The Patterns of Codon Usage between Chordates and Arthropods are Different but Co-evolving with Mutational Biases.

Different frequencies amongst codons that encode the same amino acid (i.e. synonymous codons) have been observed in multiple species. Studies focused on uncovering the forces that drive such codon usage showed that a combined effect of mutational biases and translational selection works to produce different frequencies of synonymous codons. However, only few have been able to measure and distinguish between these forces that may leave similar traces on the coding regions. Here, we have developed a codon model that allows the disentangling of mutation, selection on amino acids and synonymous codons, and GC-biased gene conversion (gBGC) which we employed on an extensive dataset of 415 chordates and 191 arthropods. We found that chordates need 15 more synonymous codon categories than arthropods to explain the empirical codon frequencies, which suggests that the extent of codon usage can vary greatly between animal phyla. Moreover, methylation at CpG sites seems to partially explain these patterns of codon usage in chordates but not in arthropods. Despite the differences between the two phyla, our findings demonstrate that in both, GC-rich codons are disfavored when mutations are GC-biased, and the opposite is true when mutations are AT-biased. This indicates that selection on the genomic coding regions might act primarily to stabilize its GC/AT content on a genome-wide level. Our study shows that the degree of synonymous codon usage varies considerably among animals, but is likely governed by a common underlying dynamic.

Mutational hotspots lead to robust but suboptimal adaptive outcomes in certain environments.

The observed mutational spectrum of adaptive outcomes can be constrained by many factors. For example, mutational biases can narrow the observed spectrum by increasing the rate of mutation at isolated sites in the genome. In contrast, complex environments can shift the observed spectrum by defining fitness consequences of mutational routes. We investigate the impact of different nutrient environments on the evolution of motility in Pseudomonas fluorescens Pf0-2x (an engineered non-motile derivative of Pf0-1) in the presence and absence of a strong mutational hotspot. Previous work has shown that this mutational hotspot can be built and broken via six silent mutations, which provide rapid access to a mutation that rescues swimming motility and confers the strongest swimming phenotype in specific environments. Here, we evolved a hotspot and non-hotspot variant strain of Pf0-2x for motility under nutrient-rich (LB) and nutrient-limiting (M9) environmental conditions. We observed the hotspot strain consistently evolved faster across all environmental conditions and its mutational spectrum was robust to environmental differences. However, the non-hotspot strain had a distinct mutational spectrum that changed depending on the nutrient environment. Interestingly, while alternative adaptive mutations in nutrient-rich environments were equal to, or less effective than, the hotspot mutation, the majority of these mutations in nutrient-limited conditions produced superior swimmers. Our competition experiments mirrored these findings, underscoring the role of environment in defining both the mutational spectrum and the associated phenotype strength. This indicates that while mutational hotspots working in concert with natural selection can speed up access to robust adaptive mutations (which can provide a competitive advantage in evolving populations), they can limit exploration of the mutational landscape, restricting access to potentially stronger phenotypes in specific environments.

Evolutionary Forces and Codon Bias in Different Flavors of Intrinsic Disorder in the Human Proteome.

In this study, we perform a systematic analysis of evolutionary forces (i.e., mutational bias and natural selection) that shape the codon usage bias of human genes encoding proteins characterized by different flavors of intrinsic disorder. Well-structured proteins are expected to be more under control by purifying natural selection than intrinsically disordered proteins because one or few mutations (even synonymous) in the genes can result in a protein that no longer folds correctly. On the contrary, intrinsically disordered proteins are thought to evolve more rapidly than well-folded proteins, due to a relaxed purifying natural selection and an increased role of mutational bias. Using different bioinformatic tools, we find evidence that codon usage in IDPs is not only affected by a basic mutational bias, but it is also more selectively constrained than the rest of the human proteome. We speculate that intrinsically disordered proteins have not only a high tolerance to mutations but also a selective propensity to preserve their structural disorder under physiological conditions. Additionally, we confirm not only that intrinsically disordered proteins are preferentially encoded by GC-rich genes, but also that they are characterized by the highest fraction of CpG sites in the sequences, implying a higher susceptibility to methylation resulting in C-T transition mutations. Overall, our results corroborate the essential role of intrinsic disorder for the evolutionary adaptability and evolvability of proteins, offering new insight about protein evolution not only in terms of functional properties and roles in diseases but also in terms of evolutionary forces they are subjected to.

The origin and underlying driving forces of the SARS-CoV-2 outbreak.

BACKGROUND: SARS-CoV-2 began spreading in December 2019 and has since become a pandemic that has impacted many aspects of human society. Several issues concerning the origin, time of introduction to humans, evolutionary patterns, and underlying force driving the SARS-CoV-2 outbreak remain unclear. METHOD: Genetic variation in 137 SARS-CoV-2 genomes and related coronaviruses as of 2/23/2020 was analyzed. RESULT: After correcting for mutational bias, the excess of low frequency mutations on both synonymous and nonsynonymous sites was revealed which is consistent with the recent outbreak of the virus. In contrast to adaptive evolution previously reported for SARS-CoV during its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation. The sequence similarity in the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression that occurred approximately 40 years ago. The current outbreak of SARS-CoV-2 was estimated to have originated on 12/11/2019 (95% HPD 11/13/2019-12/23/2019). The effective population size of the virus showed an approximately 20-fold increase from the onset of the outbreak to the lockdown of Wuhan (1/23/2020) and ceased to increase afterwards, demonstrating the effectiveness of social distancing in preventing its spread. Two mutations, 84S in orf8 protein and 251 V in orf3 protein, occurred coincidentally with human intervention. The former first appeared on 1/5/2020 and plateaued around 1/23/2020. The latter rapidly increased in frequency after 1/23/2020. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China is two times higher than those derived from the rest of the world. A network analysis found that haplotypes collected from Wuhan were interior and had more mutational connections, both of which are consistent with the observation that the SARS-CoV-2 outbreak originated in China. CONCLUSION: SARS-CoV-2 might have cryptically circulated within humans for years before being discovered. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and determine the critical steps required for effective spreading.

Three new Diplozoidae mitogenomes expose unusual compositional biases within the Monogenea class: implications for phylogenetic studies.

BACKGROUND: As the topologies produced by previous molecular and morphological studies were contradictory and unstable (polytomy), evolutionary relationships within the Diplozoidae family and the Monogenea class (controversial relationships among the Discocotylinea, Microcotylinea and Gastrocotylinea suborders) remain unresolved. Complete mitogenomes carry a relatively large amount of information, sufficient to provide a much higher phylogenetic resolution than traditionally used morphological traits and/or single molecular markers. However, their implementation is hampered by the scarcity of available monogenean mitogenomes. Therefore, we sequenced and characterized mitogenomes belonging to three Diplozoidae family species, and conducted comparative genomic and phylogenomic analyses for the entire Monogenea class. RESULTS: Taxonomic identification was inconclusive, so two of the species were identified merely to the genus level. The complete mitogenomes of Sindiplozoon sp. and Eudiplozoon sp. are 14,334 bp and 15,239 bp in size, respectively. Paradiplozoon opsariichthydis (15,385 bp) is incomplete: an approximately 2000 bp-long gap within a non-coding region could not be sequenced. Each genome contains the standard 36 genes (atp8 is missing). G + T content and the degree of GC- and AT-skews of these three mitogenome (and their individual elements) were higher than in other monogeneans. nad2, atp6 and nad6 were the most variable PCGs, whereas cox1, nad1 and cytb were the most conserved. Mitochondrial phylogenomics analysis, conducted using concatenated amino acid sequences of all PCGs, indicates that evolutionary relationships of the three genera are: (Eudiplozoon, (Paradiplozoon, Sindiplozoon)); and of the three suborders: (Discocotylinea, (Microcotylinea, Gastrocotylinea)). These intergeneric relationships were also supported by the skewness and principal component analyses. CONCLUSIONS: Our results show that nad2, atp6 and nad6 (fast-evolving) would be better candidates than cox1 (slow-evolving) for species identification and population genetics studies in Diplozoidae. Nucleotide bias and codon and amino acid usage patterns of the three diplozoid mitogenomes are more similar to cestodes and trematodes than to other monogenean flatworms. This unusual mutational bias was reflected in disproportionately long branches in the phylogram. Our study offsets the scarcity of molecular data for the subclass Polyopisthocotylea to some extent, and might provide important new insights into the evolutionary history of the three genera and three suborders.

Codon Usage Patterns of Tyrosinase Genes in Clonorchis sinensis.

Codon usage bias (CUB) is a unique property of genomes and has contributed to the better understanding of the molecular features and the evolution processes of particular gene. In this study, genetic indices associated with CUB, including relative synonymous codon usage and effective numbers of codons, as well as the nucleotide composition, were investigated in the Clonorchis sinensis tyrosinase genes and their platyhelminth orthologs, which play an important role in the eggshell formation. The relative synonymous codon usage patterns substantially differed among tyrosinase genes examined. In a neutrality analysis, the correlation between GC12 and GC3 was statistically significant, and the regression line had a relatively gradual slope (0.218). NC-plot, i.e., GC3 vs effective number of codons (ENC), showed that most of the tyrosinase genes were below the expected curve. The codon adaptation index (CAI) values of the platyhelminth tyrosinases had a narrow distribution between 0.685/0.714 and 0.797/0.837, and were negatively correlated with their ENC. Taken together, these results suggested that CUB in the tyrosinase genes seemed to be basically governed by selection pressures rather than mutational bias, although the latter factor provided an additional force in shaping CUB of the C. sinensis and Opisthorchis viverrini genes. It was also apparent that the equilibrium point between selection pressure and mutational bias is much more inclined to selection pressure in highly expressed C. sinensis genes, than in poorly expressed genes.

Phylogeny and synonymous codon usage pattern of Papaya ringspot virus coat protein gene in the sub-Himalayan region of north-east India.

Sub-Himalayan West Bengal is favorable for the production of several fruits and vegetables. Papaya is one of the common plants cultivated in the area. Most of the papaya plants of the area are susceptible to Papaya ringspot virus (PRSV). Coat protein genes of 6 PRSV isolates of the area were sequenced following RT-PCR. Phylogenetic study of the PRSV isolates showed about 80%-90% similarity with Cuban isolates. The codon usage pattern of our isolates was also analyzed, along with several other isolates. PRSV isolates of our study showed a preference for 8 putative optimal codons. Correspondence analysis of the genes of different isolates along the first 2 major axes were done, as the first 2 axes contributed more in shaping codon usage pattern. In the phylogenetic tree constructed by the neighbour-joining method, our isolates clustered together with the east Indian, north Indian, and Bangladeshi isolates. The diversity and codon usage pattern of the PRSV isolates of different regions were studied, and it was observed that the codon usage pattern of PRSV isolates is probably influenced by translational selection along with mutational bias.

Translational selection frequently overcomes genetic drift in shaping synonymous codon usage patterns in vertebrates.

Synonymous codon usage patterns are shaped by a balance between mutation, drift, and natural selection. To date, detection of translational selection in vertebrates has proven to be a challenging task, obscured by small long-term effective population sizes in larger animals and the existence of isochores in some species. The consensus is that, in such species, natural selection is either completely ineffective at overcoming mutational pressures and genetic drift or perhaps is effective but so weak that it is not detectable. The aim of this research is to understand the interplay between mutation, selection, and genetic drift in vertebrates. We observe that although variation in mutational bias is undoubtedly the dominant force influencing codon usage, translational selection acts as a weak additional factor influencing synonymous codon usage. These observations indicate that translational selection is a widespread phenomenon in vertebrates and is not limited to a few species.

System analysis of synonymous codon usage biases in archaeal virus genomes.

Recent studies of geothermally heated aquatic ecosystems have found widely divergent viruses with unusual morphotypes. Archaeal viruses isolated from these hot habitats usually have double-stranded DNA genomes, linear or circular, and can infect members of the Archaea domain. In this study, the synonymous codon usage bias (SCUB) and dinucleotide composition in the available complete archaeal virus genome sequences have been investigated. It was found that there is a significant variation in SCUB among different Archaeal virus species, which is mainly determined by the base composition. The outcome of correspondence analysis (COA) and Spearman׳s rank correlation analysis shows that codon usage of selected archaeal virus genes depends mainly on GC richness of genome, and the gene׳s function, albeit with smaller effects, also contributes to codon usage in this virus. Furthermore, this investigation reveals that aromaticity of each protein is also critical in affecting SCUB of these viral genes although it was less important than that of the mutational bias. Especially, mutational pressure may influence SCUB in SIRV1, SIRV2, ARV1, AFV1, and PhiCh1 viruses, whereas translational selection could play a leading role in HRPV1׳s SCUB. These conclusions not only can offer an insight into the codon usage biases of archaeal virus and subsequently the possible relationship between archaeal viruses and their host, but also may help in understanding the evolution of archaeal viruses and their gene classification, and more helpful to explore the origin of life and the evolution of biology.

The GC-content at the 5' ends of human protein-coding genes is undergoing mutational decay.

BACKGROUND: In vertebrates, most protein-coding genes have a peak of GC-content near their 5' transcriptional start site (TSS). This feature promotes both the efficient nuclear export and translation of mRNAs. Despite the importance of GC-content for RNA metabolism, its general features, origin, and maintenance remain mysterious. We investigate the evolutionary forces shaping GC-content at the transcriptional start site (TSS) of genes through both comparative genomic analysis of nucleotide substitution rates between different species and by examining human de novo mutations. RESULTS: Our data suggests that GC-peaks at TSSs were present in the last common ancestor of amniotes, and likely that of vertebrates. We observe that in apes and rodents, where recombination is directed away from TSSs by PRDM9, GC-content at the 5' end of protein-coding gene is currently undergoing mutational decay. In canids, which lack PRDM9 and perform recombination at TSSs, GC-content at the 5' end of protein-coding is increasing. We show that these patterns extend into the 5' end of the open reading frame, thus impacting synonymous codon position choices. CONCLUSIONS: Our results indicate that the dynamics of this GC-peak in amniotes is largely shaped by historic patterns of recombination. Since decay of GC-content towards the mutation rate equilibrium is the default state for non-functional DNA, the observed decrease in GC-content at TSSs in apes and rodents indicates that the GC-peak is not being maintained by selection on most protein-coding genes in those species.

The population frequency of human mitochondrial DNA variants is highly dependent upon mutational bias.

Next-generation sequencing can quickly reveal genetic variation potentially linked to heritable disease. As databases encompassing human variation continue to expand, rare variants have been of high interest, since the frequency of a variant is expected to be low if the genetic change leads to a loss of fitness or fecundity. However, the use of variant frequency when seeking genomic changes linked to disease remains very challenging. Here, I explored the role of selection in controlling human variant frequency using the HelixMT database, which encompasses hundreds of thousands of mitochondrial DNA (mtDNA) samples. I found that a substantial number of synonymous substitutions, which have no effect on protein sequence, were never encountered in this large study, while many other synonymous changes are found at very low frequencies. Further analyses of human and mammalian mtDNA datasets indicate that the population frequency of synonymous variants is predominantly determined by mutational biases rather than by strong selection acting upon nucleotide choice. My work has important implications that extend to the interpretation of variant frequency for non-synonymous substitutions.

Edging on Mutational Bias, Induced Natural Selection From Host and Natural Reservoirs Predominates Codon Usage Evolution in Hantaan Virus.

The molecular evolutionary dynamics that shape hantaviruses' evolution are poorly understood even now, besides the contribution of virus-host interaction to their evolution remains an open question. Our study aimed to investigate these two aspects in Hantaan virus (HTNV)-the prototype of hantaviruses and an emerging zoonotic pathogen that infects humans, causing hemorrhagic fever with renal syndrome (HFRS): endemic in Far East Russia, China, and South Korea-via a comprehensive, phylogenetic-dependent codon usage analysis. We found that host- and natural reservoir-induced natural selection is the primary determinant of its biased codon choices, exceeding the mutational bias effect. The phylogenetic analysis of HTNV strains resulted in three distinct clades: South Korean, Russian, and Chinese. An effective number of codon (ENC) analysis showed a slightly biased codon usage in HTNV genomes. Nucleotide composition and RSCU analyses revealed a significant bias toward A/U nucleotides and A/U-ended codons, indicating the potential influence of mutational bias on the codon usage patterns of HTNV. Via ENC-plot, Parity Rule 2 (PR2), and neutrality plot analyses, we would conclude the presence of both mutation pressure and natural selection effect in shaping the codon usage patterns of HTNV; however, natural selection is the dominant factor influencing its codon usage bias. Codon adaptation index (CAI), Relative codon deoptimization index (RCDI), and Similarity Index (SiD) analyses uncovered the intense selection pressure from the host (Human) and natural reservoirs (Striped field mouse and Chinese white-bellied rat) in shaping HTNV biased codon choices. Our study clearly revealed the evolutionary processes in HTNV and the role of virus-host interaction in its evolution. Moreover, it opens the door for a more comprehensive codon usage analysis for all hantaviruses species to determine their molecular evolutionary dynamics and adaptability to several hosts and environments. We believe that our research will help in a better and deep understanding of HTNV evolution that will serve its future basic research and aid live attenuated vaccines design.

Genome-Wide Analysis of Codon Usage Patterns of SARS-CoV-2 Virus Reveals Global Heterogeneity of COVID-19.

The ongoing outbreak of coronavirus disease COVID-19 is significantly implicated by global heterogeneity in the genome organization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The causative agents of global heterogeneity in the whole genome of SARS-CoV-2 are not well characterized due to the lack of comparative study of a large enough sample size from around the globe to reduce the standard deviation to the acceptable margin of error. To better understand the SARS-CoV-2 genome architecture, we have performed a comprehensive analysis of codon usage bias of sixty (60) strains to get a snapshot of its global heterogeneity. Our study shows a relatively low codon usage bias in the SARS-CoV-2 viral genome globally, with nearly all the over-preferred codons' A.U. ended. We concluded that the SARS-CoV-2 genome is primarily shaped by mutation pressure; however, marginal selection pressure cannot be overlooked. Within the A/U rich virus genomes of SARS-CoV-2, the standard deviation in G.C. (42.91% ± 5.84%) and the GC3 value (30.14% ± 6.93%) points towards global heterogeneity of the virus. Several SARS-CoV-2 viral strains were originated from different viral lineages at the exact geographic location also supports this fact. Taking all together, these findings suggest that the general root ancestry of the global genomes are different with different genome's level adaptation to host. This research may provide new insights into the codon patterns, host adaptation, and global heterogeneity of SARS-CoV-2.

Codon Usage Optimization in the Prokaryotic Tree of Life: How Synonymous Codons Are Differentially Selected in Sequence Domains with Different Expression Levels and Degrees of Conservation.

Prokaryote genomes exhibit a wide range of GC contents and codon usages, both resulting from an interaction between mutational bias and natural selection. In order to investigate the basis underlying specific codon changes, we performed a comprehensive analysis of 29 different prokaryote families. The analysis of core gene sets with increasing ancestries in each family lineage revealed that the codon usages became progressively more adapted to the tRNA pools. While, as previously reported, highly expressed genes presented the most optimized codon usage, the singletons contained the less selectively favored codons. The results showed that usually codons with the highest translational adaptation were preferentially enriched. In agreement with previous reports, a C bias in 2- to 3-fold pyrimidine-ending codons, and a U bias in 4-fold codons occurred in all families, irrespective of the global genomic GC content. Furthermore, the U biases suggested that U3-mRNA-U34-tRNA interactions were responsible for a prominent codon optimization in both the most ancestral core and the highly expressed genes. A comparative analysis of sequences that encode conserved (cr) or variable (vr) translated products, with each one being under high (HEP) and low (LEP) expression levels, demonstrated that the efficiency was more relevant (by a factor of 2) than accuracy to modeling codon usage. Finally, analysis of the third position of codons (GC3) revealed that in genomes with global GC contents higher than 35 to 40%, selection favored a GC3 increase, whereas in genomes with very low GC contents, a decrease in GC3 occurred. A comprehensive final model is presented in which all patterns of codon usage variations are condensed in four distinct behavioral groups.IMPORTANCE The prokaryotic genomes-the current heritage of the most ancient life forms on earth-are comprised of diverse gene sets, all characterized by varied origins, ancestries, and spatial-temporal expression patterns. Such genetic diversity has for a long time raised the question of how cells shape their coding strategies to optimize protein demands (i.e., product abundance) and accuracy (i.e., translation fidelity) through the use of the same genetic code in genomes with GC contents that range from less than 20 to more than 80%. Here, we present evidence on how codon usage is adjusted in the prokaryotic tree of life and on how specific biases have operated to improve translation. Through the use of proteome data, we characterized conserved and variable sequence domains in genes of either high or low expression level and quantitated the relative weight of efficiency and accuracy-as well as their interaction-in shaping codon usage in prokaryotes.

Codon Usage and Phenotypic Divergences of SARS-CoV-2 Genes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which first occurred in Wuhan (China) in December of 2019, causes a severe acute respiratory illness with a high mortality rate, and has spread around the world. To gain an understanding of the evolution of the newly emerging SARS-CoV-2, we herein analyzed the codon usage pattern of SARS-CoV-2. For this purpose, we compared the codon usage of SARS-CoV-2 with that of other viruses belonging to the subfamily of Orthocoronavirinae. We found that SARS-CoV-2 has a high AU content that strongly influences its codon usage, which appears to be better adapted to the human host. We also studied the evolutionary pressures that influence the codon usage of five conserved coronavirus genes encoding the viral replicase, spike, envelope, membrane and nucleocapsid proteins. We found different patterns of both mutational bias and natural selection that affect the codon usage of these genes. Moreover, we show here that the two integral membrane proteins (matrix and envelope) tend to evolve slowly by accumulating nucleotide mutations on their corresponding genes. Conversely, genes encoding nucleocapsid (N), viral replicase and spike proteins (S), although they are regarded as are important targets for the development of vaccines and antiviral drugs, tend to evolve faster in comparison to the two genes mentioned above. Overall, our results suggest that the higher divergence observed for the latter three genes could represent a significant barrier in the development of antiviral therapeutics against SARS-CoV-2.

Analysis of Codon Usage and Nucleotide Bias in Middle East Respiratory Syndrome Coronavirus Genes.

The Middle East Respiratory Syndrome (MERS) is an emerging disease caused by a recently identified human coronavirus (CoV). Over 2494 laboratory-confirmed cases and 858 MERS-related deaths have been reported from 27 countries. MERS-CoV has been associated with a high case fatality rate, especially in patients with pre-existing conditions. Despite the fatal nature of MERS-CoV infection, a comprehensive study to explore its evolution and adaptation in different hosts is lacking. We performed codon usage analyses on 4751 MERS-CoV genes and determined underlying forces that affect the codon usage bias in the MERS-CoV genome. The current analyses revealed a low but highly conserved, gene-specific codon usage bias in the MERS-CoV genome. The codon usage bias is mainly shaped by natural selection, while mutational pressure emerged as a minor factor affecting codon usage in some genes. Other contributory factors included CpG dinucleotide bias, physical and chemical properties of encoded proteins and gene length. Results reported in this study provide considerable insights into the molecular evaluation of MERS-CoV and could serve as a theoretical basis for optimizing MERS-CoV gene expression to study the functional relevance of various MERS-CoV proteins. Alternatively, an attenuated vaccine strain containing hundreds of silent mutations could be engineered. Codon de-optimization will not affect the amino acid sequence or antigenicity of a vaccine strain, but the sheer number of mutations would make viral reversion to a virulent phenotype extremely unlikely.

Comprehensive analysis of synonymous codon usage patterns in orf3 gene of porcine epidemic diarrhea virus in China.

The ORF3 protein of porcine epidemic diarrhea virus (PEDV) is found to function as an ion channel which influences virus virulence and production. Taking consideration of the importance of PEDV orf3 gene, we have performed comprehensive analysis to investigate its synonymous codon usage patterns. In this study, the results of base composition analysis showed A/T rich and G/C poor in PEDV orf3 genes, and the most abundant base was nucleotide T. The relative synonymous codon usage value in each codon revealed that codon usage bias existed. The mean ENC value of each gene was 48.75, indicating a low codon usage bias, as well as a relatively instable change in PEDV orf3 genes. The general correlation analysis between base composition and codon usage bias indicated that mutational bias has an impact on the PEDV codon usage bias. Neutral analysis suggested that natural selection pressure takes a more important influence than mutational bias in shaping codon usage bias. Moreover, other factors including hydrophobicity and aromaticity have been also found to influence the codon usage variation among the PEDV orf3 genes. This study not only represents the most systematic analysis of codon usage patterns in PEDV orf3 genes, but also provides a basic shaping mechanism of the codon usage bias.

COUSIN (COdon Usage Similarity INdex): A Normalized Measure of Codon Usage Preferences.

Codon Usage Preferences (CUPrefs) describe the unequal usage of synonymous codons at the gene, chromosome, or genome levels. Numerous indices have been developed to evaluate CUPrefs, either in absolute terms or with respect to a reference. We introduce the normalized index COUSIN (for COdon Usage Similarity INdex), that compares the CUPrefs of a query against those of a reference and normalizes the output over a Null Hypothesis of random codon usage. The added value of COUSIN is to be easily interpreted, both quantitatively and qualitatively. An eponymous software written in Python3 is available for local or online use (http://cousin.ird.fr). This software allows for an easy and complete analysis of CUPrefs via COUSIN, includes seven other indices, and provides additional features such as statistical analyses, clustering, and CUPrefs optimization for gene expression. We illustrate the flexibility of COUSIN and highlight its advantages by analyzing the complete coding sequences of eight divergent genomes. Strikingly, COUSIN captures a bimodal distribution in the CUPrefs of human and chicken genes hitherto unreported with such precision. COUSIN opens new perspectives to uncover CUPrefs specificities in genomes in a practical, informative, and user-friendly way.

Yeast Spontaneous Mutation Rate and Spectrum Vary with Environment.

Mutation is the ultimate genetic source of evolution and biodiversity, but to what extent the environment impacts mutation rate and spectrum is poorly understood. Past studies discovered mutagenesis induced by antibiotic treatment or starvation, but its relevance and importance to long-term evolution is unclear because these severe stressors typically halt cell growth and/or cause substantial cell deaths. Here, we quantify the mutation rate and spectrum in Saccharomyces cerevisiae by whole-genome sequencing following mutation accumulation in each of seven environments with relatively rapid cell growths and minimal cell deaths. We find the point mutation rate per generation to differ by 3.6-fold among the seven environments, generally increasing in environments with slower cell growths. This trend renders the mutation rate per year more constant than that per generation across environments, which has implications for neutral evolution and the molecular clock. Additionally, we find substantial among-environment variations in mutation spectrum, such as the transition to transversion ratio and AT mutational bias. Other main mutation types, including small insertion or deletion, segmental duplication or deletion, and chromosome gain or loss also tend to occur more frequently in environments where yeast grows more slowly. In contrast to these findings from the nuclear genome, the yeast mitochondrial mutation rate rises with the growth rate, consistent with the metabolic rate hypothesis. Together, these observations indicate that environmental changes, which are ubiquitous in nature, influence not only natural selection, but also the amount and type of mutations available to selection, and suggest that ignoring the latter impact, as is currently practiced, may mislead evolutionary inferences.

Hierarchical Assessment of Mutation Properties in Daphnia magna.

Understanding the context-dependence of spontaneous mutations is crucial to predicting evolutionary trajectories. In this experiment, the impact of genetic background and trait-type on mutational susceptibility was investigated. Mutant and non-mutant lines of six unique genotypes from two populations of Daphnia magna were phenotypically assayed using a common-garden experiment. Morphological, life-history, and behavioral traits were measured and estimates of the mutation parameters were generated. The mutation parameters varied between the populations and among genotypes, suggesting differential susceptibility to mutation depending upon genomic background. Traits also varied in their susceptibility to mutation with behavioral traits evolving more rapidly than life-history and morphological traits. These results may reflect the unique selection histories of these populations.