Mapping mutational fitness effects across the coxsackievirus B3 proteome reveals distinct profiles of mutation tolerability.

RNA viruses have notoriously high mutation rates due to error-prone replication by their RNA polymerase. However, natural selection concentrates variability in a few key viral proteins. To test whether this stems from different mutation tolerance profiles among viral proteins, we measured the effect of >40,000 non-synonymous mutations across the full proteome of coxsackievirus B3 as well as >97% of all possible codon deletions in the nonstructural proteins. We find significant variation in mutational tolerance within and between individual viral proteins, which correlated with both general and protein-specific structural and functional attributes. Furthermore, mutational fitness effects remained stable across cell lines, suggesting selection pressures are mostly conserved across environments. In addition to providing a rich dataset for understanding virus biology and evolution, our results illustrate that incorporation of mutational tolerance data into druggable pocket discovery can aid in selecting targets with high barriers to drug resistance.

The Role of Ancestral Duplicated Genes in Adaptation to Growth on Lactate, a Non-Fermentable Carbon Source for the Yeast Saccharomyces cerevisiae.

The cell central metabolism has been shaped throughout evolutionary times when facing challenges from the availability of resources. In the budding yeast, Saccharomyces cerevisiae, a set of duplicated genes originating from an ancestral whole-genome and several coetaneous small-scale duplication events drive energy transfer through glucose metabolism as the main carbon source either by fermentation or respiration. These duplicates (~a third of the genome) have been dated back to approximately 100 MY, allowing for enough evolutionary time to diverge in both sequence and function. Gene duplication has been proposed as a molecular mechanism of biological innovation, maintaining balance between mutational robustness and evolvability of the system. However, some questions concerning the molecular mechanisms behind duplicated genes transcriptional plasticity and functional divergence remain unresolved. In this work we challenged S. cerevisiae to the use of lactic acid/lactate as the sole carbon source and performed a small adaptive laboratory evolution to this non-fermentative carbon source, determining phenotypic and transcriptomic changes. We observed growth adaptation to acidic stress, by reduction of growth rate and increase in biomass production, while the transcriptomic response was mainly driven by repression of the whole-genome duplicates, those implied in glycolysis and overexpression of ROS response. The contribution of several duplicated pairs to this carbon source switch and acidic stress is also discussed.

Preservation of genetic and regulatory robustness in ancient gene duplicates of Saccharomyces cerevisiae.

Biological systems remain robust against certain genetic and environmental challenges. Robustness allows the exploration of ecological adaptations. It is unclear what factors contribute to increasing robustness. Gene duplication has been considered to increase genetic robustness through functional redundancy, accelerating the evolution of novel functions. However, recent findings have questioned the link between duplication and robustness. In particular, it remains elusive whether ancient duplicates still bear potential for innovation through preserved redundancy and robustness. Here we have investigated this question by evolving the yeast Saccharomyces cerevisiae for 2200 generations under conditions allowing the accumulation of deleterious mutations, and we put mechanisms of mutational robustness to a test. S. cerevisiae declined in fitness along the evolution experiment, but this decline decelerated in later passages, suggesting functional compensation of mutated genes. We resequenced 28 genomes from experimentally evolved S. cerevisiae lines and found more mutations in duplicates--mainly small-scale duplicates--than in singletons. Genetically interacting duplicates evolved similarly and fixed more amino acid-replacing mutations than expected. Regulatory robustness of the duplicates was supported by a larger enrichment for mutations at the promoters of duplicates than at those of singletons. Analyses of yeast gene expression conditions showed a larger variation in the duplicates' expression than that of singletons under a range of stress conditions, sparking the idea that regulatory robustness allowed a wider range of phenotypic responses to environmental stresses, hence faster adaptations. Our data support the persistence of genetic and regulatory robustness in ancient duplicates and its role in adaptations to stresses.

Fitness Trade-Offs Determine the Role of the Molecular Chaperonin GroEL in Buffering Mutations.

Molecular chaperones fold many proteins and their mutated versions in a cell and can sometimes buffer the phenotypic effect of mutations that affect protein folding. Unanswered questions about this buffering include the nature of its mechanism, its influence on the genetic variation of a population, the fitness trade-offs constraining this mechanism, and its role in expediting evolution. Answering these questions is fundamental to understand the contribution of buffering to increase genetic variation and ecological diversification. Here, we performed experimental evolution, genome resequencing, and computational analyses to determine the trade-offs and evolutionary trajectories of Escherichia coli expressing high levels of the essential chaperonin GroEL. GroEL is abundantly present in bacteria, particularly in bacteria with large loads of deleterious mutations, suggesting its role in mutational buffering. We show that groEL overexpression is costly to large populations evolving in the laboratory, leading to groE expression decline within 66 generations. In contrast, populations evolving under the strong genetic drift characteristic of endosymbiotic bacteria avoid extinction or can be rescued in the presence of abundant GroEL. Genomes resequenced from cells evolved under strong genetic drift exhibited significantly higher tolerance to deleterious mutations at high GroEL levels than at native levels, revealing that GroEL is buffering mutations in these cells. GroEL buffered mutations in a highly diverse set of proteins that interact with the environment, including substrate and ion membrane transporters, hinting at its role in ecological diversification. Our results reveal the fitness trade-offs of mutational buffering and how genetic variation is maintained in populations.

Evolutionary microbial genomics: insights into bacterial host adaptation.

Host-adapted bacteria include mutualists and pathogens of animals, plants and insects. Their study is therefore important for biotechnology, biodiversity and human health. The recent rapid expansion in bacterial genome data has provided insights into the adaptive, diversifying and reductive evolutionary processes that occur during host adaptation. The results have challenged many pre-existing concepts built from studies of laboratory bacterial strains. Furthermore, recent studies have revealed genetic changes associated with transitions from parasitism to mutualism and opened new research avenues to understand the functional reshaping of bacteria as they adapt to growth in the cytoplasm of a eukaryotic host.

The roles of whole-genome and small-scale duplications in the functional specialization of Saccharomyces cerevisiae genes.

Researchers have long been enthralled with the idea that gene duplication can generate novel functions, crediting this process with great evolutionary importance. Empirical data shows that whole-genome duplications (WGDs) are more likely to be retained than small-scale duplications (SSDs), though their relative contribution to the functional fate of duplicates remains unexplored. Using the map of genetic interactions and the re-sequencing of 27 Saccharomyces cerevisiae genomes evolving for 2,200 generations we show that SSD-duplicates lead to neo-functionalization while WGD-duplicates partition ancestral functions. This conclusion is supported by: (a) SSD-duplicates establish more genetic interactions than singletons and WGD-duplicates; (b) SSD-duplicates copies share more interaction-partners than WGD-duplicates copies; (c) WGD-duplicates interaction partners are more functionally related than SSD-duplicates partners; (d) SSD-duplicates gene copies are more functionally divergent from one another, while keeping more overlapping functions, and diverge in their sub-cellular locations more than WGD-duplicates copies; and (e) SSD-duplicates complement their functions to a greater extent than WGD-duplicates. We propose a novel model that uncovers the complexity of evolution after gene duplication.

A gene transfer agent and a dynamic repertoire of secretion systems hold the keys to the explosive radiation of the emerging pathogen Bartonella.

Gene transfer agents (GTAs) randomly transfer short fragments of a bacterial genome. A novel putative GTA was recently discovered in the mouse-infecting bacterium Bartonella grahamii. Although GTAs are widespread in phylogenetically diverse bacteria, their role in evolution is largely unknown. Here, we present a comparative analysis of 16 Bartonella genomes ranging from 1.4 to 2.6 Mb in size, including six novel genomes from Bartonella isolated from a cow, two moose, two dogs, and a kangaroo. A phylogenetic tree inferred from 428 orthologous core genes indicates that the deadly human pathogen B. bacilliformis is related to the ruminant-adapted clade, rather than being the earliest diverging species in the genus as previously thought. A gene flux analysis identified 12 genes for a GTA and a phage-derived origin of replication as the most conserved innovations. These are located in a region of a few hundred kb that also contains 8 insertions of gene clusters for type III, IV, and V secretion systems, and genes for putatively secreted molecules such as cholera-like toxins. The phylogenies indicate a recent transfer of seven genes in the virB gene cluster for a type IV secretion system from a cat-adapted B. henselae to a dog-adapted B. vinsonii strain. We show that the B. henselae GTA is functional and can transfer genes in vitro. We suggest that the maintenance of the GTA is driven by selection to increase the likelihood of horizontal gene transfer and argue that this process is beneficial at the population level, by facilitating adaptive evolution of the host-adaptation systems and thereby expansion of the host range size. The process counters gene loss and forces all cells to contribute to the production of the GTA and the secreted molecules. The results advance our understanding of the role that GTAs play for the evolution of bacterial genomes.

Two chaperonin systems in bacterial genomes with distinct ecological roles.

Bacterial chaperonins are essential to cell viability and have a role in endosymbiosis, which leads to increased biological complexity. However, the extent to which chaperonins promote ecological innovation is unknown. We screened 622 bacterial genomes for genes encoding chaperonins, and found archaeal-like chaperonins in bacteria that inhabit archaeal ecological niches. We found that chaperonins encoded in pathogenic bacteria are the most functionally divergent. We identified the molecular basis of the dramatic structural changes in mitochondrial GROEL, a highly derived chaperonin gene. Our analysis suggests that chaperonins are important capacitors of evolutionary and ecological change.

A binary interaction map between turnip mosaic virus and Arabidopsis thaliana proteomes.

Viruses are obligate intracellular parasites that have co-evolved with their hosts to establish an intricate network of protein-protein interactions. Here, we followed a high-throughput yeast two-hybrid screening to identify 378 novel protein-protein interactions between turnip mosaic virus (TuMV) and its natural host Arabidopsis thaliana. We identified the RNA-dependent RNA polymerase NIb as the viral protein with the largest number of contacts, including key salicylic acid-dependent transcription regulators. We verified a subset of 25 interactions in planta by bimolecular fluorescence complementation assays. We then constructed and analyzed a network comprising 399 TuMV-A. thaliana interactions together with intravirus and intrahost connections. In particular, we found that the host proteins targeted by TuMV are enriched in different aspects of plant responses to infections, are more connected and have an increased capacity to spread information throughout the cell proteome, display higher expression levels, and have been subject to stronger purifying selection than expected by chance. The proviral or antiviral role of ten host proteins was validated by characterizing the infection dynamics in the corresponding mutant plants, supporting a proviral role for the transcriptional regulator TGA1. Comparison with similar studies with animal viruses, highlights shared fundamental features in their mode of action.

Structural calibration of the rates of amino acid evolution in a search for Darwin in drifting biological systems.

In the last two decades, many reports of proteins under positive selection have brought the neutral theory into question. However, the methods used to detect selection have ignored the evolvability of amino acids within proteins, which is fundamental to distinguishing positive selection from the relaxed constraints caused by genetic drift. Disentangling these two counterbalancing forces is essential to test the neutral theory. Here, we calibrate rates of amino acid divergence by using structural information from the full set of crystallized proteins in bacteria. In agreement with previous reports, we show that rates of amino acid evolution correlate negatively with the number of per-amino acid atomic interactions. Calibration of the rates of evolution allows identifying signatures of selection in biological systems that evolve under strong genetic drift, such as endosymbiotic bacteria. Application of this method identifies different rates and dynamics of evolution for highly connected amino acids in the structure compared with sparsely connected ones. We also unearth patterns of Darwinian selection in fundamental cellular proteins in endosymbiotic bacteria including the cochaperonin GroES, ribosomal proteins, proteins involved in cell cycle control, DNA-binding proteins, and proteins involved in DNA replication and repair. This is, to our knowledge, the first attempt to distinguish adaptive evolution from relaxed constraints in biological systems under genetic drift.

Selection for translational robustness in Buchnera aphidicola, endosymbiotic bacteria of aphids.

Its strong intergenerational bottlenecks and effectively asexual reproduction have led Buchnera aphidicola, the endocellular symbiotic bacterium of aphids, to spectacular evolutionary and genomic changes in comparison with its free-living bacterial cousins. These changes summarize into high fixation rates of mildly deleterious destabilizing mutations. This predicts a sharp decline of its fitness and the consequent early demise of this endosymbiotic bacterium. Its survival for hundreds of millions of years casts doubt on genetic drift as the sole evolutionary force and seeks further explanation. We identify in Buchnera selection to increase the robustness of proteins to misfolding translation errors. Translational robustness varies between Buchnera lineages and protein functional categories. Metabolic proteins have been under selection for translational efficiency, whereas evolutionary rates of proteins involved in fundamental cellular processes have been largely determined by selection for translational robustness. We detect the strongest signal of translational robustness in B. aphidicola Cinara cedri with a very similar pattern to that inferred for the most common symbiotic ancestor of Buchnera lineages. This indicates that B. aphidicola Cinara cedri lineage may have probably reached the minimum evolutionary stable gene composition for endosymbiotic lifestyle. The evolutionary patterns from the comparative genomic analyses of these endosymbionts support a paradoxically complex dynamic for apparently simple genomes.

Genome-wide functional divergence after the symbiosis of proteobacteria with insects unraveled through a novel computational approach.

Symbiosis has been among the most important evolutionary steps to generate biological complexity. The establishment of symbiosis required an intimate metabolic link between biological systems with different complexity levels. The strict endo-cellular symbiotic bacteria of insects are beautiful examples of the metabolic coupling between organisms belonging to different kingdoms, a eukaryote and a prokaryote. The host (eukaryote) provides the endosymbiont (prokaryote) with a stable cellular environment while the endosymbiont supplements the host's diet with essential metabolites. For such communication to take place, endosymbionts' genomes have suffered dramatic modifications and reconfigurations of proteins' functions. Two of the main modifications, loss of genes redundant for endosymbiotic bacteria or the host and bacterial genome streamlining, have been extensively studied. However, no studies have accounted for possible functional shifts in the endosymbiotic proteomes. Here, we develop a simple method to screen genomes for evidence of functional divergence between two species clusters, and we apply it to identify functional shifts in the endosymbiotic proteomes. Despite the strong effects of genetic drift in the endosymbiotic systems, we unexpectedly identified genes to be under stronger selective constraints in endosymbionts of aphids and ants than in their free-living bacterial relatives. These genes are directly involved in supplementing the host's diet with essential metabolites. A test of functional divergence supports a strong relationship between the endosymbiosis and the functional shifts of proteins involved in the metabolic communication with the insect host. The correlation between functional divergence in the endosymbiotic bacterium and the ecological requirements of the host uncovers their intimate biochemical and metabolic communication and provides insights on the role of symbiosis in generating species diversity.

Evolution from Free-Living Bacteria to Endosymbionts of Insects: Genomic Changes and the Importance of the Chaperonin GroEL.

Major insect lineages have independently acquired bacterial species, mainly from Gamma-proteobacteria and Bacteroidetes class, which could be nutritional mutualistic factories, facultative mutualists that protect against biotic and abiotic stresses, or reproductive manipulators (which alter the fertility of the host species in its benefit). Some of them are enclosed in bacteriocytes to assure their maternal transmission over generations. All of them show an increased level of genetic drift due to the small population size and the continuous population bottlenecking at each generation, processes that have shaped their genome, proteome, and morphology. Depending on the nature of the relationship, the degree of genome plasticity varies, i.e., obligate nutritional mutualistic symbionts have extremely small genomes lacking mobile elements, bacteriophages, or recombination machinery. Under these conditions, endosymbionts face high mutational pressures that may drive to extinction or symbiont replacement. How do then they survive for such long evolutionary time, and why do they show a genome stasis? In this chapter, after a brief introduction to the problem, we will focus on the genome changes suffered by these endosymbionts, and on the mutational robustness mechanisms, including the moonlighting chaperone GroEL that could explain their long prevalence from an evolutionary perspective by comparing them with free-living bacteria.

GWideCodeML: A Python Package for Testing Evolutionary Hypotheses at the Genome-Wide Level.

One of the most widely used programs for detecting positive selection, at the molecular level, is the program codeml, which is implemented in the Phylogenetic Analysis by Maximum Likelihood (PAML) package. However, it has a limitation when it comes to genome-wide studies, as it runs on a gene-by-gene basis. Furthermore, the size of such studies will depend on the number of orthologous genes the genomes have income and these are often restricted to only account for instances where a one-to-one relationship is observed between the genomes. In this work, we present GWideCodeML, a Python package, which runs a genome-wide codeml with the option of parallelization. To maximize the number of analyzed genes, the package allows for a variable number of taxa in the alignments and will automatically prune the topology to fit each of them, before running codeml.

Transcriptional Rewiring, Adaptation, and the Role of Gene Duplication in the Metabolism of Ethanol of Saccharomyces cerevisiae.

Ethanol is the main by-product of yeast sugar fermentation that affects microbial growth parameters, being considered a dual molecule, a nutrient and a stressor. Previous works demonstrated that the budding yeast arose after an ancient hybridization process resulted in a tier of duplicated genes within its genome, many of them with implications in this ethanol "produce-accumulate-consume" strategy. The evolutionary link between ethanol production, consumption, and tolerance versus ploidy and stability of the hybrids is an ongoing debatable issue. The implication of ancestral duplicates in this metabolic rewiring, and how these duplicates differ transcriptionally, remains unsolved. Here, we study the transcriptomic adaptive signatures to ethanol as a nonfermentative carbon source to sustain clonal yeast growth by experimental evolution, emphasizing the role of duplicated genes in the adaptive process. As expected, ethanol was able to sustain growth but at a lower rate than glucose. Our results demonstrate that in asexual populations a complete transcriptomic rewiring was produced, strikingly by downregulation of duplicated genes, mainly whole-genome duplicates, whereas small-scale duplicates exhibited significant transcriptional divergence between copies. Overall, this study contributes to the understanding of evolution after gene duplication, linking transcriptional divergence with duplicates' fate in a multigene trait as ethanol tolerance.IMPORTANCE Gene duplication events have been related with increasing biological complexity through the tree of life, but also with illnesses, including cancer. Early evolutionary theories indicated that duplicated genes could explore alternative functions due to relaxation of selective constraints in one of the copies, as the other remains as ancestral-function backup. In unicellular eukaryotes like yeasts, it has been demonstrated that the fate and persistence of duplicates depend on duplication mechanism (whole-genome or small-scale events), shaping their actual genomes. Although it has been shown that small-scale duplicates tend to innovate and whole-genome duplicates specialize in ancestral functions, the implication of duplicates' transcriptional plasticity and transcriptional divergence on environmental and metabolic responses remains largely obscure. Here, by experimental adaptive evolution, we show that Saccharomyces cerevisiae is able to respond to metabolic stress (ethanol as nonfermentative carbon source) due to the persistence of duplicated genes. These duplicates respond by transcriptional rewiring, depending on their transcriptional background. Our results shed light on the mechanisms that determine the role of duplicates, and on their evolvability.

Genomic instability in an interspecific hybrid of the genus Saccharomyces: a matter of adaptability.

Ancient events of polyploidy have been linked to huge evolutionary leaps in the tree of life, while increasing evidence shows that newly established polyploids have adaptive advantages in certain stress conditions compared to their relatives with a lower ploidy. The genus Saccharomyces is a good model for studying such events, as it contains an ancient whole-genome duplication event and many sequenced Saccharomyces cerevisiae are, evolutionary speaking, newly formed polyploids. Many polyploids have unstable genomes and go through large genome erosions; however, it is still unknown what mechanisms govern this reduction. Here, we sequenced and studied the natural S. cerevisiae × Saccharomyces kudriavzevii hybrid strain, VIN7, which was selected for its commercial use in the wine industry. The most singular observation is that its nuclear genome is highly unstable and drastic genomic alterations were observed in only a few generations, leading to a widening of its phenotypic landscape. To better understand what leads to the loss of certain chromosomes in the VIN7 cell population, we looked for genetic features of the genes, such as physical interactions, complex formation, epistatic interactions and stress responding genes, which could have beneficial or detrimental effects on the cell if their dosage is altered by a chromosomal copy number variation. The three chromosomes lost in our VIN7 population showed different patterns, indicating that multiple factors could explain the mechanisms behind the chromosomal loss. However, one common feature for two out of the three chromosomes is that they are among the smallest ones. We hypothesize that small chromosomes alter their copy numbers more frequently as a low number of genes is affected, meaning that it is a by-product of genome instability, which might be the chief driving force of the adaptability and genome architecture of this hybrid.

Genome structure reveals the diversity of mating mechanisms in Saccharomyces cerevisiae x Saccharomyces kudriavzevii hybrids, and the genomic instability that promotes phenotypic diversity.

Interspecific hybridization has played an important role in the evolution of eukaryotic organisms by favouring genetic interchange between divergent lineages to generate new phenotypic diversity involved in the adaptation to new environments. This way, hybridization between Saccharomyces species, involving the fusion between their metabolic capabilities, is a recurrent adaptive strategy in industrial environments. In the present study, whole-genome sequences of natural hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii were obtained to unveil the mechanisms involved in the origin and evolution of hybrids, as well as the ecological and geographic contexts in which spontaneous hybridization and hybrid persistence take place. Although Saccharomyces species can mate using different mechanisms, we concluded that rare-mating is the most commonly used, but other mechanisms were also observed in specific hybrids. The preponderance of rare-mating was confirmed by performing artificial hybridization experiments. The mechanism used to mate determines the genomic structure of the hybrid and its final evolutionary outcome. The evolution and adaptability of the hybrids are triggered by genomic instability, resulting in a wide diversity of genomic rearrangements. Some of these rearrangements could be adaptive under the stressful conditions of the industrial environment.

The evolution of the flagellar assembly pathway in endosymbiotic bacterial genomes.

Genome shrinkage is a common feature of most intracellular pathogens and symbionts. Reduction of genome sizes is among the best-characterized evolutionary ways of intracellular organisms to save and avoid maintaining expensive redundant biological processes. Endosymbiotic bacteria of insects are examples of biological economy taken to completion because their genomes are dramatically reduced. These bacteria are nonmotile, and their biochemical processes are intimately related to those of their host. Because of this relationship, many of the processes in these bacteria have been either lost or have suffered massive remodeling to adapt to the intracellular symbiotic lifestyle. An example of such changes is the flagellum structure that is essential for bacterial motility and infectivity. Our analysis indicates that genes responsible for flagellar assembly have been partially or totally lost in most intracellular symbionts of gamma-Proteobacteria. Comparative genomic analyses show that flagellar genes have been differentially lost in endosymbiotic bacteria of insects. Only proteins involved in protein export within the flagella assembly pathway (type III secretion system and the basal body) have been kept in most of the endosymbionts, whereas those involved in building the filament and hook of flagella have only in few instances been kept, indicating a change in the functional purpose of this pathway. In some endosymbionts, genes controlling protein-export switch and hook length have undergone functional divergence as shown through an analysis of their evolutionary dynamics. Based on our results, we suggest that genes of flagellum have diverged functionally as to specialize in the export of proteins from the bacterium to the host.

Computational biology methods and their application to the comparative genomics of endocellular symbiotic bacteria of insects.

Comparative genomics has become a real tantalizing challenge in the postgenomic era. This fact has been mostly magnified by the plethora of new genomes becoming available in a daily bases. The overwhelming list of new genomes to compare has pushed the field of bioinformatics and computational biology forward toward the design and development of methods capable of identifying patterns in a sea of swamping data noise. Despite many advances made in such endeavor, the ever-lasting annoying exceptions to the general patterns remain to pose difficulties in generalizing methods for comparative genomics. In this review, we discuss the different tools devised to undertake the challenge of comparative genomics and some of the exceptions that compromise the generality of such methods. We focus on endosymbiotic bacteria of insects because of their genomic dynamics peculiarities when compared to free-living organisms.

Comparative Genomics Between Saccharomyces kudriavzevii and S. cerevisiae Applied to Identify Mechanisms Involved in Adaptation.

Yeasts belonging to the Saccharomyces genus play an important role in human-driven fermentations. The species S. cerevisiae has been widely studied because it is the dominant yeast in most fermentations and it has been widely used as a model eukaryotic organism. Recently, other species of the Saccharomyces genus are gaining interest to solve the new challenges that the fermentation industry are facing. One of these species is S. kudriavzevii, which exhibits interesting physiological properties compared to S. cerevisiae, such as a better adaptation to grow at low temperatures, a higher glycerol synthesis and lower ethanol production. The aim of this study is to understand the molecular basis behind these phenotypic differences of biotechnological interest by using a species-based comparative genomics approach. In this work, we sequenced, assembled and annotated two new genomes of S. kudriavzevii. We used a combination of different statistical methods to identify functional divergence, signatures of positive selection and acceleration of substitution rates at specific amino acid sites of proteins in S. kudriavzevii when compared to S. cerevisiae, and vice versa. We provide a list of candidate genes in which positive selection could be acting during the evolution of both S. cerevisiae and S. kudriavzevii clades. Some of them could be related to certain important differences in metabolism previously reported by other authors such us DAL3 and ARO4, involved in nitrogen assimilation and amino acid biosynthesis. In addition, three of those genes (FBA1, ZIP1, and RQC2) showed accelerated evolutionary rates in Sk branch. Finally, genes of the riboflavin biosynthesis were also among those genes with a significant higher rate of nucleotide substitution and those proteins have amino acid positions contributing to functional divergence.