The polyphyletic origins of glycyl-tRNA synthetase and lysyl-tRNA synthetase and their implications.

I analyzed the polyphyletic origin of glycyl-tRNA synthetase (GlyRS) and lysyl-tRNA synthetase (LysRS), making plausible the following implications. The fact that the genetic code needed to evolve aminoacyl-tRNA synthetases (ARSs) only very late would be in perfect agreement with a late origin, in the main phyletic lineages, of both GlyRS and LysRS. Indeed, as suggested by the coevolution theory, since the genetic code was structured by biosynthetic relationships between amino acids and as these occurred on tRNA-like molecules which were evidently already loaded with amino acids during its structuring, this made possible a late origin of ARSs. All this corroborates the coevolution theory of the origin of the genetic code to the detriment of theories which would instead predict an early intervention of the action of ARSs in organizing the genetic code. Furthermore, the assembly of the GlyRS and LysRS protein domains in main phyletic lineages is itself at least evidence of the possibility that ancestral genes were assembled using pieces of genetic material that coded these protein domains. This is in accordance with the exon theory of genes which postulates that ancestral exons coded for protein domains or modules that were assembled to form the first genes. This theory is exemplified precisely in the evolution of both GlyRS and LysRS which occurred through the assembly of protein domains in the main phyletic lineages, as analyzed here. Furthermore, this late assembly of protein domains of these proteins into the two main phyletic lineages, i.e. a polyphyletic origin of both GlyRS and LysRS, appears to corroborate the progenote evolutionary stage for both LUCA and at least the first part of the evolutionary stages of the ancestor of bacteria and that of archaea. Indeed, this polyphyletic origin would imply that the genetic code was still evolving because at least two ARSs, i.e. proteins that make the genetic code possible today, were still evolving. This would imply that the evolutionary stages involved were characterized not by cells but by protocells, that is, by progenotes because this is precisely the definition of a progenote. This conclusion would be strengthened by the observation that both GlyRS and LysRS originating in the phyletic lineages leading to bacteria and archaea, would demonstrate that, more generally, proteins were most likely still in rapid and progressive evolution. Namely, a polyphyletic origin of proteins which would qualify at least the initial phase of the evolutionary stage of the ancestor of bacteria and that of archaea as stages belonging to the progenote.

Theories of the origin of the genetic code: Strong corroboration for the coevolution theory.

I analyzed all the theories and models of the origin of the genetic code, and over the years, I have considered the main suggestions that could explain this origin. The conclusion of this analysis is that the coevolution theory of the origin of the genetic code is the theory that best captures the majority of observations concerning the organization of the genetic code. In other words, the biosynthetic relationships between amino acids would have heavily influenced the origin of the organization of the genetic code, as supported by the coevolution theory. Instead, the presence in the genetic code of physicochemical properties of amino acids, which have also been linked to the physicochemical properties of anticodons or codons or bases by stereochemical and physicochemical theories, would simply be the result of natural selection. More explicitly, I maintain that these correlations between codons, anticodons or bases and amino acids are in fact the result not of a real correlation between amino acids and codons, for example, but are only the effect of the intervention of natural selection. Specifically, in the genetic code table we expect, for example, that the most similar codons - that is, those that differ by only one base - will have more similar physicochemical properties. Therefore, the 64 codons of the genetic code table ordered in a certain way would also represent an ordering of some of their physicochemical properties. Now, a study aimed at clarifying which physicochemical property of amino acids has influenced the allocation of amino acids in the genetic code has established that the partition energy of amino acids has played a role decisive in this. Indeed, under some conditions, the genetic code was found to be approximately 98% optimized on its columns. In this same work, it was shown that this was most likely the result of the action of natural selection. If natural selection had truly allocated the amino acids in the genetic code in such a way that similar amino acids also have similar codons - this, not through a mechanism of physicochemical interaction between, for example, codons and amino acids - then it might turn out that even different physicochemical properties of codons (or anticodons or bases) show some correlation with the physicochemical properties of amino acids, simply because the partition energy of amino acids is correlated with other physicochemical properties of amino acids. It is very likely that this would inevitably lead to a correlation between codons (or anticodons or bases) and amino acids. In other words, since the codons (anticodons or bases) are ordered in the genetic code, that is to say, some of their physicochemical properties should also be ordered by a similar order, and given that the amino acids would also appear to have been ordered in the genetic code by selection natural, then it should inevitably turn out that there is a correlation between, for example, the hydrophobicity of anticodons and that of amino acids. Instead, the intervention of natural selection in organizing the genetic code would appear to be highly compatible with the main mechanism of structuring the genetic code as supported by the coevolution theory. This would make the coevolution theory the only plausible explanation for the origin of the genetic code.

The time of appearance of the genetic code.

I support the hypothesis that the origin of the genetic code occurred simultaneously with the evolution of cellularity. That is to say, I favour the hypothesis that the origin of the genetic code is a very, very late event in the history of life on Earth. I corroborate this hypothesis with observations favouring the progenote's stage for the Last Universal Common Ancestor (LUCA), for the ancestor of bacteria and that of archaea. Indeed, these progenotic stages would imply that - at that time - the origin of the genetic code was still ongoing simply because this origin would fall within the very definition of progenote. Therefore, if the evolution of cellularity had truly been coeval with the origin of the genetic code - at least in its terminal part - then this would favour theories such as the coevolution theory of the origin of the genetic code because this theory would postulate that this origin must have occurred in extremely complex protocellular conditions and not concerning stereochemical or physicochemical interactions having to do with other stages of the origin of life. In this sense, the coevolution theory would be corroborated while the stereochemical and physicochemical theories would be damaged. Therefore, the origin of the genetic code would be linked to the origin of the cell and not to the origin of life as sometimes asserted. Therefore, I will discuss the late hypothesis of the origin of the genetic code in the context of the theories proposed to explain this origin and more generally of its implications for the early evolution of life.

The absence of the evolutionary state of the Prokaryote would imply a polyphyletic origin of proteins and that LUCA, the ancestor of bacteria and that of archaea were progenotes.

I analysed the similarity gradient observed in protein families - of phylogenetically deep fundamental traits - of bacteria and archaea, ranging from cases such as the core of the DNA replication apparatus where there is no sequence similarity between the proteins involved, to cases in which, as in the translation initiation factors, only some proteins involved would be homologs, to cases such as for aminoacyl-tRNA synthetases in which most of the proteins involved would be homologs. This pattern of similarity between bacteria and archaea would seem to be a very clear indication of a transitional evolutionary stage that preceded both the Last Bacterial Common Ancestor and the Last Archaeal Common Ancestor, i.e. progenotic stages. Indeed, this similarity pattern would seem to exemplify an ongoing transition as all the evolutionary phases would be represented in it. Instead, in the cellular stage it is expected that these evolutionary phases should have already been overcome, i.e. completed, and therefore no longer detectable. In fact, if we had really been in the presence of the prokaryotic stage then we should not have observed this similarity pattern in proteins involved in defining the ancestral characters of bacteria and archaea, as the completion of the different cellular structures should have required a very low number of proteins to be late evolved in lineages leading to bacteria and archaea. Indeed, the already reached state of the Prokaryote would have determined complete cellular structures therefore a total absence of proteins to evolve independently in the two main phyletic lineages and able to complete the evolution of a particular character already evidently in a definitive state, which, on the other hand, does not appear to have been the case. All this would have prevented the formation of this pattern of similarity which instead would appear to be real. In conclusion, the existence of this pattern of similarity observed in the families of homologous proteins of bacteria and archaea would imply the absence of the evolutionary stage of the Prokaryote and consequently a progenotic status to be assigned to the LUCA. Indeed, the LUCA stage would have been a stage of evolutionary transition because it is belatedly marked by the presence of all the different evolutionary phases, evidently more easily interpretable within the definition of progenote than that of genote precisely because they are inherent in an evolutionary transition and not to an evolution that has already been achieved. Finally, I discuss the importance of these arguments for the polyphyletic origin of proteins.

The error minimization of the genetic code would have been determined by natural selection and not by a neutral evolution.

I discuss the mechanisms by which the error minimization observed in the genetic code would have been produced; that is, the ability of the genetic code to buffer, for example, the deleterious effects of translation errors. Here, I analyse whether the error minimization was produced by the intervention of natural selection or whether it is an emergent, that is, neutral property; in other words, whether it is a by-product of another mechanism that was structuring the genetic code. In particular, I criticize Massey's simulations (2008) - favouring the neutral hypothesis - which, containing elements of natural selection, would render his conclusions at least partly tautological. Furthermore, I criticize some of Koonin's (2017) interpretations regarding Massey's simulations. Finally, I criticize the opinion of Janzen et al. (2022) according to which their self-aminoacylating ribozyme system would have been capable of generating an error minimization of the genetic code as its emergent property. That is to say, I criticize, more generally, a neutral origin of error minimization. Indeed, any mechanism for structuring the genetic code would be capable of generating, in theory, such an emergent property. The problem is that to demonstrate this, it would be necessary to show that the level of optimization achieved by the genetic code would be that expected under the neutral hypothesis, the one that Janzen et al. (2022) instead they did not make. Therefore, their view is only a hypothesis and is very far from being corroborated by their results. Instead, in the literature there is a strong evidence that the level of optimization achieved by the genetic code is so high that it would imply, per se, an intervention of natural selection in the origin of error minimization of the genetic code. On the other hand, this level of optimization would be very far from what might have been produced by a neutral process.

The origins of the cell membrane, the progenote, and the universal ancestor (LUCA).

I analyse the consequences that the diversity of cell membranes present in bacteria and archaea would have for the stage reached by the evolution of cellularity in the Last Universal Common Ancestor (LUCA). Regardless of how the bacterial and archaeal membranes were distributed in the LUCA, the conclusion that would seem to emerge is always the same. That is to say, it is more likely that LUCA was a progenote rather than a genote. Indeed, if LUCA hosted both types of membranes then this would have been a transitional condition which would imply, in itself, that LUCA was a progenote. In other words, the two types of membranes evidently had to segregate in that of bacteria and that of archaea, and this segregation would imply enormous changes not expected for a cellular stage but the norm for that of progenote. Instead, in the case in which LUCA hosted only one type of membrane, for example that of archaea, then the evolution of the other type of membrane - presumably in the ancestor of bacteria - would imply a progenotic LUCA precisely because such a late origin would seem be an expression, at that time, of a still rapid and progressive evolution because it involves a fundamental and profound genetic trait, and should therefore reflect the progenotic stage. Finally, in the event that LUCA had been devoid of a membrane, then the late origin of the two types of membranes - in the ancestor of archaea and in that of bacteria - would seem to be a clear expression of a rapid and progressive evolution typical of progenote. Indeed, this would imply, for example, that all mechanisms and functions associated with the two cell membranes still had to evolve which would express the progenotic stage precisely because it would seem to be an evolution still in progress, and therefore typical of the stage of progenote. Therefore, LUCA was most likely a progenote also because there would be no strong arguments in favour of the hypothesis that LUCA had instead reached the evolutionary stage of cellularity.

Arguments against the stereochemical theory of the origin of the genetic code.

I support the hypothesis that stereochemical theory is unnatural because it is based on artificial and not simple mechanisms as required for a good theory. Indeed, for stereochemical theory the origin of the genetic code requires, in the first place, a primary interaction, for example, between a codon and an amino acid on a proto-tRNA. But this interaction is a necessary but not sufficient condition, because the evolution of the mRNA molecule, which would really define the genetic code, is still necessary for the complete origin of the genetic code. In other words, the need for two molecules, tRNA and mRNA, to define the genetic code, with their at least partial independence would testify to an artificial mechanism typical of stereochemical theory because it would not guarantee that amino acid-codon (or -anticodon) assignments realized in the first phase of the origin of the genetic code, would necessarily be maintained also in the second phase of its completion. Furthermore, the genetic code encodes for amino acids but amino acids are not the truly functional aspect, they are only intermediaries, of their final products, proteins, which are the only true entities actually coded by genes. Therefore, it would not be immediately clear from the point of view of stereochemical theory, to say why it is the amino acids and not the proteins that are involved in the primary stereochemical interactions that would have led to the origin of the genetic code. Hence, at least some of the stereochemical theory models would be not very credible, not being able to say much about the coding of proteins by genes. Finally, I inspected the genetic code table following the logic that more closely similar amino acids should - according to stereochemical theory - be coded by highly similar codons, finding that only a few pairs of amino acids actually satisfy this logic, further discretizing the stereochemical theory.

The RNase P, LUCA, the ancestors of the life domains, the progenote, and the tree of life.

I have tried to interpret the phylogenetic distribution of the RNase P with the aim of helping to clarify the stage reached by the evolution of cellularity in the Last Universal Common Ancestor (LUCA); that is to say, if the evolutionary stage of the LUCA was represented by a protocell (progenote) or by a complete cell (genote). Since there are several arguments that lead one to believe that only the RNA moiety of the RNase P was present in the LUCA, this might imply that this evolutionary stage was actually the RNA world. If true this would imply that the LUCA was a progenote because the RNA world being a world subject to multiple evolutionary transitions that would involve a high noise at many its levels, which would fall within the definition of the progenote. Furthermore, since RNA-mediated catalysis is much less efficient than protein-mediated catalysis, then the only RNA moiety that was present in the LUCA could imply - by per se, without invoking the existence of the RNA world - that the LUCA was a progenote because an inefficient catalysis might have characterized this evolutionary stage. This evolutionary stage would still fall under the definition of the progenote. In addition, the observation that the protein moieties of the RNase P of bacteria and archaea are not-homologs would imply that these originated independently in the two main phyletic lineages. In turn, this would imply the progenotic nature of the ancestors of both archaea and bacteria. Indeed, it is admissible that such a late origin - in the main phyletic lineages - of the protein moieties of the RNase P is witness to an evolutionary transition towards a more efficient catalysis, evidently made clear precisely by the evolution of the protein moieties of the RNase P which would have helped the RNA of the RNase P to a more efficient catalysis. Hence, this would date that evolutionary moment as a transition to a much more efficient catalysis and consequently would imply which in that evolutionary stage there was the actual transition from the progenotic to genotic status. Finally, this late origin of the RNase P protein moieties in the bacterial and archaeal domains per se could imply the presence of a progenotic stage for their ancestors, or at least that a cell stage would have been much less likely. In fact, it is true that genes can originate both in a cellular and in a progenotic stage, but they mainly typify the latter because they are, by definition, in formation. Then it is expected that in the evolutionary stage of the formation of the main phyletic lineages - that is to say, in an evolutionary time in which the formation of genes might be expected - that the origin of proteins is to be related to a rapid and progressive evolution typical of the progenote precisely because in such an evolutionary stage the origin of genes is more easily and simply explained as reflecting a progenotic rather than a genotic stage. Indeed, if instead the evolutionary stage of the ancestors of bacteria and archaea had been the cellular one, then observing the origin of the protein moieties of the RNase P would have been, to some extent, anomalous because this completion should have already occurred, simply because the transformation of a ribozyme into an enzyme should have already taken place precisely because it falls within the very definition of the cellular status. The conclusion is that both the LUCA and the ancestor of archaea and that of bacteria may have been progenotes. If these arguments were true then either the tree of life as commonly understood would not exist and therefore the main phyletic lineages would have originated directly from the LUCA, or there would have been at least two different populations of progenotes that would have finally defined the domain of bacteria and that of archaea.

The genetic code is very close to a global optimum in a model of its origin taking into account both the partition energy of amino acids and their biosynthetic relationships.

We used the Moran's I index of global spatial autocorrelation with the aim of studying the distribution of the physicochemical or biological properties of amino acids within the genetic code table. First, using this index we are able to identify the amino acid property - among the 530 analyzed - that best correlates with the organization of the genetic code in the set of amino acid permutation codes. Considering, then, a model suggested by the coevolution theory of the genetic code origin - which in addition to the biosynthetic relationships between amino acids took into account also their physicochemical properties - we investigated the level of optimization achieved by these properties either on the entire genetic code table, or only on its columns or only on its rows. Specifically, we estimated the optimization achieved in the restricted set of amino acid permutation codes subject to the constraints derived from the biosynthetic classes of amino acids, in which we identify the most optimized amino acid property among all those present in the database. Unlike what has been claimed in the literature, it would appear that it was not the polarity of amino acids that structured the genetic code, but that it could have been their partition energy instead. In actual fact, it would seem to reach an optimization level of about 96% on the whole table of the genetic code and 98% on its columns. Given that this result has been obtained for amino acid permutation codes subject to biosynthetic constraints, that is to say, for a model of the genetic code consistent with the coevolution theory, we should consider the following conclusions reasonable. (i) The coevolution theory might be corroborated by these observations because the model used referred to the biosynthetic relationships between amino acids, which are suggested by this theory as having been fundamental in structuring the genetic code. (ii) The very high optimization on the columns of the genetic code would not only be compatible but would further corroborate the coevolution theory because this suggests that, as the genetic code was structured along its rows by the biosynthetic relationships of amino acids, on its columns strong selective pressure might have been put in place to minimize, for example, the deleterious effects of translation errors. (iii) The finding that partition energy could be the most optimized property of amino acids in the genetic code would in turn be consistent with one of the main predictions of the coevolution theory. Since the partition energy is reflective of the protein structure and therefore of the enzymatic catalysis, the latter might really have been the main selective pressure that would have promoted the origin of the genetic code. Indeed, we observe that the ß-strands show an optimization percentage of 95.45%; so it is possible to hypothesize that they might have become the object of selection during the origin of the genetic code, conditioning the choice of biosynthetic relationships between amino acids. (iv) The finding that the polarity of amino acids is less optimized than their partition energy in the genetic code table might be interpreted against the physicochemical theories of the origin of the genetic code because these would suggest, for example, that a very high optimization of the polarity of amino acids in the code could be an expression of interactions between amino acids and codons or anticodons, which would have promoted its origin. This might now become less sustainable, given the very high optimization that is instead observed in favor of the partition energy but not polarity. Finally, (v) the very high optimization of the partition energy of amino acids would seem to make a neutral origin of error minimization, i.e. of the ability of the genetic code to buffer, for example, the deleterious effects of translation errors, very unlikely. Indeed, an optimization of about 100% would seem that it might not have been achieved by a simple neutral process, but this ability should probably have been generated instead by the intervention of natural selection. In actual fact, we show that the neutral theory of the origin of error minimization has been falsified for the model analyzed here. Therefore, we will discuss our observations within the theories proposed to explain the origin of the organization of the genetic code, reaching the conclusion that the coevolution theory is the most strongly corroborated theory.

The phylogenetic distribution of the cell division system would not imply a cellular LUCA but a progenotic LUCA.

The stage reached by the evolution of cellularity in the Last Universal Common Ancestor (LUCA) has not yet been identified. In actual fact, it has not been clarified whether the LUCA was a cell (genote) or a protocell (progenote). Recently, Pende et al. (2021) analysed the phylogenetic distribution of the cell division system present in bacteria and archaea reaching the conclusion that LUCA was a cell and not a progenote. I find this conclusion unreasonable with respect to the observations they presented. One of the points is that the presence in the domains of life of many genes - some paralogs - which would define the membrane-remodeling superfamily would seem to imply a tempo and a mode of evolution for the LUCA more typical of the progenote than the genote. Indeed, the simultaneous presence of different genes - in a given evolutionary stage and with functions that are also partially correlated - would seem to define a heterogeneity that would appear to be the expression of a rapid and progressive evolution precisely because this evolution would have taken place in the diversification of all these genes. Furthermore, the presence of different genes coding for the function of cell division and related functions could reflect a progenotic status in LUCA, precisely because these functions might have originated from a single ancestral gene instead coding for a protein (or proteins) with multiple functions, and therefore an expression of a rapid and progressive evolution typical of the progenote. I also criticize other aspects of considerations made by Pende at al. (2021). The arguments presented here together with those existing in the literature make the hypothesis of a cellular LUCA favoured by Pende et al. (2021) unlikely.

The evolutionary stages of the complexity of biological catalysts mark and clarify the phases of the origin of the genetic code: A model for the origin of the reading frame with codons from proto-mRNAs with different frames.

I analyse the origin of the genetic code in the light of the evolution of biological catalysts. I discuss the rudimentary forms that the genetic code assumed in the presence of a catalysis performed by ions or by low molecular weight molecules, such as nucleotide coenzymes. However, it is only with the advent of a mixed polymer made of RNA and peptides - covalently linked - that the genetic code took on a clearer form. Indeed, the first true form of coding appeared. Furthermore, interacting peptidated RNAs promoted an extremely rudimentary form of protein synthesis. This stage evolved into a stage in which proto-mRNAs guided interactions among peptidated RNAs aimed at the synthesis of peptidated RNAs having an active catalytic function. Finally, the invasion of aminoacylated proto-tRNAs with specific amino acids, coming from amino acid metabolism, and recognising only three bases on these proto-mRNAs with reading frames larger than three bases, would have triggered the birth of actual mRNAs, i.e. the origin of codons. All this would have linked the metabolism of amino acids to the origin of mRNAs and therefore to the origin of the organization of the genetic code, as maintained by the coevolution theory of the genetic code.

Errors of the ancestral translation, LUCA, and nature of its direct descendants.

I analyzed the implications of the observation that the methyltransferases, Trm5 and TrmD, which perform the methylation of the 37th base (m1G37) in tRNAs of bacteria and archaea respectively, are not homologous proteins. The first implication is that these methyltransferases originated very late only when the fundamental lineages leading to bacteria and archaea had separated, otherwise the two methyltransferases would have been homologous enzymes, which they are not. The conclusion that Trm5 and TrmD originated only when the main lineages were defined would imply that at least some aspects of the translation, such as +1 frameshifting, were still in rapid and progressive evolution, that is, they were still originating. This would in itself imply a high rate of translation errors because the absence of m1G37 from tRNAs could have determined a high rate of +1 translational frameshifting in the reading of mRNAs, identifying this stage as that of a phase of the origin of the genetic code. Furthermore, the observation that the frameshifting mechanism was still in rapid and progressive evolution in such an advanced evolutionary stage would imply that other mechanisms concerning translation were still rapidly evolving simply because it would be very unique if only the frameshifting mechanism were the only one still originating. Importantly, the observation that in archaea m1G37 also acts as a determinant of the identity of the tRNACysGCA would imply in itself that some aspects of the origin of the genetic code were still originating, greatly strengthening the hypothesis that other aspects of the translation apparatus were still in rapid and progressive evolution. Then, all this would imply a status of progenote for LUCA and ancestors of archaea and bacteria because a high rate of translation errors would fall within the definition of progenote.

The origin of the genetic code and origin of ideas.

Inouye et al. (2020) use the observation that Ser is coded in the genetic code by two blocks of codons that differ on more than one base to understand some aspects of the origin of the genetic code organization. I argue instead that this observation per se cannot be used to understand any aspect of the origin of the genetic code, unless it is accompanied by other assumptions concerning in the specific case: (i) the ancestrality of some amino acids, (ii) the hypothesis that the first mRNA to be translated was poly-G, which can be translated into poly-Gly, and (iii) an evolutionary mechanism for the genetic code origin based on the duplication of tRNAs. However, both the tRNA duplication mechanism and the existence of poly-G as the first mRNA to be translated are not corroborated as mechanisms through which the genetic code would have been structured. For example, the origin of the actual mRNA should have been preceded by the evolution of a proto-mRNA which evidently already coded for more than one amino acid. Therefore, when it evolved from proto-mRNA, the mRNA should already have coded for more than one amino acid. In other words, poly-G as mRNA would most likely never have existed because the first mRNAs already had to code for more than one amino acid. On the contrary, all these assumptions would have been operational if the observations of Inouye et al. (2020) had been discussed within the coevolution theory of the origin of the genetic code, which they do not.

The late appearance of DNA, the nature of the LUCA and ancestors of the domains of life.

It has been firmly observed that replicative DNA polymerases of bacteria, archaea and eukaryotes are not homologous proteins. This lack of homology in the replication apparatus among the domains of life is not only compatible with but would seem to imply the view that the emergence of DNA occurred in the fundamental cellular lineages. In consequence, this diversity of DNA polymerase would go back to the level of ancestors of the domains of life and to the evolutionary time in which the DNA emerged. Therefore, the presumed evolutionary stage linked to the RNA- > DNA transition would have occurred only at the level of ancestors of the main lineages of the tree of life. Thus, the high noise associated with this major evolutionary transition and the impossibility for a cellular stage to generate different fundamental genetically profound traits - such as the different replication apparatuses of bacteria, archaea and eukaryotes - would imply not only that the last universal common ancestor (LUCA) was a progenote but that the ancestors of the domains of life were also at this evolutionary stage. So, I criticize the hypotheses which want, instead, that completely different cells - such as, bacteria and archaea - could have originated from a cellular LUCA.

LUCA as well as the ancestors of archaea, bacteria and eukaryotes were progenotes: Inference from the distribution and diversity of the reading mechanism of the AUA and AUG codons in the domains of life.

Here I use the rationale assuming that if of a certain trait that exerts its function in some aspect of the genetic code or, more generally, in protein synthesis, it is possible to identify the evolutionary stage of its origin then it would imply that this evolutionary moment would be characterized by a high translational noise because this trait would originate for the first time during that evolutionary stage. That is to say, if this trait had a non-marginal role in the realization of the genetic code, or in protein synthesis, then the origin of this trait would imply that, more generally, it was the genetic code itself that was still originating. But if the genetic code were still originating - at that precise evolutionary stage - then this would imply that there was a high translational noise which in turn would imply that it was in the presence of a protocell, i.e. a progenote that was by definition characterized by high translational noise. I apply this rationale to the mechanism of modification of the base 34 of the anticodon of an isoleucine tRNA that leads to the reading of AUA and AUG codons in archaea, bacteria and eukaryotes. The phylogenetic distribution of this mechanism in these phyletic lineages indicates that this mechanism originated only after the evolutionary stage of the last universal common ancestor (LUCA), namely, during the formation of cellular domains, i.e., at the stage of ancestors of these main phyletic lineages. Furthermore, given that this mechanism of modification of the base 34 of the anticodon of the isoleucine tRNA would result to emerge at a stage of the origin of the genetic code - despite in its terminal phases - then all this would imply that the ancestors of bacteria, archaea and eukaryotes were progenotes. If so, all the more so, the LUCA would also be a progenote since it preceded these ancestors temporally. A consequence of all this reasoning might be that since these three ancestors were of the progenotes that were different from each other, if at least one of them had evolved into at least two real and different cells - basically different from each other - then the number of cellular domains would not be three but it would be greater than three.

The phylogenetic distribution of the glutaminyl-tRNA synthetase and Glu-tRNAGln amidotransferase in the fundamental lineages would imply that the ancestor of archaea, that of eukaryotes and LUCA were progenotes.

The function of the glutaminyl-tRNA synthetase and Glu-tRNAGln amidotransferase might be related to the origin of the genetic code because, for example, glutaminyl-tRNA synthetase catalyses the fundamental reaction that makes the genetic code. If the evolutionary stage of the origin of these two enzymes could be unambiguously identified, then the genetic code should still have been originating at that particular evolutionary stage because the fundamental reaction that makes the code itself was still evidently evolving. This would result in that particular evolutionary moment being attributed to the evolutionary stage of the progenote because it would have a relationship between the genotype and the phenotype not yet fully realized because the genetic code was precisely still originating. I then analyzed the distribution of the glutaminyl-tRNA synthetase and Glu-tRNAGln aminodotrasferase in the main phyletic lineages. Since in some cases the origin of these two enzymes can be related to the evolutionary stages of ancestors of archaea and eukaryotes, this would indicate these ancestors as progenotes because at that evolutionary moment the genetic code was evidently still evolving, thus realizing the definition of progenote. The conclusion that the ancestor of archaea and that of eukaryotes were progenotes would imply that even the last universal common ancestor (LUCA) was a progenote because it appeared, on the tree of life, temporally before these ancestors.

An RNA Ring was Not the Progenitor of the tRNA Molecule.

I analyzed the model that suggests that an RNA ring might have been the progenitor of the tRNA molecule (Demongeot and Moreira in J Theor Biol 249:314-324, 2007; Demongeot and Seligmann in J Mol Evol 1-23, 2019a; Demongeot and Norris in Life 9(2):51, 2019). In particular, I analyze three ways in which this precursor, especially in its RNA hairpin form, could have evolved into the complete tRNA molecule. These three modalities are based on multiple duplication events, and therefore, appear to be less parsimonious than that which assumes that this molecule originated through one duplication of a single hairpin structure. The conclusion is, therefore, that the latter model appears to be preferable with respect to that of the RNA ring, also because there are many independent observations and some of a historical nature that would corroborate it in an extraordinary way.

Common ancestry of eukaryotes and Asgardarchaeota: Three, two or more cellular domains of life?

There is much evidence that eukaryotes have many traits in common with archaea and in phylogenetic analyses they are closely linked. In particular, it has been suggested that Asgardarchaeota would be part of the same clade of eukaryotes. If so - and being the difference between Asgardarchaeota and eukaryotes very large - then all this would imply that their common ancestor was a progenote, i.e. a protocell in which the relationship between genotype and phenotype was still evolving. This, in turn, would imply that true cells would appear on the tree of life only later, that is to say, only when the ancestor of Asgardarchaeota and the one of eukaryotes appeared. However, this way of seeing would define these ancestors as primary fundamental cells, namely, as cellular domains of life because it would be in this evolutionary stage that true cells would appear for the first time. Finally, the Asgardarchaeota-eukaryote transition is discussed, that is, some aspects of eukaryogenesis and the taxonomic rank of eukaryotes are analyzed.

A comparison between two models for understanding the origin of the tRNA molecule.

I respond to criticisms raised by Kim et al. (2018) to my model concerning the origin of the tRNA molecule. In particular, their model would hypothesize the tRNA originated due to the ligation of three hairpin structures followed by two deletions, while my model predicts that this molecule derived from the assembly of only two hairpin-like structures. Thus, using the Ockham razor, the latter model would be chosen because it required fewer hypotheses. Furthermore, the predictions on homology between the different regions of the tRNA molecule as predicted by my model would be statistically more significant than those predicted by their model. Moreover, it would be above all the existence of molecular fossils - i.e. the split tRNA genes - to corroborate the model of the assembly of only two hairpins. These fossils would be completely absent from the Kim et al. (2018) model.

The key role of the elongation factors in the origin of the organization of the genetic code.

I suggest a model based on primordial elongation factors able to explain a relevant aspect of the organization of the code itself: why the biosynthetic relationships between amino acids are mainly allocated on the rows of the genetic code. I hypothesize the existence of specific primordial elongation factors able to recognize two aspects of the ancestral aminoacylated-tRNA: the third base of the anticodon and a distinctive recognition sequence, i.e., specific for pre-tRNAs of early amino acids. If, according to the coevolution theory, it is assumed that the biosynthetic transformations of amino acids occurred on pre-tRNAs, then a duplication of the pre-tRNA of the biosynthetic progenitor followed both by a point mutation in the third base of the anticodon and evolution of the specific elongation factor so as to make it capable of recognizing this new third anticodon base. All these events were sufficient to allow the progenitor pre-tRNA to acquire other codons in a new row of the genetic code. The conquest of further codons in the same row might occur very easily, requiring only point mutations in the first two bases of the anticodon. Since the biosynthetic transformations occurred on pre-tRNAs loaded by amino acids, new amino acids produced by biosynthetic pathways were able to easily acquire the codons of their amino acid precursors. The consequence of all this is that the evolution of biosynthetic families of amino acids would have occurred almost exclusively along the rows of the genetic code. Experimental evidence in favor of the model is represented by specific elongation factors such as that of selenocysteine, which specifically recognizes Sec-tRNASec and brings it to the ribosome. Such elongation factors and their mechanism of recognition would represent molecular fossils of the mechanism hypothesized by the model and these would be a very strong corroboration for it.