Robustness of predictions of extremely thermally stable proteins in ancient organisms

A number of studies have addressed the environmental temperatures experienced by ancient life. Computational studies using a nonhomogeneous evolution model have estimated ancestral G + C contents of ribosomal RNAs and the amino acid compositions of ancestral proteins, generating hypotheses regarding the mesophilic last universal common ancestor. In contrast, our previous study computationally reconstructed ancestral amino acid sequences of nucleoside diphosphate kinases using a homogeneous model and then empirically resurrected the ancestral proteins. The thermal stabilities of these ancestral proteins were equivalent to or greater than those of extant homologous thermophilic proteins, supporting the thermophilic universal ancestor theory. In this study, we reinferred ancestral sequences using a dataset from which hyperthermophilic sequences were excluded. We also reinferred ancestral sequences using a nonhomogeneous evolution model. The newly reconstructed ancestral proteins are still thermally stable, further supporting the hypothesis that the ancient organisms contained thermally stable proteins and therefore that they were thermophilic.     

The molecular signal for the adaptation to cold temperature during early life on Earth

Several lines of evidence such as the basal location of thermophilic lineages in large-scale phylogenetic trees and the ancestral sequence reconstruction of single enzymes or large protein concatenations support the conclusion that the ancestors of the bacterial and archaeal domains were thermophilic organisms which were adapted to hot environments during the early stages of the Earth. A parsimonious reasoning would therefore suggest that the last universal common ancestor (LUCA) was also thermophilic. Various authors have used branch-wise non-homogeneous evolutionary models that better capture the variation of molecular compositions among lineages to accurately reconstruct the ancestral G + C contents of ribosomal RNAs and the ancestral amino acid composition of highly conserved proteins. They confirmed the thermophilic nature of the ancestors of Bacteria and Archaea but concluded that LUCA, their last common ancestor, was a mesophilic organism having a moderate optimal growth temperature. In this letter, we investigate the unknown nature of the phylogenetic signal that informs ancestral sequence reconstruction to support this non-parsimonious scenario. We find that rate variation across sites of molecular sequences provides information at different time scales by recording the oldest adaptation to temperature in slow-evolving regions and subsequent adaptations in fast-evolving ones.     

About the last common ancestor, the universal life-tree and lateral gene transfer: a reappraisal

An organismal tree rooted in the bacterial branch and derived from a hyperthermophilic last common ancestor (LCA) is still widely assumed to represent the path followed by evolution from the most primeval cells to the three domains recognized among contemporary organisms: Bacteria, Archaea and Eucarya. In the past few years, however, more and more discrepancies between this pattern and individual protein trees have been brought to light. There has been an overall tendency to attribute these incongruities to widespread lateral gene transfer. However, recent developments, a reappraisal of earlier evidence and considerations of our own lead us to a quite different view. It would appear (i) that the role of lateral gene transfer was overemphasized in recent discussions of molecular phylogenies; (ii) that the LCA was probably a non-thermophilic protoeukaryote from which both Archaea and Bacteria emerged by reductive evolution but not as sister groups, in keeping with a current evolutionary scheme for the biosynthesis of membrane lipids; and (iii) that thermophilic Archaea may have been the first branch to diverge from the ancestral line.     

Gaining and losing the thermophilic adaptation in prokaryotes

We studied the evolution of thermophily in prokaryotes using the phylogenetic relationships between 279 bacteria and archaea and their thermophilic amino acid composition signature. Our findings suggest several examples in which the capacity of thermophilic adaptation has been gained or lost over relatively short evolutionary periods throughout the evolution of prokaryotes.     

On the Origin of Operons and Their Possible Role in Evolution Toward Thermophily

We present a hypothesis suggesting that close linkage of functionally related anabolic genes and their ultimate integration into operons developed under selective pressure as a molecular strategy which contributed to the viability of ancestral thermophilic cells. Cotranslation of functionally related proteins is viewed as having facilitated the formation of multienzyme complexes channeling thermolabile substrates and the mutual stabilization of inherently thermolabile proteins. In this perspective, the evolutionary scheme considered the most probable is the evolution of both Bacteria and Archaea by thermoreduction (Forterre 1995) from a mesophilic, protoeukaryotic last common ancestor (LCA) endowed with appreciable genetic redundancy.     

Independent contrasts succeed where ancestor reconstruction fails in a known bacteriophage phylogeny

Abstract.— Methods of ancestor reconstruction are important tools for evolutionary inference that are difficult to test empirically because ancestral states are rarely known with certainty. We evaluated reconstruction methods for continuous phenotypic characters using taxa from an experimentally generated bacteriophage phylogeny. Except for one slowly evolving character, the estimated ancestral states of continuous phenotypic characters were highly inaccurate and biased, even when including a known ancestor at the root. This error was caused by a directional trend in character evolution and by rapid rates of character evolution. Computer simulations confirmed that such factors affect reconstruction of continuous characters in general. We also used phenotypic viral characters to evaluate two methods that attempt to estimate the correlation between characters during evolution. Whereas a nonphylogenetic regression was relatively inaccurate and biased, independent contrasts accurately estimated the correlation between characters with little bias.     

Ancestral sequence reconstruction in primate mitochondrial DNA: compositional bias and effect on functional inference

Reconstruction of ancestral DNA and amino acid sequences is an important means of inferring information about past evolutionary events. Such reconstructions suggest changes in molecular function and evolutionary processes over the course of evolution and are used to infer adaptation and convergence. Maximum likelihood (ML) is generally thought to provide relatively accurate reconstructed sequences compared to parsimony, but both methods lead to the inference of multiple directional changes in nucleotide frequencies in primate mitochondrial DNA (mtDNA). To better understand this surprising result, as well as to better understand how parsimony and ML differ, we constructed a series of computationally simple "conditional pathway" methods that differed in the number of substitutions allowed per site along each branch, and we also evaluated the entire Bayesian posterior frequency distribution of reconstructed ancestral states. We analyzed primate mitochondrial cytochrome b (Cyt-b) and cytochrome oxidase subunit I (COI) genes and found that ML reconstructs ancestral frequencies that are often more different from tip sequences than are parsimony reconstructions. In contrast, frequency reconstructions based on the posterior ensemble more closely resemble extant nucleotide frequencies. Simulations indicate that these differences in ancestral sequence inference are probably due to deterministic bias caused by high uncertainty in the optimization-based ancestral reconstruction methods (parsimony, ML, Bayesian maximum a posteriori). In contrast, ancestral nucleotide frequencies based on an average of the Bayesian set of credible ancestral sequences are much less biased. The methods involving simpler conditional pathway calculations have slightly reduced likelihood values compared to full likelihood calculations, but they can provide fairly unbiased nucleotide reconstructions and may be useful in more complex phylogenetic analyses than considered here due to their speed and flexibility. To determine whether biased reconstructions using optimization methods might affect inferences of functional properties, ancestral primate mitochondrial tRNA sequences were inferred and helix-forming propensities for conserved pairs were evaluated in silico. For ambiguously reconstructed nucleotides at sites with high base composition variability, ancestral tRNA sequences from Bayesian analyses were more compatible with canonical base pairing than were those inferred by other methods. Thus, nucleotide bias in reconstructed sequences apparently can lead to serious bias and inaccuracies in functional predictions.     

A Universal Trend among Proteomes Indicates an Oily Last Common Ancestor

Despite progresses in ancestral protein sequence reconstruction, much needs to be unraveled about the nature of the putative last common ancestral proteome that served as the prototype of all extant lifeforms. Here, we present data that indicate a steady decline (oil escape) in proteome hydrophobicity over species evolvedness (node number) evident in 272 diverse proteomes, which indicates a highly hydrophobic (oily) last common ancestor (LCA). This trend, obtained from simple considerations (free from sequence reconstruction methods), was corroborated by regression studies within homologous and orthologous protein clusters as well as phylogenetic estimates of the ancestral oil content. While indicating an inherent irreversibility in molecular evolution, oil escape also serves as a rare and universal reaction-coordinate for evolution (reinforcing Darwin''s principle of Common Descent), and may prove important in matters such as (i) explaining the emergence of intrinsically disordered proteins, (ii) developing composition- and speciation-based “global” molecular clocks, and (iii) improving the statistical methods for ancestral sequence reconstruction.     

Evolutionary Triplet Models of Structured RNA

The reconstruction and synthesis of ancestral RNAs is a feasible goal for paleogenetics. This will require new bioinformatics methods, including a robust statistical framework for reconstructing histories of substitutions, indels and structural changes. We describe a “transducer composition” algorithm for extending pairwise probabilistic models of RNA structural evolution to models of multiple sequences related by a phylogenetic tree. This algorithm draws on formal models of computational linguistics as well as the 1985 protosequence algorithm of David Sankoff. The output of the composition algorithm is a multiple-sequence stochastic context-free grammar. We describe dynamic programming algorithms, which are robust to null cycles and empty bifurcations, for parsing this grammar. Example applications include structural alignment of non-coding RNAs, propagation of structural information from an experimentally-characterized sequence to its homologs, and inference of the ancestral structure of a set of diverged RNAs. We implemented the above algorithms for a simple model of pairwise RNA structural evolution; in particular, the algorithms for maximum likelihood (ML) alignment of three known RNA structures and a known phylogeny and inference of the common ancestral structure. We compared this ML algorithm to a variety of related, but simpler, techniques, including ML alignment algorithms for simpler models that omitted various aspects of the full model and also a posterior-decoding alignment algorithm for one of the simpler models. In our tests, incorporation of basepair structure was the most important factor for accurate alignment inference; appropriate use of posterior-decoding was next; and fine details of the model were least important. Posterior-decoding heuristics can be substantially faster than exact phylogenetic inference, so this motivates the use of sum-over-pairs heuristics where possible (and approximate sum-over-pairs). For more exact probabilistic inference, we discuss the use of transducer composition for ML (or MCMC) inference on phylogenies, including possible ways to make the core operations tractable.     

Bacterial community analysis of Indonesian hot springs

We report the first attempts to describe thermophilic bacterial communities in Indonesia's thermal springs using molecular phylogenetic analyses. 16S rRNA genes from laboratory cultures and DNA directly amplified from three hot springs in West Java were sequenced. The 22 sequences obtained were assignable to the taxa Proteobacteria, Bacillus and Flavobacterium, including a number of clades not normally associated with thermophily.     

Extremely thermophilic translation system in the common ancestor commonote: ancestral mutants of Glycyl-tRNA synthetase from the extreme thermophile Thermus thermophilus

Based on phylogenetic analysis of 16 S and 18 S rRNAs, the common ancestor of all organisms (Commonote) was proposed to be hyperthermophilic. We have previously tested this hypothesis using enzymes with ancestral residues that are inferred by molecular phylogenetic analysis. The ancestral mutant enzymes involved in metabolic systems show higher thermal stability than wild-type enzymes, consistent with the hyperthermophile common ancestor hypothesis. Here, we have extended the experiments to include an enzyme of the translation system, glycyl-tRNA synthetase (GlyRS). The translation system often shows a phylogenetic tree that is similar to the rRNA tree. Thus, it is likely that the tree represents the evolutionary route of the organisms. The maximum-likelihood tree of alpha(2) type GlyRS was constructed. From this analysis the ancestral sequence of GlyRS was deduced and individual or pairs of ancestral residues were introduced into Thermus thermophilus GlyRS. The ancestral mutants were expressed in Escherichia coli, purified and activity measured. The thermostability of eight mutated proteins was evaluated by CD (circular dichroism) measurements. Six mutants showed higher thermostability than wild-type enzyme and seven mutants showed higher activity than wild-type enzyme at 70 degrees C, suggesting an extremely thermophilic translation system in the common ancestor Commonote.     

LIKELIHOOD OF ANCESTOR STATES IN ADAPTIVE RADIATION

Theories of ecological diversification make predictions about the timing and ordering of character state changes through history. These theories are testable by “reconstructing” ancestor states using phylogenetic trees and measurements of contemporary species. Here we use maximum likelihood to estimate and evaluate the accuracy of ancestor reconstructions. We present likelihoods of discrete ancestor states and derive probability distributions for continuous ancestral traits. The methods are applied to several examples: diets of ancestral Darwin's finches; origin of inquilinism in gall wasps; microhabitat partitioning and body size evolution in scrubwrens; digestive enzyme evolution in artiodactyl mammals; origin of a sexually selected male trait, the sword, in platies and swordtails; and evolution of specialization in Anolis lizards. When changes between discrete character states are rare, the maximum-likelihood results are similar to parsimony estimates. In this case the accuracy of estimates is often high, with the exception of some nodes deep in the tree. If change is frequent then reconstructions are highly uncertain, especially of distant ancestors. Ancestor states for continuous traits are typically highly uncertain. We conclude that measures of uncertainty are useful and should always be provided, despite simplistic assumptions about the probabilistic models that underlie them. If uncertainty is too high, reconstruction should be abandoned in favor of approaches that fit different models of trait evolution to species data and phylogenetic trees, taking into account the range of ancestor states permitted by the data.     

The universal ancestor was a thermophile or a hyperthermophile.

By exploiting the correlation between the optimal growth temperature of organisms and a thermophily index based on the propensity of amino acids to enter thermophile/hyperthermophile proteins, an analysis is conducted in order to establish whether the last universal common ancestor (LUCA) was a mesophile or a (hyper)thermophile. This objective is reached by using maximum parsimony and maximum likelihood to reconstruct the ancestral sequences of the LUCA for two pairs of sets of paralogous protein sequences by means of the phylogenetic tree topology derived from the small subunit ribosomal RNA, even if this is rooted in all three possible ways. The thermophily index of all the reconstructed ancestral sequences of the LUCA belongs to the set of the thermophile/hyperthermophile sequences, thus supporting the hypotheses that see the LUCA as a thermophile or a hyperthermophile.     

Reconstruction of ancestral FGLamide-type insect allatostatins: a novel approach to the study of allatostatin function and evolution

Allatostatins (ASTs) are a class of regulatory neuropeptides, with diverse functions, found in an array of invertebrate phyla. ASTs have complex gene structure, in which individual ASTs are cleaved from a precursor peptide. Little is known about the molecular evolution of AST structure and function, even in extensively studied groups such as cockroaches. This paper presents the application of a novel technique for the analysis of this system, that of ancestral reconstruction, whereby ancestral amino acid sequences are resurrected in the laboratory. We inferred the ancestral sequences of a well-characterized peptide, AST 7, for the insect ancestor, as well as several cockroach ancestors. Peptides were assayed for in vitro inhibition of JH production in Diploptera punctata and Periplaneta americana. Our results surprisingly, indicate a decrease in potency of the ancestral cockroach AST7 peptide in comparison with more ancient ones such as the ancestral insect peptide, as well as more recently evolved cockroach peptides. We propose that this unexpected decrease in peptide potency at the cockroach ancestor may be related to the concurrent increase in peptide copy number in the lineages leading to cockroaches. This model is consistent with current physiological data, and may be linked to the increased role of ASTs in the regulation of reproductive processes in the cockroaches.     

Phylogenetic and Morphological Evolution of Green Euglenophytes Based on 18S rRNA

Green euglenophytes are a group of eukaryotes with ancient origin. In order to understand the evolution of the group, it is interesting to know which characteristics are more primitive. Here, a phylogenetic tree of green euglenophytes based on the 18S rRNA gene was constructed, and ancestral states were reconstructed based on eight morphological characters. This research clarifies the phylogenetic relationships of green euglenophytes and provides a basis for the study of the origin of these plants. The phylogenetic tree, which was constructed by Bayesian inference, revealed that: Eutreptia and Eutreptiella were sister groups and that Lepocinclis, Phacus, and Discoplastis were close relatives; Euglena, Cryptoglena, Monomorphina, and Colacium were closely related in addition to Trachelomonas and Strombomonas; and Euglena was not monophyletic. An ancestral reconstruction based on morphological characters revealed seven primitive character states: ductile surface, spirally striated, slightly narrowing or sharp elongated cauda, absence of a lorica, chloroplast lamellar, shield or large discoid, pyrenoid with sheath, and with many small paramylon grains. However, the ancestral state of the length of the flagellum could not be inferred. Euglena and Euglenaria, which both possessed all of the ancestral character states, might represent the most ancient lineages of green euglenophytes.     

Is increased maternal basking an adaptation or a pre-adaptation to viviparity in lizards?

Pregnant females modify their thermoregulatory behaviour in many species of viviparous (live-bearing) reptiles, typically maintaining higher and more stable body temperatures at this time. Such modifications often have been interpreted as adaptations to viviparity, functioning to accelerate embryonic development and/or modify phenotypic traits of hatchlings. An alternative possibility is that similar maternal thermophily may be widespread also in oviparous species and if so, would be a pre-adaptation (rather than an adaptation) to viviparity. Because eggs are retained in utero for a significant proportion of development even in oviparous reptiles, maternal thermophily might confer similar advantages in oviparous as in viviparous taxa. Experimental trials on montane oviparous scincid lizards (Bassiana duperreyi) support the pre-adaptation hypothesis. First, captive females (both reproductive and non-reproductive) selected higher temperatures than males. Second, experimentally imposing thermal regimes on pregnant females significantly affected their oviposition dates and the phenotypic traits (body shape, running speed) of their hatchlings. Thus, as for many other behavioural correlates of pregnancy in viviparous reptiles, maternal thermophily likely may have already been present in the ancestral oviparous taxa that gave rise to present-day viviparous forms.     

Assessing the accuracy of ancestral protein reconstruction methods

The phylogenetic inference of ancestral protein sequences is a powerful technique for the study of molecular evolution, but any conclusions drawn from such studies are only as good as the accuracy of the reconstruction method. Every inference method leads to errors in the ancestral protein sequence, resulting in potentially misleading estimates of the ancestral protein's properties. To assess the accuracy of ancestral protein reconstruction methods, we performed computational population evolution simulations featuring near-neutral evolution under purifying selection, speciation, and divergence using an off-lattice protein model where fitness depends on the ability to be stable in a specified target structure. We were thus able to compare the thermodynamic properties of the true ancestral sequences with the properties of “ancestral sequences” inferred by maximum parsimony, maximum likelihood, and Bayesian methods. Surprisingly, we found that methods such as maximum parsimony and maximum likelihood that reconstruct a “best guess” amino acid at each position overestimate thermostability, while a Bayesian method that sometimes chooses less-probable residues from the posterior probability distribution does not. Maximum likelihood and maximum parsimony apparently tend to eliminate variants at a position that are slightly detrimental to structural stability simply because such detrimental variants are less frequent. Other properties of ancestral proteins might be similarly overestimated. This suggests that ancestral reconstruction studies require greater care to come to credible conclusions regarding functional evolution. Inferred functional patterns that mimic reconstruction bias should be reevaluated.     

Modeling Olfactory Bulb Evolution through Primate Phylogeny

Adaptive characterizations of primates have usually included a reduction in olfactory sensitivity. However, this inference of derivation and directionality assumes an ancestral state of olfaction, usually by comparison to a group of extant non-primate mammals. Thus, the accuracy of the inference depends on the assumed ancestral state. Here I present a phylogenetic model of continuous trait evolution that reconstructs olfactory bulb volumes for ancestral nodes of primates and mammal outgroups. Parent-daughter comparisons suggest that, relative to the ancestral euarchontan, the crown-primate node is plesiomorphic and that derived reduction in olfactory sensitivity is an attribute of the haplorhine lineage. The model also suggests a derived increase in olfactory sensitivity at the strepsirrhine node. This oppositional diversification of the strepsirrhine and haplorhine lineages from an intermediate and non-derived ancestor is inconsistent with a characterization of graded reduction through primate evolution.     

The effects of multiple ancestral residues on the Thermus thermophilus 3-isopropylmalate dehydrogenase

Previously, we showed that mutants of Thermus thermophilus 3-isopropylmalate dehydrogenase (IPMDH) each containing a residue (ancestral residue) that had been predicted to exist in a postulated common ancestor protein often have greater thermal stabilities than does the contemporary wild-type enzyme. In this study, the combined effects of multiple ancestral residues were analyzed. Two mutants, containing multiple mutations, Sup3mut (Val181Thr/Pro324Thr/Ala335Glu) and Sup4mut (Leu134Asn/Val181Thr/Pro324Thr/Ala335Glu) were constructed and show greater thermal stabilities than the wild-type and single-point mutant IPMDHs do. Most of the mutants have similar or improved catalytic efficiencies at 70 degrees C when compared with the wild-type IPMDH.