Understanding in an evolutionary context is not like understanding in a general (modern) biological context. It is nature, not the biologist, that does the evolutionary experiment, and essentially all such experiments have been done in the distant past and certainly cannot be repeated in the laboratory. Such limitations require that evolutionary understanding draw heavily upon inference and inspired conjecture. Therefore, if we are to reconstruct the history of life on this planet and understand the process by which it arose, theories capable of relating and interpreting the available facts in broad, meaningful ways, capable of defining and focusing scientific thinking on particular ideas, will have to be developed. In this concluding section, we try to do exactly this, i.e., to use the aminoacyl-tRNA synthetases to help construct a tentative picture of what the primary lineages are and what the evolutionary process that produced them is. The goal is to develop a clearer concept of how modern cells evolved and to identify various stages in that process.
Because of their modular nature (see above) (51
), the aminoacyl-tRNA synthetases readily undergo horizontal gene transfer. Also, because their functions are ancient and universal, these transfers are expected to be broad in scope (which they are), to occur throughout the recorded evolutionary course (which they appear to do), and to be largely selectively neutral in character (which is certainly the simplest explanation, for example, for the fact that the same organism can harbor genes for two very different but functionally equivalent subtypes of a given synthetase). Because the 20 charging enzymes are in essence alike in function (and presumably alike in their relationships to the cell as a whole), one would expect the horizontal gene transfer profiles of the 20 synthetases all to be similar in some general respect (although in their details, of course, each would be unique). However, this is clearly not the case: the horizontal gene transfer profiles of the different synthetases can be qualitatively different. Nevertheless, these profiles seem to be of several general types, which are crudely distinguished by the extent to which and ways in which they manifest the canonical pattern.
Evolutionary Significance of the Canonical Pattern
What is the significance of the canonical pattern; at what stage in the evolution of the cell did the canonical pattern arise? The horizontal gene transfer patterns of the aminoacyl-tRNA synthetases begin to provide answers here, but far too few data now exist to treat these answers as anything but theoretical conjectures requiring more thorough testing in the future.
It is essentially self-evident that, other factors being equal, most horizontal gene transfers will be taxonomically local (the donor and recipient would be close neighbors on the phylogenetic tree), for the simple reason that in such cases, horizontal displacement of an indigenous gene by a foreign equivalent would be minimally perturbing of cellular function, in that the indigenous and foreign genes are relatively similar in sequence. With the caveat that the more taxonomically local the horizontal gene replacement, the harder it is to recognize (the more data are needed), we nevertheless think that the data available at present suggest that horizontal gene transfers of the aminoacyl-tRNA synthetases are not predominantly local. The horizontal displacements that have occurred deep in the phylogenetic tree, involving transfers from one major taxon to another major taxon, appear to be unexpectedly prevalent.
The majority of transfers that introduce synthetases of the archaeal genre into the bacteria appear to involve a gene that arose deep in the common archaeal/eukaryotic lineage and transferred to the ancestor of some major bacterial taxon. These can only have been very ancient evolutionary events. Also, the fact that they appear to dominate the landscape of aminoacyl-tRNA relationships suggests that the dynamic of horizontal gene transfer has not remained constant over the evolutionary course covered by the universal phylogenetic tree.
We assume that the primary determinant of the nature of horizontal transfer is the nature of the recipient cell. If this is so, a dynamic of horizontal gene transfer that changes over the evolutionary course means that the nature of cells has changed over that course. Our theory, then, is that the deeper branching in the universal phylogenetic tree corresponds to evolutionary stages when the evolution of the cell was not yet complete, when the modern cell(s) had yet to emerge. In other words, the primary branchings in the phylogenetic tree (the canonical pattern) and the deep branchings in each of the domains involved primitive entities that were in the process of evolving to become modern cells. This would easily explain why the sequences of bacterial and archaeal aminoacyl-tRNA synthetases (different genres) differ so dramatically from one another while the sequences within each of the domains differ in relatively trivial ways (reference 68
offers a more detailed discussion of this matter).
The complex, highly refined cells of today obviously came from primitive counterparts that were far simpler and more rudimentary. Not only were the subsystems of the cell—the translation and transcription mechanisms, the genome and its replication apparatus, and so on—simpler and less accurate in their functions than today's versions thereof, but also the primitive cell as a whole was more loosely and less hierarchically organized and its states were fewer and less well defined (72
). Such simple systems, loosely and simply organized and defined, can undergo changes of a more dramatic sort than would be permissible in complex, highly integrated modern cells. Horizontal transfer of genes would be less disruptive of these primitive cells and their subsystems, and so the process of horizontal gene transfer would be of a very different nature, i.e., more pervasive, widespread, and spectacular, than it now is. Indeed, at very early times, horizontal gene transfer could have encompassed all aspects of the cell, all its genes (67
). The process would dominate the primitive evolutionary dynamic; most of the evolutionary innovation would be horizontally acquired. At such early evolutionary stages, life (no matter how varied at the level of the individual organism) can be looked at as communal in the evolutionary sense, for it is only through horizontally shared innovation that the evolution leading to modern life was possible; i.e., the community evolved as a whole, not as individual organismal lineages (69
; see also discussions of earlier forms of the idea in references 36
Gradually, then, as the subsystems of the cell over time became increasingly complex and refined and as they became more intricately interwoven into the evolving fabric of the cell, horizontal gene transfers would become increasingly restricted: foreign parts tend not to be compatible with complex, precisely defined machines. Of course, the first systems in the cell to become so refined, which probably were the information-processing mechanisms—in particular translation (72
)—would be the first to become refractory to horizontal gene transfer.
What the basal canonical pattern then represents is an early stage when the first (the more complex) subsystems of the cell became more or less refractory to horizontal gene transfer and the universal ancestor had differentiated into the communities that would spawn the primary organismal groupings. At that early time, the eukaryotic and archaeal lineages were still communally unified (at least in terms of the information processing systems) and all aspects of the cell had long evolutionary developments ahead of them.
The aminoacyl-tRNA synthetases that exhibit the canonical phylogenetic pattern are therefore those whose organismal taxonomic distributions became fixed at this early stage in cellular evolution. In their evolutionary profiles, these enzymes retain some record of the evolutions of the three basic cell types. Conversely, the aminoacyl-tRNA synthetases whose evolutionary profiles show little or no canonical pattern were still in evolutionary flux at this early stage; their organismal distributions would not stabilize until much later in the evolutionary course, after modern cells had evolved in some cases. The last synthetases to achieve their present taxonomic distribution, we think, are the members of the gemini group, to which we now return our attention.
The gemini group exemplifies the evolution of the amino acid charging systems. Multiple ways of associating amino acids with their tRNAs have always existed—as the existence of the two main synthetase structural classes implies. A considerable body of experimental data now shows that structures derived from RNA alone can both chemically discriminate between amino acids and catalyze aminoacylation with a high degree of substrate specificity (77
). This ability of RNA to mimic some of the essential characteristics of contemporary aminoacyl-tRNA synthetases strongly supports older ideas that direct interactions between nucleic acids and amino acids (or peptides) contributed to the origin of translation (25
The evolutionary field is strewn with the relics of apparent takeover (replacement) battles among the tRNA-charging systems. Long before the universal ancestor gave rise to the primary organismal groupings, the ancestor of the LeuRS, IleRS, and ValRS, say, spawned some variant that came to displace earlier versions of the leucine, isoleucine, and valine-charging systems. We know these to be early events because the LeuRS, IleRS, and ValRS evolutionary profiles retain semblances of the canonical pattern. At that early time or before, the two unrelated enzymes of the glycine-charging system as well as the two lysine-charging enzyme also existed, but from their evolutionary profiles (i.e., from the lack of canonical pattern shown), it would appear that the takeover battles in these cases continued to some extent into the modern evolutionary era.
Serine and cysteine represent takeovers in which one of the two systems has achieved almost complete dominance. There seems to have been an archaeal type for both systems, quite different from the corresponding bacterial type. However, these archaeal types have now largely been displaced by their bacterial counterparts. The lack of canonical pattern in these cases is prima facie evidence that the spread of the bacterial types occurred late in the evolutionary course, probably after the major branchings in each of the primary organismal groupings had begun to form.
The final two aminoacyl-tRNA synthetases in the gemini group, glutamine and asparagine, add a solid time point to the developing picture of the evolutionary course. For each of these amino acids, the aminoacyl-tRNA synthetase has arisen from within the cluster defined by the synthetases of the corresponding dicarboxylic acid, and for both, the ancestral source has been a GluRS or AspRS of the archaeal/eukaryotic genre. (As seen above, the GluRS and AspRS enzymes exhibit strongly canonical pattern in their own right; i.e., they are ancient in both origin and taxonomic distribution.)
GlnRS arises specifically from the eukaryotic GluRS lineage (see the glutamine section above). Although AsnRS shows clear affinity with the archaeal and eukaryotic AspRSs (to the exclusion of bacterial AsnRSs), no convincing specific affinity to either the eukaryotic or archaeal AspRS is evident. This could be explained by the AsnRS arising somewhat prior to the GlnRS, which is also consistent with the wider taxonomic distribution of the former. Given its ancestry, the modern GlnRS, unlike its parental GluRS, had to have come into being after the three organismal domains themselves arose; the enzyme achieved its taxonomic distribution subsequently. We think that the lack of canonical pattern shown by all synthetases in the gemini group has a similar explanation, i.e., that all their taxonomic distributions, like that of GlnRS, were established relatively late in the evolutionary course, well after the domains themselves had arisen and their major branchings had begun to coalesce.
Some General Evolutionary Matters
It is one thing to note that certain bacterial taxa contain one or more aminoacyl-tRNA synthetases of the archaeal genre or that there are two very dissimilar types of a bacterial synthetase that are nevertheless of the same genre; e.g., TyrRS. It is another to understand how these came to be. In some cases, the source of the enzyme can be satisfactorily localized. Several examples exist (see above) in which the bacterial enzyme is not only of the archaeal genre but distinctly Pyrococcus
-like. Such localizations will refine with the sequencing of additional archaeal genomes, just as will the precise source of the (plasmid-borne) mupirocin-resistant IleRS recently acquired by some Staphylococcus
) (currently localized phylogenetically only to the general vicinity of the major gram-positive bacterial taxon represented by C. acetobutylicum
[see the isoleucine section above]).
However, in a number of cases a bacterial synthetase is obviously of the general archaeal genre but resembles neither the archaeal nor the eukaryotic version specifically. This immediately raises a nontrivial question about the source of the synthetase. The same general question arises in the context of the bacteria alone when two very disparate versions of a synthetase exist, both bacterial in genre. The reflex answer to such questions, of course, is that undiscovered deeply branching lineages in either the Archaea or the Bacteria are the sources. Such explanations serve only to trivialize the questions. What are these hypothetical lineages? In the archaeal cases, some of them would need to branch not from the archaeal stem itself but from the common stem shared by the archaea and eukaryotes, in which case they would represent undiscovered organismal domains! Obviously, microbiologists have yet to uncover a great deal of the diversity in the microbial world, but the idea that in this day and age they have missed entire domains stretches credulity somewhat.
A more satisfying explanation may lie in the classical evolutionists' concept of evolutionary radiations, which again involves the tempo-mode relationship (55
). The fossil record holds key evidence that otherwise would not be available, i.e., evidence of extinct lineages. In the world of macroscopic organisms, an evolution radiation (which is a period of rapid evolution) has three important characteristics: (i) accelerated evolutionary tempo, (ii) remarkable and remarkably diverse evolutionary invention, and (iii) creation of new lineages, most of which are short-lived (on the evolutionary timescale).
This last characteristic offers a conceptually challenging explanation for the point under discussion: the evolutions that underlay the development of the Bacteria and the Archaea (and Eucarya) were not the simple straightforward courses one might naively infer from the ancestral stems on phylogenetic trees. Rather, these ostensibly bare linear stems actually correspond to periods of great evolutionary turmoil, invention, and radiation. However, the evidence for almost all of this is gone (especially on the organismal level); the lineages are extinct. However, what the aminoacyl-tRNA synthetases may be telling us is that not all traces of extinct lineages are necessarily erased. Horizontal gene transfer makes it possible for some of the genes, some of the evolutionary inventions that occurred in these extinct lineages, to survive today, preserved by their transfer to lineages that have persisted.
As mentioned above, the horizontal transfer of aminoacyl-tRNA synthetase genes between Archaea and Bacteria is asymmetric in the sense that transfers of archaeal-type synthetases into the bacteria are common, both early on and after the organismal domains coalesced (and their major taxa had begun to emerge). Transfers of bacterial synthetases into the Archaea appear not to have occurred (at least early on [see the discussion of the gemini group above]). This asymmetry has no satisfactory explanation right now. It is not because archaea are refractory to the transfer of aminoacyl-tRNA synthetase (or any other) genes. We have seen ample evidence above that intra-archaeal exchanges occur. The explanation could be that the archaeal cell type and the bacterial cell type are of different natures, the one more permissive as regards accepting foreign genetic material than the other. Alternatively, the archaeal and bacterial cell types may not have matured evolutionarily (become modern cells) at the same time, so that transfer of genes occurred from a relatively mature archaeal type of cell to a relatively immature bacterial type of cell, which would therefore be more receptive to foreign genetic material.
It is not intended that the reader accept the above hypothetical scheme as truth; nor do we wish him or her to view it as idle and so useless speculation. The ideas we have put forth in this section are simply part of our attempt to paint a picture of the course of cellular evolution and to generate a theory that has the consistency, explanatory power, and conceptual power to inspire and inform studies of deep evolutionary questions.