Thomas Cavalier-Smith

“ Thus, using Kuhnian language, we could call the shift, which I hope is underway, of considering the root to be between neomura and eubacteria to accepting it among negibacteria a paradigm shift, because it affects our perspectives in so many ways and also meets the usual intense resistance associated with such a major shift. But the proper way to effect such a shift is not philosophic discussion but by evaluating the evidence critically and in detail. In evolutionary biology both refutation and confirmation have a role. ”

“ The greater simplicity of the chlorobacterial OM, with neither lipopolysaccharides nor apparently an Omp85 protein-targeting mechanism, is what one expects of the earliest negibacteria, and is unlikely to have been caused by secondary loss of Omp85 alone while retaining an OM; there is no evidence that such loss has ever occurred in the whole history of life. Although the unique absence of a well-conserved homologue of Omp85 from the OM of Chlorobacteria is strong evidence that they are the most ancient negibacteria, it does not by itself prove that they are the most ancient eubacteria of all. ”

“ In essence, we are caught between the Scylla of strongly systematically-biased molecules that give the wrong root with high confidence and the Charybdis of less-biased, weakly-resolving molecules that give the right and several wrong versions of the root with too little support to distinguish them [ [1] ; transition analysis, if critically applied, can pilot us into safer waters. Although it may not give the absolute certainty that some crave, it can allow us to reconstruct the past history of life with much higher confidence than anyone would have dreamed of a few decades ago. ”

“ Under the evolutionary scheme proposed by Cavalier-Smith, bacteria are a kingdom and eukaryotes are nested within bacteria. Yet, instead of being considered a single phylum, as "archaebacteria" are, eukaryotes are divided in six different kingdoms. How can a taxonomic category contain sub-elements that are ranked at the same initial level? I understand that eukaryotes have a complex morphology and features. However, archaea do possess very different features too. ”

“ To do that we must also exclude the root from all Posibacteria; proteasome evolution excludes it only from Actinomycetales and neomura, and thus left open the possibility that it might be beside or within Endobacteria (or even among early actinobacteria) . As argued above, negibacterial envelope biogenesis is so complex as to polarise the transition from negibacteria, not the reverse, and thus exclude the root from all unibacteria. My new arguments concerning flagella evolution also clearly place the root within negibacteria, specifically among Glidobacteria. ”

“ Transition analysis, if imaginative and critical, often clearly polarizes change unambiguously in the complete absence of a fossil record. Fossils are static and discontinuous; they do not show transitions or continuity directly and can be interpreted properly only by critical transition analysis, which is therefore the most fundamental way to polarize the direction of evolutionary change. For vertebrate evolution the fossil record is a valuable extra, inessential benefit. It is important to note that not all transitions can be clearly polarized when studied individually. ”

“ Rejecting any of the names eubacteria, prokaryotes or bacteria just because these groups are paraphyletic would be extremely stupid and very harmful to our ability to communicate sensibly about evolution and cell diversity. ”

“ I hope that I have convinced the reader that the root should be on the branch from Chloroflexus to the rest of eubacteria on both the protochlorophyllide and chlorophyllide reductase subtrees. Yet both put it between the two green bacterial phyla with chlorosomes and proteobacteria. In the past this has been misinterpreted as evidence for lateral gene transfer between the green bacteria. It is not. It is just a bad tree, dimensionally hugely distorted by quantum evolution and misrooted by paralogue rooting that is necessarily misled by gross long-branch artefacts. ”

“ Genes can survive only by coding for real physical structures of interacting macromolecules, whose evolution can and must be studied by transition analysis of their phenotypes, not just of their gene sequences. ”

“ It is a mistake to think that sequence trees are the only way of testing phylogenetic history. Nonetheless, they are important. Several features of the universal tree presented here, especially the relationships among eurybacteria, should be tested by taxonomically much more extensive gene sequencing and phylogenetic analysis of scores of the most conservative genes (with proper controls against lateral gene transfer) using methods best adapted to avoiding those systematic biases that can make it increasingly likely that you will get the wrong tree with high confidence as more genes are added. ”

“ I am not referring to poorly shaped progenotes there, as your last common ancestor seemed itself fairly evolved. In fact, could an organism such as the last ancestor of all life, "a non-flagellate, non-spore-forming, eubacterium with acyl-ester membrane lipids, a murein peptidoglycan wall, and fully developed eubacterial molecular biology and cell division mechanisms"p.101, have really been alone on Earth, immune to any character exchanges with contemporaries? If not, why would we like to trace back all the living beings characters to such a single root? ”

“ It is the subset of changes that have a sufficient degree of complexity to allow unambiguous polarization that are of key importance for rooting trees. The key question that decides the utility of a particular character for this purpose is whether its evolution has enough evidence of directionality, which may be inherent in the process of evolutionary change itself or deducible by comparison between an evolved state and its putative precursors and knowledge of their phylogenetic distribution. Without evidence of directionality a character cannot be used clearly to polarize the tree. ”

“ Despite great advances in clarifying the family tree of life, it is still not agreed where its root is or what properties the most ancient cells possessed – the most difficult problems in phylogeny. Protein paralogue trees can theoretically place the root, but are contradictory because of tree-reconstruction artefacts or poor resolution; ribosome-related and DNA-handling enzymes suggested one between neomura (eukaryotes plus archaebacteria) and eubacteria, whereas metabolic enzymes often place it within eubacteria but in contradictory places. ”

“ Contrary to molecular pessimism that neglects organismal properties of bacteria [173] , one can plant the tree of life; in my view the arguments in this and earlier papers [ [1] , [5] , [174] together unambiguously establish that the universal root is within negibacteria, and that it is most probably among the non-flagellate, photosynthetic, gliding negibacteria, and most likely of all among or beside the chlorobacteria. For understanding their evolution, bacteria (=prokaryotes) must be thought of as organisms [ [1] , not disembodied genes [ [173] . ”

“ The greater simplicity of the type III system stimulated the suggestion that it might be ancestral to flagella; as the system is also present in a few free-living bacteria it could be more ancient than eukaryotes, against which the best studied examples are all directed, so that suggestion is not totally implausible. If flagella did evolve from type III secretion injectors, it would place the root of the whole tree of life within Exoflagellata, as only they are known to possess it. ”

“ How did you rule out – if you did – that having a simpler molecular organization could not actually correspond to a refinement, an optimization going away from a more complex/heavy structure? If there is no reason that there should be a unique tendency in evolution (i.e. toward complexification all along the TOL from the root) , should we expect that living beings would experiment different adaptations?
In the case of the HslV, which selective advantages were brought in by the addition of alpha and beta subunits, each with partitioned functions only? ”

“ Thus loss played a much bigger role than innovation in the origin of archaebacteria. In terms of major innovations of protein structure, as well as in cell structure and cell biology, the dichotomy between prokaryotes and eukaryotes is immensely more important than that between eubacteria and archaebacteria. Ribosomal and DNA-handling proteins underwent episodic accelerated evolution during the neomuran revolution, as did rRNA [ [1] , giving the impression on sequence trees that archaebacteria are much more distant from eubacteria than the vast majority of proteins indicates to be the case. ”

“ You cannot and should not attempt to establish clades a priori. But seeking independent evidence for relationships is desirable. But the key polarizations are independent of the uncertainties you mention. Thus, for example, the logic of polarization from negibacteria to posibacteria is independent of whether Posibacteria are paraphyletic or polyphyletic: my arguments show that neither endobacteria nor actinobacteria can be ancestral to negibacteria as both have the same type of flagellum and neither has either of the two probable precursors of flagella. ”

“ Fourthly, single-gene trees are always unrooted, lacking inherent evidence of direction; any nucleotide can substitute reversibly for any other. These severe limitations of sequence trees emphatically do not mean that they are worthless. On the contrary, they are indispensable, but they must be interpreted critically and supplemented by cladistic, transition analysis and congruence testing, and by critical palaeontology, in order to produce a reliable and comprehensive picture. ”

“ The fact that his genomic evidence does not even suggest a eukaryote relationship with archaebacteria, which all biologists accept, proves the severe limitations of such crude 'genome-wide' criteria, unless critically interpreted by explicit evolutionary reasoning such as transition analysis of key characters. Without this genomics is often unilluminating and sometimes positively misleading; it risks becoming a substitute for critical thought. It is essential to recognize that molecular evolution is grossly non-uniform across genomes, lineages, and time. ”

“ A recent unduly pessimistic discussion of the logical problems involved in rooting the tree of life [187] suggested that "'transition analysis' of the structural evolution of the cytoplasmic membrane might be helpful"; I have shown here that evolution of the OM is more helpful. However, transition analysis of a single character is insufficient, for transition analysis does not place the root in one fell swoop as protein paralogue trees were mistakenly thought to. It provides a systematic step-by-step approach. ”

“ Let me summarize: proteasome evolution excludes the root from the proteate clade; cell envelope evolution and flagella evolution independently of each other exclude it from Posibacteria; eubacterial flagellar evolution also excludes it from Gracilicutes and Eurybacteria, and places it among the non-flagellate eubacteria that have gliding motility by slime secretion from junctional pore complexes (i.e. Glidobacteria) . Later sections will present two further transition analyses that narrow down the position of the root still further within the Glidobacteria. ”

“ Search for congruence among multiple lines of evidence – the more diverse the better and resolving apparent contradictions by weighing up the evidence is not special to evolutionary biology but fundamental to all science. ”

“ The tree of life is an organismal phylogeny. All cellular inheritance in prokaryotes is vertical, not horizontal, as cell fusion never occurs except occasionally in Streptomyces. It is meaningful to ask whether the last common ancestor of life had eubacterial or archaebacterial membrane lipids, whether it had one or two bounding membranes, whether it had flagella or not, whether it was photosynthetic or not. ”

“ The theoretical base enounced at the beginning of the manuscript to tackle the directionality of evolutionary change and suggest a position for the root of the tree of life appears most reasonable. It proposes a combination, or rather an interpretation, of gene phylogenies with cladistics, transition analysis, congruence testing and critical palaeontology. If rigorously applied, this strategy should provide the best approximation that scientists can dream of to the history of life. ”

“ Saying that polarizing phylogeny is 'incredibly difficult' is an exaggeration, but not one that in any way contradicts my assertion that positioning the root and establishing the properties of the most ancient cells is the most difficult phylogenetic problem. ”

“ Before discovering it I seriously considered that the root might be within Posibacteria as you (like Gupta earlier) suggest. However, I find it hard to believe that the similarities of the distal domain of TonB and OmpA can be convergent or that this apparently chimaeric molecule TonB could have first evolved in a posibacterium with no OM and happened by chance to have a distal region perfectly preadapted to be the ancestor of all OM β-barrel proteins that now function only in a membrane that had not yet evolved. Thus I cannot see how evolution could have gone in the opposite direction. ”

“ Before starting my detailed review of this paper, I want to express my personal esteem for T. Cavalier-Smith repeated contributions to the field of evolutionary biology. All along the years, he has often put forward important ideas and original hypotheses that have prompted many debates about the evolution of life, all domains included. Multiple tests of his works have taught us that even if T. Cavalier-Smith's claims were not necessarily right, his thought provocative publications always deserved the consideration of the scientific community. ”

“ Study of the biogenesis of their outer membrane is particularly important for understanding early cell evolution and for testing whether it might be derived by simplification from that of other negibacteria, rather than the primitive state as argued here. Characterizing the many lineages known only from environmental rRNA sequences, establishing the full metabolic diversity of the group, and extensive genomic and evolutionary studies to determine whether it is holophyletic or paraphyletic are all essential for finally pinpointing the root of the tree of life. ”

“ Proteasome, flagella, murein wall, and outer membrane evolution now strongly corroborate this. They show that posibacteria, actinomycetes, and neomura all arose after the primary radiation of life, probably all substantially later. In my view the widespread assumption that archaebacteria are among the most ancient life forms [ [19] is profoundly mistaken [ [1] , [127] .
A root within eubacteria offers a simple perspective on the pattern of protein fold superfamilies across the three domains [117] . ”