Codon Substitution Models Research Articles

Next-generation development and application of codon model in evolution.

To date, numerous nucleotide, amino acid, and codon substitution models have been developed to estimate the evolutionary history of any sequence/organism in a more comprehensive way. Out of these three, the codon substitution model is the most powerful. These models have been utilized extensively to detect selective pressure on a protein, codon usage bias, ancestral reconstruction and phylogenetic reconstruction. However, due to more computational demanding, in comparison to nucleotide and amino acid substitution models, only a few studies have employed the codon substitution model to understand the heterogeneity of the evolutionary process in a genome-scale analysis. Hence, there is always a question of how to develop more robust but less computationally demanding codon substitution models to get more accurate results. In this review article, the authors attempted to understand the basis of the development of different types of codon-substitution models and how this information can be utilized to develop more robust but less computationally demanding codon substitution models. The codon substitution model enables to detect selection regime under which any gene or gene region is evolving, codon usage bias in any organism or tissue-specific region and phylogenetic relationship between different lineages more accurately than nucleotide and amino acid substitution models. Thus, in the near future, these codon models can be utilized in the field of conservation, breeding and medicine.

Adaptive evolution of scn4aa in Takifugu and Tetraodon

Tetrodotoxin (TTX) is a potent neurotoxin firstly discovered in the ovary of pufferfish. The genetic basis of voltage-gated sodium channel resistance to TTX has been widely studied, but it remains unclear in the evolution history of voltage-gated sodium channel resistance to TTX in pufferfish with different TTX concentrations. In this study, six scn4aa coding sequences of pufferfish were firstly cloned and sequenced, then used to investigate the adaptive evolution of scn4aa associated with TTX concentration and reconstruct ancestral sequences with seven scn4aa of other fishes. The result of CODEML (codon substitution model) program from the PAML (phylogenetic analysis by maximum likelihood) package shows only in the genus of Takifugu, which contains TTX highly in the liver, under positive selection. The result also indicates that three of four positively selected sites are located in the intracellular regions, which may compensate for normal function. The ancestral sequence reconstruction may suggest that the replacements providing weak toxin resistance might have appeared first in scn4aa, then the genus Takifugu evolved stronger resistance to TTX later. These results contribute to the explanation of the evolutional history of voltage-gated sodium channel resistance to TTX in pufferfish.

Detecting signals of adaptive evolution in grape plastomes with a focus on the Cretaceous-Palaeogene (K/Pg) transition.

Although plastid genes are widely used in phylogenetic studies, signals of positive selection have been scarcely investigated in the grape family. The plastomes from 91 accessions of Vitaceae were examined to understand the extent to which positive selection is present and to identify which genes are involved. Moreover, the changes through time of genes under episodic positive selection were investigated and the hypothesis of an adaptive process following the Cretaceous-Palaeogene (K/Pg) transition about 66 million years ago was tested. Different codon-substitution models were used to assess pervasive and episodic positive selection events on 70 candidate plastid genes. Divergence times between lineages were estimated and stochastic character mapping analysis was used to simulate variation over time of the genes found to be under episodic positive selection. A total of 20 plastid genes (29 %) showed positive selection. Among them, 14 genes showed pervasive signatures of positive selection and nine genes showed episodic signatures of positive selection. In particular, four of the nine genes (psbK, rpl20, rpoB, rps11) exhibited a similar pattern showing an increase in the rate of variation close to the K/Pg transition. Multiple analyses have shown that the grape family has experienced ancient and recent positive selection events and that the targeted genes are involved in essential functions such as photosynthesis, self-replication and metabolism. Our results are consistent with the idea that the K/Pg transition has favoured an increased rate of change in some genes. Intense environmental perturbations have influenced the rapid diversification of certain lineages, and new mutations arising on some plastid genes may have been fixed by natural selection over the course of many generations.

Signatures of Adaptation in Mitochondrial Genomes of Palearctic Subterranean Voles (Arvicolinae, Rodentia).

This study evaluates signatures of selection in the evolution of the mitochondrial DNA of voles, subfamily Arvicolinae, during the colonization of subterranean environments. The comparative sequence analysis of mitochondrial protein-coding genes of eight subterranean vole species (Prometheomys schaposchnikowi, three species of the genus Ellobius: Ellobius talpinus, Ellobius fuscocapillus and Ellobius lutescens, two species of the genus Terricola: Terricola subterraneus and Terricola daghestanicus, Lasiopodomys mandarinus, and Hyperacrius fertilis) and their closest aboveground relatives was applied using codon-substitution models. The highest number of selection signatures was detected in genes ATP8 and CYTB. The relaxation of selection was observed in most mitochondrial DNA protein-coding genes for subterranean species. The largest amount of relaxed genes is discovered in mole voles (genus Ellobius). The number of selection signatures was found to be independent of the evolutionary age of the lineage but fits the degree of specialization to the subterranean niche. The common trends of selective pressures were observed among the evolutionary ancient and highly specialized subterranean rodent families and phylogenetically young lineages of voles. It suggests that the signatures of adaptation in individual mitochondrial protein-coding genes associated with the colonization of the subterranean niche may appear within a rather short evolutionary timespan.

Extra base hits: Widespread empirical support for instantaneous multiple-nucleotide changes

Despite many attempts to introduce evolutionary models that permit substitutions to instantly alter more than one nucleotide in a codon, the prevailing wisdom remains that such changes are rare and generally negligible or are reflective of non-biological artifacts, such as alignment errors. Codon models continue to posit that only single nucleotide change have non-zero rates. Here, we develop and test a simple hierarchy of codon-substitution models with non-zero evolutionary rates for only one-nucleotide (1H), one- and two-nucleotide (2H), or any (3H) codon substitutions. Using over 42, 000 empirical alignments, we find widespread statistical support for multiple hits: 61% of alignments prefer models with 2H allowed, and 23%-with 3H allowed. Analyses of simulated data suggest that these results are not likely to be due to simple artifacts such as model misspecification or alignment errors. Further modeling reveals that synonymous codon island jumping among codons encoding serine, especially along short branches, contributes significantly to this 3H signal. While serine codons were prominently involved in multiple-hit substitutions, there were other common exchanges contributing to better model fit. It appears that a small subset of sites in most alignments have unusual evolutionary dynamics not well explained by existing model formalisms, and that commonly estimated quantities, such as dN/dS ratios may be biased by model misspecification. Our findings highlight the need for continued evaluation of assumptions underlying workhorse evolutionary models and subsequent evolutionary inference techniques. We provide a software implementation for evolutionary biologists to assess the potential impact of extra base hits in their data in the HyPhy package and in the Datamonkey.org server.

Holocentric Chromosomes Probably Do Not Prevent Centromere Drive in Cyperaceae.

Centromere drive model describes an evolutionary process initiated by centromeric repeats expansion, which leads to the recruitment of excess kinetochore proteins and consequent preferential segregation of an expanded centromere to the egg during female asymmetric meiosis. In response to these selfish centromeres, the histone protein CenH3, which recruits kinetochore components, adaptively evolves to restore chromosomal parity and counter the detrimental effects of centromere drive. Holocentric chromosomes, whose kinetochores are assembled along entire chromosomes, have been hypothesized to prevent expanded centromeres from acquiring a selective advantage and initiating centromere drive. In such a case, CenH3 would be subjected to less frequent or no adaptive evolution. Using codon substitution models, we analyzed 36 CenH3 sequences from 35 species of the holocentric family Cyperaceae. We found 10 positively selected codons in the CenH3 gene [six codons in the N-terminus and four in the histone fold domain (HFD)] and six branches of its phylogeny along which the positive selection occurred. One of the positively selected codons was found in the centromere targeting domain (CATD) that directly interacts with DNA and its mutations may be important in centromere drive suppression. The frequency of these positive selection events was comparable to the frequency of positive selection in monocentric clades with asymmetric female meiosis. Taken together, these results suggest that preventing centromere drive is not the primary adaptive role of holocentric chromosomes, and their ability to suppress it likely depends on their kinetochore structure in meiosis.

Link Your Sites (LYS) Scripts: Automated Search of Protein Structures and Mapping of Sites Under Positive Selection Detected by PAML

The visualization of the molecular context of an amino acid mutation in a protein structure is crucial for the assessment of its functional impact and the understanding of its evolutionary implications. Currently, searches for fast evolving amino acid positions using codon substitution models like those implemented in PAML (Yang and Nielsen in Estimating synonymous and nonsynonymous substitution rates under realistic evolutionary models. Mol Biol Evol 17(1):32–43, 2000; Zhang et al. in Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level. Mol Biol Evol 22(12):2472–2479, 2005) are done in almost complete proteomes, generating large numbers of candidate proteins making the analysis of individual protein structures and models very time-consuming. Here we present the package Link Your Sites (LYS) that can be used to reduce the number of analysed targets to those for which structural information can be retrieved. LYS consists of two python wrapper scripts, where the first one (i) mines the RCSB database (Berman et al. in The protein data bank. Nucleic Acids Res 28(1):235–242, 2000) using the BLAST alignment tool to find the best matching homologous sequences, (ii) fetches their domain positions by using Prosites (Hamelryck and Manderick in Pdb file parser and structure class implemented in python. Bioinformatics 19(17):2308–2310, 2003; Sigrist et al. in Prosite: a documented database using patterns and profiles as motif descriptors. Brief Bioinf 3(3):265–274, 2002; Sigrist et al. in New and continuing developments at prosite. Nucleic Acids Res 41(D1):D344–D347, 2012), (iii) parses the output of PAML extracting the positional information of fast-evolving sites and transforms them into the coordinate system of the protein structure, (iv) outputs one file per gene with the equivalence among the positions in the input sequence and homologous structure. The second script produces figures to be used in publications highlighting the positively selected sites mapped on regions that are known to have functional relevance.

Delineation of the Crucial Evolutionary Amino Acid Sites in Trehalose-6-Phosphate Synthase From Higher Plants.

Trehalose-6-phosphate synthase (TPS) is a key enzyme in the biosynthesis of trehalose, with its direct product, trehalose-6-phosphate, playing important roles in regulating whole-plant carbohydrate allocation and utilization. Genes encoding TPS constitute a multigene family in which functional divergence appears to have occurred repeatedly. To identify the crucial evolutionary amino acid sites of TPS in higher plants, a series of bioinformatics tools were applied to investigate the phylogenetic relationships, functional divergence, positive selection, and co-evolution of TPS proteins. First, we identified 150 TPS genes from 13 higher plant species. Phylogenetic analysis placed these TPS proteins into 2 clades: clades A and B, of which clade B could be further divided into 4 subclades (B1-B4). This classification was supported by the intron-exon structures, with more introns present in clade A. Next, detection of the critical functionally divergent amino acid sites resulted in the isolation of a total of 286 sites reflecting nonredundant radical shifts in amino acid properties with a high posterior probability cutoff among subclades. In addition, positively selected sites were identified using a codon substitution model, from which 46 amino acid sites were isolated as exhibiting positive selection at a significant level. Moreover, 18 amino acid sites were highlighted both for functional divergence and positive selection; these may thus potentially represent crucial evolutionary sites in the TPS family. Further co-evolutionary analysis revealed 3 pairs of sites: 11S and 12H, 33S and 34N, and 109G and 110E as demonstrating co-evolution. Finally, the 18 crucial evolutionary amino acid sites were mapped in the 3-dimensional structure. A total of 77 sites harboring functionally and structurally important residues of TPS proteins were found by using the CLIPS-4D online tool; notably, no overlap was observed with the identified crucial evolutionary sites, providing positive evidence supporting their designation. A total of 18 sites were isolated as key amino acids by using multiple bioinformatics tools based on their concomitant functional divergence and positive selection. Almost all these key sites are located in 2 domains of this protein family where they exhibit no overlap with the structurally and functionally conserved sites. These results will provide an improved understanding of the complexity of the TPS gene family and of its function and evolution in higher plants. Moreover, this knowledge may facilitate the exploitation of these sites for protein engineering applications.

A Phenotype-Genotype Codon Model for Detecting Adaptive Evolution.

A central objective in biology is to link adaptive evolution in a gene to structural and/or functional phenotypic novelties. Yet most analytic methods make inferences mainly from either phenotypic data or genetic data alone. A small number of models have been developed to infer correlations between the rate of molecular evolution and changes in a discrete or continuous life history trait. But such correlations are not necessarily evidence of adaptation. Here, we present a novel approach called the phenotype-genotype branch-site model (PG-BSM) designed to detect evidence of adaptive codon evolution associated with discrete-state phenotype evolution. An episode of adaptation is inferred under standard codon substitution models when there is evidence of positive selection in the form of an elevation in the nonsynonymous-to-synonymous rate ratio $\omega$ to a value $\omega > 1$. As it is becoming increasingly clear that $\omega > 1$ can occur without adaptation, the PG-BSM was formulated to infer an instance of adaptive evolution without appealing to evidence of positive selection. The null model makes use of a covarion-like component to account for general heterotachy (i.e., random changes in the evolutionary rate at a site over time). The alternative model employs samples of the phenotypic evolutionary history to test for phenomenological patterns of heterotachy consistent with specific mechanisms of molecular adaptation. These include 1) a persistent increase/decrease in $\omega$ at a site following a change in phenotype (the pattern) consistent with an increase/decrease in the functional importance of the site (the mechanism); and 2) a transient increase in $\omega$ at a site along a branch over which the phenotype changed (the pattern) consistent with a change in the site's optimal amino acid (the mechanism). Rejection of the null is followed by post hoc analyses to identify sites with strongest evidence for adaptation in association with changes in the phenotype as well as the most likely evolutionary history of the phenotype. Simulation studies based on a novel method for generating mechanistically realistic signatures of molecular adaptation show that the PG-BSM has good statistical properties. Analyses of real alignments show that site patterns identified post hoc are consistent with the specific mechanisms of adaptation included in the alternate model. Further simulation studies show that the covarion-like component of the PG-BSM plays a crucial role in mitigating recently discovered statistical pathologies associated with confounding by accounting for heterotachy-by-any-cause. [Adaptive evolution; branch-site model; confounding; mutation-selection; phenotype-genotype.].

A63 Quantifying the dynamics of evolutionary rates through time

The availability of evolutionary rate estimates in recent years led to the observation that they may depend on the time-scale on which they are measured. Specifically, RNA virus evolutionary rates are frequently estimated to be low towards the past and high towards the present. This time-dependent rate phenomenon (TDRP) has important implications for evolutionary studies as it could severely bias divergence time estimates. While recent studies are providing insights into the relationship between viral evolutionary rate and time, formal probabilistic models to draw inference under TDRP scenarios remain lacking. Here, we adopt epoch-modelling to develop a Bayesian model of discrete rate changes through time in an unknown evolutionary history and combine this with a log-linear parameterization of rates as a function of times in the past. We provide an implementation for nucleotide substitution rates as well as for nonsynonymous rates change in a codon substitution model. Using a foamy virus dataset for which internal node calibrations can be applied based on host-virus co-divergence, we estimate a significant decline in evolutionary rates as a function of time into the past for nucleotide substitutions as well as for non-synonymous substitutions in a codon model. We also estimate a deep evolutionary history for primate Lentiviruses by combining an HIV-1 group M node calibration and a biogeographic calibration for viruses in drill monkeys in the TDRP model. Our analyses lead to the conclusion that studies of evolutionary timescales require a reconsideration of substitution rates, in either codon and nucleotide substitution model, as a dynamic feature of molecular evolution.

Physicochemical Amino Acid Properties Better Describe Substitution Rates in Large Populations.

Substitutions between chemically distant amino acids are known to occur less frequently than those between more similar amino acids. This knowledge, however, is not reflected in most codon substitution models, which treat all nonsynonymous changes as if they were equivalent in terms of impact on the protein. A variety of methods for integrating chemical distances into models have been proposed, with a common approach being to divide substitutions into radical or conservative categories. Nevertheless, it remains unclear whether the resulting models describe sequence evolution better than their simpler counterparts. We propose a parametric codon model that distinguishes between radical and conservative substitutions, allowing us to assess if radical substitutions are preferentially removed by selection. Applying our new model to a range of phylogenomic data, we find differentiating between radical and conservative substitutions provides significantly better fit for large populations, but see no equivalent improvement for smaller populations. Comparing codon and amino acid models using these same data shows that alignments from large populations tend to select phylogenetic models containing information about amino acid exchangeabilities, whereas the structure of the genetic code is more important for smaller populations. Our results suggest selection against radical substitutions is, on average, more pronounced in large populations than smaller ones. The reduced observable effect of selection in smaller populations may be due to stronger genetic drift making it more challenging to detect preferences. Our results imply an important connection between the life history of a phylogenetic group and the model that best describes its evolution.

Looking for Darwin in Genomic Sequences: Validity and Success Depends on the Relationship Between Model and Data.

Codon substitution models (CSMs) are commonly used to infer the history of natural section for a set of protein-coding sequences, often with the explicit goal of detecting the signature of positive Darwinian selection. However, the validity and success of CSMs used in conjunction with the maximum likelihood (ML) framework is sometimes challenged with claims that the approach might too often support false conclusions. In this chapter, we use a case study approach to identify four legitimate statistical difficulties associated with inference of evolutionary events using CSMs. These include: (1) model misspecification, (2) low information content, (3) the confounding of processes, and (4) phenomenological load, or PL. While past criticisms of CSMs can be connected to these issues, the historical critiques were often misdirected, or overstated, because they failed to recognize that the success of any model-based approach depends on the relationship between model and data. Here, we explore this relationship and provide a candid assessment of the limitations of CSMs to extract historical information from extant sequences. To aid in this assessment, we provide a brief overview of: (1) a more realistic way of thinking about the process of codon evolution framed in terms of population genetic parameters, and (2) a novel presentation of the ML statistical framework. We then divide the development of CSMs into two broad phases of scientific activity and show that the latter phase is characterized by increases in model complexity that can sometimes negatively impact inference of evolutionary mechanisms. Such problems are not yet widely appreciated by the users of CSMs. These problems can be avoided by using a model that is appropriate for the data; but, understanding the relationship between the data and a fitted model is a difficult task. We argue that the only way to properly understand that relationship is to perform in silico experiments using a generating process that can mimic the data as closely as possible. The mutation-selection modeling framework (MutSel) is presented as the basis of such a generating process. We contend that if complex CSMs continue to be developed for testing explicit mechanistic hypotheses, then additional analyses such as those described in here (e.g., penalized LRTs and estimation of PL) will need to be applied alongside the more traditional inferential methods.

Evolutionary Analysis of the Highly Conserved Insect Odorant Coreceptor (Orco) Revealed a Positive Selection Mode, Implying Functional Flexibility.

Odorant coreceptor (Orco) represents one of the essential genes in the insect olfactory system, which facilitates signal transduction and heterodimerization with different odorant receptors (Ors) in the insect antennal dendritic membrane. Evolutionary analysis by detecting positive selection is important to examine the functional flexibility of Orco that potentially supports insect survival. The maximum likelihood codon substitution model was applied using CODEML program as implemented in PAML ver 4.9e package across 59 Orco codon sequences available from GenBank. These sequences represented five major insect orders and two reproductive systems (holometabola and nonholometabola). In the site model that identified common ω values for Orco, it was clearly shown that Orco was under strong purifying selection, indicated by the ω value that was far from 1 (ω: 0.03). However, in to the branch model, positive selection was detected to be acting on Dipteran lineages, whereas in the branch-site model, several sites were under significant positive selection occurring in the following four clades: Coleoptera, Diptera, Lepidoptera, and Psocodea. The typical evolutionary mode acting on Orco was consistent with the entropy value [H(x)], confirming that 48.9% of the Orco site was under conservation (H(x) < 0.5), whereas 26.9% of the Orco sites was under high variation (H(x) > 1). These findings confirmed that Orco genes are generally highly conserved and can possibly be used for the manipulation of insect pest control programs. However, positive selection that acts on certain lineages suggested future adaptive evolutionary ability of Orco to anticipate flexible functions for successful olfactory processes.

Introduction of Viral Hemorrhagic Septicemia Virus into Freshwater Cultured Rainbow Trout Is Followed by Bursts of Adaptive Evolution.

Viral hemorrhagic septicemia virus (VHSV), a rhabdovirus infecting teleost fish, has repeatedly crossed the boundary from marine fish species to freshwater cultured rainbow trout. These naturally replicated cross-species transmission events permit the study of general and repeatable evolutionary events occurring in connection with viral emergence in a novel host species. The purpose of the present study was to investigate the adaptive molecular evolution of the VHSV glycoprotein, one of the key virus proteins involved in viral emergence, following emergence from marine species into freshwater cultured rainbow trout. A comprehensive phylogenetic reconstruction of the complete coding region of the VHSV glycoprotein was conducted, and adaptive molecular evolution was investigated using a maximum likelihood approach to compare different codon substitution models allowing for heterogeneous substitution rate ratios among amino acid sites. Evidence of positive selection was detected at six amino acid sites of the VHSV glycoprotein, within the signal peptide, the confirmation-dependent major neutralizing epitope, and the intracellular tail. Evidence of positive selection was found exclusively in rainbow trout-adapted virus isolates, and amino acid combinations found at the six sites under positive selection pressure differentiated rainbow trout- from non-rainbow trout-adapted isolates. Furthermore, four adaptive sites revealed signs of recurring identical changes across phylogenetic groups of rainbow trout-adapted isolates, suggesting that repeated VHSV emergence in freshwater cultured rainbow trout was established through convergent routes of evolution that are associated with immune escape.IMPORTANCE This study is the first to demonstrate that VHSV emergence from marine species into freshwater cultured rainbow trout has been accompanied by bursts of adaptive evolution in the VHSV glycoprotein. Furthermore, repeated detection of the same adaptive amino acid sites across phylogenetic groups of rainbow trout-adapted isolates indicates that adaptation to rainbow trout was established through parallel evolution. In addition, signals of convergent evolution toward the maintenance of genetic variation were detected in the conformation-dependent neutralizing epitope or in close proximity to disulfide bonds involved in the structural conformation of the neutralizing epitope, indicating adaptation to immune response-related genetic variation across freshwater cultured rainbow trout.

Multiple Factors Confounding Phylogenetic Detection of Selection on Codon Usage.

Detecting selection on codon usage (CU) is a difficult task, since CU can be shaped by both the mutational process and selective constraints operating at the DNA, RNA, and protein levels. Yang and Nielsen (2008) developed a test (which we call CUYN) for detecting selection on CU using two competing mutation-selection models of codon substitution. The null model assumes that CU is determined by the mutation bias alone, whereas the alternative model assumes that both mutation bias and/or selection act on CU. In applications on mammalian-scale alignments, the CUYN test detects selection on CU for numerous genes. This is surprising, given the small effective population size of mammals, and prompted us to use simulations to evaluate the robustness of the test to model violations. Simulations using a modest level of CpG hypermutability completely mislead the test, with 100% false positives. Surprisingly, a high level of false positives (56.1%) resulted simply from using the HKY mutation-level parameterization within the CUYN test on simulations conducted with a GTR mutation-level parameterization. Finally, by using a crude optimization procedure on a parameter controlling the CpG hypermutability rate, we find that this mutational property could explain a very large part of the observed mammalian CU. Altogether, our work emphasizes the need to evaluate the potential impact of model violations on statistical tests in the field of molecular phylogenetic analysis. The source code of the simulator and the mammalian genes used are available as a GitHub repository (https://github.com/Simonll/LikelihoodFreePhylogenetics.git).

Evolution of methicillin-resistant Staphylococcus aureus: Evidence of positive selection in a penicillin-binding protein (PBP) 2a coding gene mecA

Methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA) represents more and more S. aureus infections. MecA, the novel coding gene of penicillin-binding protein (PBP) 2a of MRSA, is the key resistance factor of β-lactam, but little is known about the evolution of this gene. Given the crucial role of mecA in S. aureus physiology and β-lactam resistance, the selective forces may contribute to adaptation of the bacteria to the special environments such as its host or antibiotics. To understand the evolution of this gene, we screened GenBank database and analyzed mecA of 249 S. aureus strains. Twenty-nine unique alleles with 26 unique amino acid sequences were identified. Phylogenetic analysis showed three main groups of mecA in the global S. aureus strains. Analysis of these alleles using codon-substitution models (M8, M3, and M2a) and likelihood ratio tests (LRTs) of the codeML package and a random-effects likelihood (REL) method of HyPhy package for the site-specific ratio of nonsynonymous to synonymous substitution rates suggested that fourteen sites in the allosteric domain of PBP2a have been subjected to strong positive selection pressure. Mutations of two positive selection sites (N146K and E239K) were reported to be essential for ceftaroline- or L-695, 256-resistant. Further study indicated that the positive selection pressure might be more likely related to the host's inflammatory or immune response during S. aureus infection. Our studies provide the first evidence of positive Darwinian selection in the mecA of S. aureus, contributing to a better understanding of the adaptive mechanism of this bacterium.

Large Scale Analyses and Visualization of Adaptive Amino Acid Changes Projects

When changes at few amino acid sites are the target of selection, adaptive amino acid changes in protein sequences can be identified using maximum-likelihood methods based on models of codon substitution (such as codeml). Although such methods have been employed numerous times using a variety of different organisms, the time needed to collect the data and prepare the input files means that tens or hundreds of coding regions are usually analyzed. Nevertheless, the recent availability of flexible and easy to use computer applications that collect relevant data (such as BDBM) and infer positively selected amino acid sites (such as ADOPS), means that the entire process is easier and quicker than before. However, the lack of a batch option in ADOPS, here reported, still precludes the analysis of hundreds or thousands of sequence files. Given the interest and possibility of running such large-scale projects, we have also developed a database where ADOPS projects can be stored. Therefore, this study also presents the B+ database, which is both a data repository and a convenient interface that looks at the information contained in ADOPS projects without the need to download and unzip the corresponding ADOPS project file. The ADOPS projects available at B+ can also be downloaded, unzipped, and opened using the ADOPS graphical interface. The availability of such a database ensures results repeatability, promotes data reuse with significant savings on the time needed for preparing datasets, and effortlessly allows further exploration of the data contained in ADOPS projects.

Genomic signatures of adaptation to wine biological ageing conditions in biofilm-forming flor yeasts.

The molecular and evolutionary processes underlying fungal domestication remain largely unknown despite the importance of fungi to bioindustry and for comparative adaptation genomics in eukaryotes. Wine fermentation and biological ageing are performed by strains of S.cerevisiae with, respectively, pelagic fermentative growth on glucose and biofilm aerobic growth utilizing ethanol. Here, we use environmental samples of wine and flor yeasts to investigate the genomic basis of yeast adaptation to contrasted anthropogenic environments. Phylogenetic inference and population structure analysis based on single nucleotide polymorphisms revealed a group of flor yeasts separated from wine yeasts. A combination of methods revealed several highly differentiated regions between wine and flor yeasts, and analyses using codon-substitution models for detecting molecular adaptation identified sites under positive selection in the high-affinity transporter gene ZRT1. The cross-population composite likelihood ratio revealed selective sweeps at three regions, including in the hexose transporter gene HXT7, the yapsin gene YPS6 and the membrane protein coding gene MTS27. Our analyses also revealed that the biological ageing environment has led to the accumulation of numerous mutations in proteins from several networks, including Flo11 regulation and divalent metal transport. Together, our findings suggest that the tuning of FLO11 expression and zinc transport networks are a distinctive feature of the genetic changes underlying the domestication of flor yeasts. Our study highlights the multiplicity of genomic changes underlying yeast adaptation to man-made habitats and reveals that flor/wine yeast lineage can serve as a useful model for studying the genomics of adaptive divergence.

Standard Codon Substitution Models Overestimate Purifying Selection for Nonstationary Data.

Estimation of natural selection on protein-coding sequences is a key comparative genomics approach for de novo prediction of lineage-specific adaptations. Selective pressure is measured on a per-gene basis by comparing the rate of nonsynonymous substitutions to the rate of synonymous substitutions. All published codon substitution models have been time-reversible and thus assume that sequence composition does not change over time. We previously demonstrated that if time-reversible DNA substitution models are applied in the presence of changing sequence composition, the number of substitutions is systematically biased towards overestimation. We extend these findings to the case of codon substitution models and further demonstrate that the ratio of nonsynonymous to synonymous rates of substitution tends to be underestimated over three data sets of mammals, vertebrates, and insects. Our basis for comparison is a nonstationary codon substitution model that allows sequence composition to change. Goodness-of-fit results demonstrate that our new model tends to fit the data better. Direct measurement of nonstationarity shows that bias in estimates of natural selection and genetic distance increases with the degree of violation of the stationarity assumption. Additionally, inferences drawn under time-reversible models are systematically affected by compositional divergence. As genomic sequences accumulate at an accelerating rate, the importance of accurate de novo estimation of natural selection increases. Our results establish that our new model provides a more robust perspective on this fundamental quantity.

Positive selection in cathelicidin host defense peptides: adaptation to exogenous pathogens or endogenous receptors?

The cause of adaptive protein evolution includes internal (for example, co-evolution of ligand-receptor pairs) and external (for example, adaptation to different ecological niches) mechanisms. Host defense peptides (HDPs) are a class of vertebrate-specific cationic antimicrobial peptides evolving under positive selection. Besides their antibiotic activity, HDPs also exert an effect on multiple host immune cells, thus providing an ideal model to study selective agents driving their evolution. On the basis of a combination of computational and experimental approaches, we studied the evolution of LL-37-type HDPs in mammals, the mature peptide of cathelicidin CAP18 (herein termed CAP18-MP) and investigated the driving force behind the evolution. Using codon-substitution maximum likelihood models, we analyzed CAP18-MPs in 40 species belonging to nine mammalian Orders and identified four positively selected sites (PSSs) that all are located on two terminal unordered regions of CAP18-MPs. Grafting the two positively selected regions of human or whale CAP18-MP to the α-helical scaffold of a rabbit homolog (substituting its corresponding parts) led to no alterations in antibacterial activity, spectrum and action mode. Likewise, further deletion of the two terminal regions did not alter its functional features. Evolutionary conservation analysis of mammalian FPR2, a receptor known to interact with the C-terminal positively selected region of LL-37, revealed high evolutionary variability in its ligand-binding extracellular loop domains, matching sequence diversity of the unordered regions in CAP18-MPs. This is the first report describing that the signature of positive selection of cathelicidins is not associated with their direct bactericidal activity, but rather with the evolutionary variability of their endogenous receptors.