of these, such as nitrogenase (74% identity), are of the sort that could obviously offer great selective advantage. Others, such as serine tRNA-amino acyl synthetase (51% identity), are likely to be simple displacements. The fact that the list as it stands now is not populated in the region of 75–100% identity argues that ‘rampant’ is too strong a term to describe the amount of lateral gene transfer that has occurred between the Archaeal and Bacterial lineages.
4. The container approach If agreement cannot be reached on the general ancestry of all the genes in extant organisms, is it possible we may find some consensus about the cells that contain them, as exemplified by the architecture of their cell walls, membranes, and organelles? Although the nature of the containers is naturally dictated by genes, it may be that those particular genes are the ones that can provide a reasonable perspective. Let me sketch a greatly oversimplified scenario of some early events, knowing full well that there are countless variations possible.
Let us start with some ill-defined community of primitive organisms, the simple genomes of which are composed of assorted small chromosomes of RNA [14]. Ribosomes are the dominant features in these cells. Let the cells be abundant over all the Earth, and, as proposed by others, let there be a free exchange of genes. Now let some small set of these cells discover the means to use DNA as the genetic material. Obviously, this complicated shift must have involved a large number of incremental steps, important aspects of which included reverse transcription and the provision of deoxyribonucleotides. Let us simply accept the genetic advantages of a DNA-based genome. The bifurcation into DNA-based and RNAbased genomes could mark the earliest division of the prokaryotic and eukaryotic lineages [14], and the suggestion has long been around that some of the more primitive cells could have survived and independently evolved another advantage: namely, the cytoskeleton that is characteristic of eukaryotes [7, 17]. The foraging lifestyle of eukaryotes depended on the cytoskeleton for both mobility and endocytosis for engulfing a food supply. As such, the cytoskeleton is the very basis of the endosymbiont origin of organelles [7]. It has been conjectured that the nucleus may have been the first of these transformed symbionts [12]. In this regard, the mechanics of endocytosis leading to a nucleus must have differed from those involved in organelle formation. Thus, although both the nucleus and endosymbionts have double-layered membranes, the outer membrane of the nucleus is continuous
88
Doolittle
with the membranous structure of the host cell, and only the inner membrane should ‘belong’ to the invader. In contrast, both membranes of mitochondria are attributed to the symbiont. Reasonably, then, the symbiont destined to become the mitochondrion was itself doublemembraned, as are its proteobacterial ancestors. The nucleus, on the other hand, if it really did begin as an endosymbiont, ought to be the relic of a single-membraned symbiont; either a Gram-positive eubacterium or an archaebacterium would fit the description. Ribosomal RNA comparisons early on led to the suggestion that the symbiont was an Archaeon [16]. If this were the case, however, it might be expected that the inner nuclear membrane would use ether-based lipids, something that is not observed. The case for there being two distinct endosymbiotic events on the lineage leading to all extant eucarya, the one giving rise to a DNAbased nucleus and the second giving rise to the mitochondrion, could largely explain the three groups of gene products found among the Eucarya (figure 2). First, there are those proteins vertically descended from the time of the first major pre-DNA bifurcation. These would include the principal cytoskeletal proteins, such as tubulin and actin [3]. Second, there are those gene products that may have been brought in by the hypothetical ancestor of the nucleus, including elongation factors and other proteins shared by Archaea and Eucarya, such as fibrillarin and ubiquitin. Finally, there are all those gene products brought in by the ancestor of the mitochondrion. This scenario could explain the three different phylogenies for different genes and allow us to count properly the individual lateral transfers discussed above.
5. What happened to the Archezoa? Cavalier-Smith [1] realized that if the organelles of eukaryotes arose by engulfment of prokaryotes, then perhaps some very early lineages of eukaryotes diverged before the engulfment and are still extant. Although he was fully aware that some extant eukaryotes lacking mitochondria had probably lost them, he tem-
Figure 2. Tree of life that accommodates some of the most often mentioned conundrums, including those shown in Kandler’s triangle (figure 1).
porarily assigned all amitochondriates to a new class called Archezoa, until such time as evidence supporting loss should become available. Alas, much evidence has been accumulated at this point, and there is now question as to whether any bona fide Archezoa remain. Much of the evidence has centered on the existence of genes for heat shock protein 70 and a chaperonin protein called cpn60 [15], both of which are generally characteristic of mitochondria and their forebearers among the a-proteobacteria.
6. Prospects and frustrations Are there relic organisms that could possibly support such a scenario? It is perhaps too much to hope that an RNA-based cellular organism still lurks in some exotic habitat on this Earth, and the community would be happy to settle for a genuine archezoan. For a while, hopes were pinned on the protist Pelomyxa palustris, an exotic amoeba that lacks mitochondria but which contains several obligate endosymbionts, including Bacteria that generate H2 and CO2 and Archaea that consume those byproducts and put out methane [13]. These hopes were dampened recently when it was reported that sequence analysis of the rRNA from Pelomyxa
Searching for the common ancestor
puts them high on the eukaryotic tree (L. Morin and J.-P. Mignot, 1996, unpublished findings cited in ref. [2]). Being desperate, we should wait to see the data before jettisoning all hope for Pelomyxa as a link to the past. Even if that report is upheld, other primitive eukaryotes may still be lingering somewhere on Earth that might illuminate the sequence of events. Roger Stanier liked to remind us that there is a metaphysical aspect to evolutionary speculation [17]. In this vein, as we look back on the 20 years since recombinant DNA took hold, many well-entrenched positions have had to be reversed in the light of newly reported facts. Vast amounts of sequence data notwithstanding, there are many things about early life on Earth that are not yet known.
References [1] Cavalier-Smith T., Endosymbiotic origin of the mitochondrial envelope, in: Schwemmler W., Schenk H.E.A. (Eds.), Endocytobiology II, DeGruyter, Berlin, 1983. [2] Cavalier-Smith T., A revised six-kingdom system of life, Biol. Rev. 73 (1998) 203–266. [3] Doolittle R.F., The origins and evolution of eukaryotic proteins, Phil. Trans. R. Soc. London B 349 (1995) 235–240. [4] Doolittle W.F., Phylogenetic classification and the universal tree, Science 284 (1999) 2124–2128. [5] Feng D.F., Cho G., Doolittle R.F., Determining divergence times with a protein clock: update and reevaluation, Proc. Natl. Acad. Sci. USA 94 (1998) 13028–13033.
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[6] Gupta R.S., Life’s third domain (Archaea): an established fact or an endangered paradigm?, Theor. Popul. Biol. 54 (1998) 91–104. [7] Hartman H., The origin of the eukaryotic cell, Speculations Sci. Technol. 7 (1984) 77–81. [8] Jain R., Rivera M.C., Lake J.A., Horizontal transfer among genomes: the complexity hypothesis, Proc. Natl. Acad. Sci. USA 96 (1999) 3801–3806. [9] Kandler O., Cell wall biochemistry and three-domain concept of life, Syst. Appl. Microbiol. 16 (1994) 501–509. [10] Kandler O., The early diversification of life, in: Bengston S. (Ed.), Early Life on Earth, Nobel Symposium No. 84, Columbia University Press, 1994. [11] Kandler O., Hoppe H., Lack of peptidoglycan in the cell walls of Methanosarcina barkeri, Arch. Microbiol. 113 (1977) 57–60. [12] Lake J.A., Rivera M.C., Was the nucleus the first endosymbiont?, Proc. Natl. Acad. Sci. USA 91 (1994) 2880–2881. [13] Lee J.L., Soldo A.T., Reisser W., Lee M.J., Jeon K.W., Gortz H.D., The extent of algal and bacteriological endosymbioses in protozoa, J. Protozool. 32 (1985) 391–403. [14] Poole A.M., Jeffares D.C., Penny D., The path from the RNA world, J. Mol. Evol. 46 (1999) 1–17. [15] Roger A.J., Svard S.G., Tovar J., Clark C.G., Smith M.W., Gillin F.D., Sogin M.L., A mitochondrial-like chaperonin 60 gene in Giardia lamblia: evidence that diplomonads once harbored an endosymbiont related to the progenitor of mitochondria, Proc. Natl. Acad. Sci. USA 95 (1998) 229–234. [16] Sogin M., Early evolution and the origin of eukaryotes, Curr. Opin. Genet. Dev. 1 (1991) 457–463. [17] Stanier R.Y., Some aspects of the biology of cells and their possible evolutionary significance, Symp. Soc. Gen. Microbiol. 20 (1970) 1–38. [18] Woese C., The universal ancestor, Proc. Natl. Acad. Sci. USA 95 (1998) 6854–6859. [19] Woese C.R., Fox G.E., Phylogenetic structure of the prokaryotic domain: the primary kingdoms, Proc. Natl. Acad. Sci. USA 75 (1977) 5088–5090. [20] Woese C., Kandler O., Wheelis M.L., Towards a natural system of organisms: proposals for the domains Archaea, Bacteria and Eucarya, Proc. Natl. Acad. Sci. USA 87 (1990) 4576–4579.
Searching for the common ancestor Russell F. Doolittle* Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0634, USA
Abstract — In this brief mini-review I address the current controversy surrounding the nature of the last common ancestor of all life on Earth. The pros and cons of the various positions have been hotly debated of late with no sign of the tumult subsiding. As such, I could not possibly do justice to all the varied and opposing views, nor even cite them. Let me just say at the outset that my own views on the subject at hand have been greatly influenced by the writings of T. Cavalier-Smith, W.F. Doolittle, P. Gogarten, R. Gupta, H. Hartman, O. Kandler, E. Koonin, J. Lake, W. Martin, M. Sogin, and C. Woese, inter alia. I hope they will forgive me if my depiction of events does not do full justice to their contributions. © 2000 Éditions médicales et scientifiques Elsevier SAS anomalous phylogenies / horizontal gene transfers / endosymbiont imports
1. The sequence revolution Forty years ago, the prospect of being able to determine amino acid sequences lent hope to the notion that the history of life on Earth could in large part be reconstructed. Twenty years ago, the advent of DNA sequencing momentarily made that hope border on certainty. The problem is that the more we have learned, the more the details have mattered. Dealing with ancient events of necessity involves conjecture. Controversy has dogged us every step of the way, whether it be the origin of spliceasomal introns, horizontal gene transfers, the technical limits of phylogenetic reconstruction, or, now the most sacred of Darwinian constructs, the notion of the last common ancestor. Certainly no thinking biologist doubts that all living organisms on the Earth today are genetically related. It is the nature of that relationship that is under fire. For the first three-quarters of the 20th century, all cellular life was categorized
* Tel.: +1 858 534 4417; fax: +1 858 534 4985; [email protected]
as being either prokaryotic or eukaryotic [17]. A membrane-enshrouded nucleus was either present or not. Then, in 1977, two lines of evidence suggested that the prokaryotes came in two kinds. The distinction was made on the basis of ribosomal RNA sequences [19] on the one hand, and the nature of cell walls on the other [11]. The now three groups of organisms were initially designated Archaebacteria, Eubacteria, and Eukaryotes, but subsequently renamed Archaea, Bacteria, and Eucarya [20]. Given the limited data at hand, it was not possible to root a tree of life for the three domains. Did the bacterial nature of the Archaea and Bacteria imply recent common ancestry, as many still believe, or, as many others propose, have the Archaea shared a more recent common ancestry with Eucarya than they have with Bacteria? There is also the third possibility that Bacteria are nearer kin to Eucarya. Evidence favoring each of these ‘trees of life’ has been posed by Kandler [9] in the form of a sphynxian triangle (figure 1). Thus, Archaea and Bacteria both have circular genomes but Eucarya do not. Bacteria and Eucarya have ester-based lipids, while Archaea use ethers; but Archaea and Eucarya are clearly
86
Doolittle
Figure 1. Symbolic triangle posed by Kandler [9] showing paradoxical affinities of the three domains of life.
united by the kind of elongation factors they possess. Which of these properties is telling the truth? It was assumed that amino acid sequences would eventually be able to resolve the matter, especially when several groups were able to use the device of rooting the phylogeny by paralogous gene products. The robust tree that emerged from those studies had the eukaryotes appearing as an offshoot of archaebacteria. By 1990, this tree of life was the generally accepted model and served as the basis for the nomenclature change mentioned above [20]. As the last decade of the millenium progressed, however, the model came under increasing fire because of the large number of discrepancies that have arisen, many of which have come to light as the result of completely sequenced microbial genomes. Some of these have struck at the very heart of the three-domain concept. The use of ‘signature sequences’, for example, has led to the almost heretical proposal that the Archaea are really kin to Gram-positive eubacteria [6]. (I say almost only because I recall that CavalierSmith had at one point suggested that Archaea and posibacteria shared a common ancestor which had lost the outer membrane common to more primitive bacteria.)
2. Was there a common ancestor? Serious doubt about the existence of a specific last universal common ancestor was first raised
by Kandler, who suggested that the three different lineages began at a time when life was still colonial and undifferentiated [10]. As more modern traits were added and exchanged, the tree of life became more bush-like. The idea was extended by Woese [18], mostly in an effort to explain the large numbers of lateral gene transfers that were being uncovered by the large-scale genome comparisons. W. Ford Doolittle [4] pushed the idea even further, raising the prospect that we will never be able to trace organismal lineages as such, an idea that has provoked a storm of comment from others. If the rate of lateral gene transfer exceeds the rate of species diversification, he suggested, then it is possible we will never be able to reconstruct a true tree of life.
3. How rampant is rampant? Although it is not a simple matter to assess the frequency of horizontal gene transfer, there are some approaches to consider. First, we should categorize the different kinds of transfer. To that end, let us put aside for a moment all the transfers that might have occurred as a consequence of organellar import. These will be considered in the next section. The remainder can be divided into two major kinds: a) those that confer some significant new advantage on the recipient; and b) those that merely displace an equivalent gene in the recipient. The latter kind typically involve stand-alone enzymes [8]. In a kind of neutral evolution mode, the frequency of occurrence for these equivalent displacements may be relatively high, but the fraction surviving may be small. In contrast, transfers offering new opportunities may be rare, but the chances of survival will be great. One way to get a handle on the frequency of transfer is from simple measures of sequence identity. For example, if a gene were transferred very recently, say within the last million years, its protein sequence in the new setting (recipient) ought to be virtually identical with what it was in the donor organism. We can ask, then, how similar are the most similar proteins that occur in Archaea and Bacteria? As it happens,
Searching for the common ancestor
87
Table I. Strongest sequence resemblances between Archaea and Bacteria. Protein Nitrogenase HS70 Enolase HMG-CoA reductase Nucleotide diP kinase Gapdh B Serine RS Dihydrolip. Dh Glu-NH3 synthase
Archaea
Bacteria
Percent ID
Methanosarcina barkeri Methanococcus mazeii Methanococcus jannaschii Archaeoglobus fulgidus Methanococcus jannaschii Haloarcula vallismortis Haloarcula vallismortis Halobacterium volcanii Methanococcus voltae
Clostridium pasteurianum Clostridium acetobutyl. Zymomonas mobilis Pseudomonas sp. Staphylococcus aureus Bacillus subtilis Bacillus subtilis Bacillus subtilis Bacillus cereus
74.0 67.7 59.7 57.0 56.1 51.8 50.6 49.9 48.0
the most similar pair of sequences that I have been able to find, where one member is from the Archaea and the other from Bacteria, is 74% identical (table I). In contrast, in an earlier survey of 34 enzymes common to Archaea and Bacteria, the average resemblance was found to be 38% identical [5]. In fact, the most similar sequences between any two species of Archaea and Bacteria (table I) are all candidates for horizontal transfers, as shown by phylogenetic tree construction. Some of these, such as nitrogenase (74% identity), are of the sort that could obviously offer great selective advantage. Others, such as serine tRNA-amino acyl synthetase (51% identity), are likely to be simple displacements. The fact that the list as it stands now is not populated in the region of 75–100% identity argues that ‘rampant’ is too strong a term to describe the amount of lateral gene transfer that has occurred between the Archaeal and Bacterial lineages.
4. The container approach If agreement cannot be reached on the general ancestry of all the genes in extant organisms, is it possible we may find some consensus about the cells that contain them, as exemplified by the architecture of their cell walls, membranes, and organelles? Although the nature of the containers is naturally dictated by genes, it may be that those particular genes are the ones that can provide a reasonable perspective. Let me sketch a greatly oversimplified scenario of some early events, knowing full well that there are countless variations possible.
Let us start with some ill-defined community of primitive organisms, the simple genomes of which are composed of assorted small chromosomes of RNA [14]. Ribosomes are the dominant features in these cells. Let the cells be abundant over all the Earth, and, as proposed by others, let there be a free exchange of genes. Now let some small set of these cells discover the means to use DNA as the genetic material. Obviously, this complicated shift must have involved a large number of incremental steps, important aspects of which included reverse transcription and the provision of deoxyribonucleotides. Let us simply accept the genetic advantages of a DNA-based genome. The bifurcation into DNA-based and RNAbased genomes could mark the earliest division of the prokaryotic and eukaryotic lineages [14], and the suggestion has long been around that some of the more primitive cells could have survived and independently evolved another advantage: namely, the cytoskeleton that is characteristic of eukaryotes [7, 17]. The foraging lifestyle of eukaryotes depended on the cytoskeleton for both mobility and endocytosis for engulfing a food supply. As such, the cytoskeleton is the very basis of the endosymbiont origin of organelles [7]. It has been conjectured that the nucleus may have been the first of these transformed symbionts [12]. In this regard, the mechanics of endocytosis leading to a nucleus must have differed from those involved in organelle formation. Thus, although both the nucleus and endosymbionts have double-layered membranes, the outer membrane of the nucleus is continuous
88
Doolittle
with the membranous structure of the host cell, and only the inner membrane should ‘belong’ to the invader. In contrast, both membranes of mitochondria are attributed to the symbiont. Reasonably, then, the symbiont destined to become the mitochondrion was itself doublemembraned, as are its proteobacterial ancestors. The nucleus, on the other hand, if it really did begin as an endosymbiont, ought to be the relic of a single-membraned symbiont; either a Gram-positive eubacterium or an archaebacterium would fit the description. Ribosomal RNA comparisons early on led to the suggestion that the symbiont was an Archaeon [16]. If this were the case, however, it might be expected that the inner nuclear membrane would use ether-based lipids, something that is not observed. The case for there being two distinct endosymbiotic events on the lineage leading to all extant eucarya, the one giving rise to a DNAbased nucleus and the second giving rise to the mitochondrion, could largely explain the three groups of gene products found among the Eucarya (figure 2). First, there are those proteins vertically descended from the time of the first major pre-DNA bifurcation. These would include the principal cytoskeletal proteins, such as tubulin and actin [3]. Second, there are those gene products that may have been brought in by the hypothetical ancestor of the nucleus, including elongation factors and other proteins shared by Archaea and Eucarya, such as fibrillarin and ubiquitin. Finally, there are all those gene products brought in by the ancestor of the mitochondrion. This scenario could explain the three different phylogenies for different genes and allow us to count properly the individual lateral transfers discussed above.
5. What happened to the Archezoa? Cavalier-Smith [1] realized that if the organelles of eukaryotes arose by engulfment of prokaryotes, then perhaps some very early lineages of eukaryotes diverged before the engulfment and are still extant. Although he was fully aware that some extant eukaryotes lacking mitochondria had probably lost them, he tem-
Figure 2. Tree of life that accommodates some of the most often mentioned conundrums, including those shown in Kandler’s triangle (figure 1).
porarily assigned all amitochondriates to a new class called Archezoa, until such time as evidence supporting loss should become available. Alas, much evidence has been accumulated at this point, and there is now question as to whether any bona fide Archezoa remain. Much of the evidence has centered on the existence of genes for heat shock protein 70 and a chaperonin protein called cpn60 [15], both of which are generally characteristic of mitochondria and their forebearers among the a-proteobacteria.
6. Prospects and frustrations Are there relic organisms that could possibly support such a scenario? It is perhaps too much to hope that an RNA-based cellular organism still lurks in some exotic habitat on this Earth, and the community would be happy to settle for a genuine archezoan. For a while, hopes were pinned on the protist Pelomyxa palustris, an exotic amoeba that lacks mitochondria but which contains several obligate endosymbionts, including Bacteria that generate H2 and CO2 and Archaea that consume those byproducts and put out methane [13]. These hopes were dampened recently when it was reported that sequence analysis of the rRNA from Pelomyxa
Searching for the common ancestor
puts them high on the eukaryotic tree (L. Morin and J.-P. Mignot, 1996, unpublished findings cited in ref. [2]). Being desperate, we should wait to see the data before jettisoning all hope for Pelomyxa as a link to the past. Even if that report is upheld, other primitive eukaryotes may still be lingering somewhere on Earth that might illuminate the sequence of events. Roger Stanier liked to remind us that there is a metaphysical aspect to evolutionary speculation [17]. In this vein, as we look back on the 20 years since recombinant DNA took hold, many well-entrenched positions have had to be reversed in the light of newly reported facts. Vast amounts of sequence data notwithstanding, there are many things about early life on Earth that are not yet known.
References [1] Cavalier-Smith T., Endosymbiotic origin of the mitochondrial envelope, in: Schwemmler W., Schenk H.E.A. (Eds.), Endocytobiology II, DeGruyter, Berlin, 1983. [2] Cavalier-Smith T., A revised six-kingdom system of life, Biol. Rev. 73 (1998) 203–266. [3] Doolittle R.F., The origins and evolution of eukaryotic proteins, Phil. Trans. R. Soc. London B 349 (1995) 235–240. [4] Doolittle W.F., Phylogenetic classification and the universal tree, Science 284 (1999) 2124–2128. [5] Feng D.F., Cho G., Doolittle R.F., Determining divergence times with a protein clock: update and reevaluation, Proc. Natl. Acad. Sci. USA 94 (1998) 13028–13033.
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[6] Gupta R.S., Life’s third domain (Archaea): an established fact or an endangered paradigm?, Theor. Popul. Biol. 54 (1998) 91–104. [7] Hartman H., The origin of the eukaryotic cell, Speculations Sci. Technol. 7 (1984) 77–81. [8] Jain R., Rivera M.C., Lake J.A., Horizontal transfer among genomes: the complexity hypothesis, Proc. Natl. Acad. Sci. USA 96 (1999) 3801–3806. [9] Kandler O., Cell wall biochemistry and three-domain concept of life, Syst. Appl. Microbiol. 16 (1994) 501–509. [10] Kandler O., The early diversification of life, in: Bengston S. (Ed.), Early Life on Earth, Nobel Symposium No. 84, Columbia University Press, 1994. [11] Kandler O., Hoppe H., Lack of peptidoglycan in the cell walls of Methanosarcina barkeri, Arch. Microbiol. 113 (1977) 57–60. [12] Lake J.A., Rivera M.C., Was the nucleus the first endosymbiont?, Proc. Natl. Acad. Sci. USA 91 (1994) 2880–2881. [13] Lee J.L., Soldo A.T., Reisser W., Lee M.J., Jeon K.W., Gortz H.D., The extent of algal and bacteriological endosymbioses in protozoa, J. Protozool. 32 (1985) 391–403. [14] Poole A.M., Jeffares D.C., Penny D., The path from the RNA world, J. Mol. Evol. 46 (1999) 1–17. [15] Roger A.J., Svard S.G., Tovar J., Clark C.G., Smith M.W., Gillin F.D., Sogin M.L., A mitochondrial-like chaperonin 60 gene in Giardia lamblia: evidence that diplomonads once harbored an endosymbiont related to the progenitor of mitochondria, Proc. Natl. Acad. Sci. USA 95 (1998) 229–234. [16] Sogin M., Early evolution and the origin of eukaryotes, Curr. Opin. Genet. Dev. 1 (1991) 457–463. [17] Stanier R.Y., Some aspects of the biology of cells and their possible evolutionary significance, Symp. Soc. Gen. Microbiol. 20 (1970) 1–38. [18] Woese C., The universal ancestor, Proc. Natl. Acad. Sci. USA 95 (1998) 6854–6859. [19] Woese C.R., Fox G.E., Phylogenetic structure of the prokaryotic domain: the primary kingdoms, Proc. Natl. Acad. Sci. USA 75 (1977) 5088–5090. [20] Woese C., Kandler O., Wheelis M.L., Towards a natural system of organisms: proposals for the domains Archaea, Bacteria and Eucarya, Proc. Natl. Acad. Sci. USA 87 (1990) 4576–4579.