MOTIVATION: The selection among substitution models of molecular evolution is fundamental for obtaining accurate phylogenetic inferences. At the protein level, evolutionary analyses are traditionally based on empirical substitution models but these models make unrealistic assumptions and are being surpassed by structurally constrained substitution (SCS) models. The SCS models often consider site-dependent evolution, a process that provides realism but complicates their implementation into likelihood functions that are commonly used for substitution model selection. RESULTS: We present a method to perform selection among site-dependent SCS models, also among empirical and site-dependent SCS models, based on the approximate Bayesian computation (ABC) approach and its implementation into the computational framework ProteinModelerABC. The framework implements ABC with and without regression adjustments and includes diverse empirical and site-dependent SCS models of protein evolution. Using extensive simulated data, we found that it provides selection among SCS and empirical models with acceptable accuracy. As illustrative examples, we applied the framework to analyze a variety of protein families observing that SCS models fit them better than the corresponding best-fitting empirical substitution models. AVAILABILITY AND IMPLEMENTATION: ProteinModelerABC is freely available from https://github.com/DavidFerreiro/ProteinModelerABC, can run in parallel and includes a graphical user interface. The framework is distributed with detailed documentation and ready-to-use examples.

The evolution of protein-coding genes is usually driven by selective processes, which favor some evolutionary trajectories over others, optimizing the subsequent protein stability and activity. The analysis of selection in this type of genetic data is broadly performed with the metric nonsynonymous/synonymous substitution rate ratio (dN/dS). However, most of the well-established methodologies to estimate this metric make crucial assumptions, such as lack of recombination or invariable codon frequencies along genes, which can bias the estimation. Here, we review the most relevant biases in the dN/dS estimation and provide a detailed guide to estimate this metric using state-of-the-art procedures that account for such biases, along with illustrative practical examples and recommendations. We also discuss the traditional interpretation of the estimated dN/dS emphasizing the importance of considering complementary biological information such as the role of the observed substitutions on the stability and function of proteins. This review is oriented to help evolutionary biologists that aim to accurately estimate selection in protein-coding sequences.

MOTIVATION: The evolutionary processes of mutation and recombination, upon which selection operates, are fundamental to understand the observed molecular diversity. Unlike nucleotide sequences, the estimation of the recombination rate in protein sequences has been little explored, neither implemented in evolutionary frameworks, despite protein sequencing methods are largely used. RESULTS: In order to accommodate this need, here I present a computational framework, called ProteinEvolverABC, to jointly estimate recombination and substitution rates from alignments of protein sequences. The framework implements the approximate Bayesian computation approach, with and without regression adjustments and includes a variety of substitution models of protein evolution, demographics and longitudinal sampling. It also implements several nuisance parameters such as heterogeneous amino acid frequencies and rate of change among sites and, proportion of invariable sites. The framework produces accurate coestimation of recombination and substitution rates under diverse evolutionary scenarios. As illustrative examples of usage, I applied it to several viral protein families, including coronaviruses, showing heterogeneous substitution and recombination rates. AVAILABILITY AND IMPLEMENTATION: ProteinEvolverABC is freely available from https://github.com/miguelarenas/proteinevolverabc, includes a graphical user interface for helping the specification of the input settings, extensive documentation and ready-to-use examples. Conveniently, the simulations can run in parallel on multicore machines. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: The nonsynonymous/synonymous substitution rate ratio (dN/dS) is a commonly used parameter to quantify molecular adaptation in protein-coding data. It is known that the estimation of dN/dS can be biased if some evolutionary processes are ignored. In this concern, common ML methods to estimate dN/dS assume invariable codon frequencies among sites, despite this characteristic is rare in nature, and it could bias the estimation of this parameter. RESULTS: Here we studied the influence of variable codon frequencies among genetic regions on the estimation of dN/dS. We explored scenarios varying the number of genetic regions that differ in codon frequencies, the amount of variability of codon frequencies among regions and the nucleotide frequencies at each codon position among regions. We found that ignoring heterogeneous codon frequencies among regions overall leads to underestimation of dN/dS and the bias increases with the level of heterogeneity of codon frequencies. Interestingly, we also found that varying nucleotide frequencies among regions at the first or second codon position leads to underestimation of dN/dS while variation at the third codon position leads to overestimation of dN/dS. Next, we present a methodology to reduce this bias based on the analysis of partitions presenting similar codon frequencies and we applied it to analyze four real datasets. We conclude that accounting for heterogeneous codon frequencies along sequences is required to obtain realistic estimates of molecular adaptation through this relevant evolutionary parameter. AVAILABILITY AND IMPLEMENTATION: The applied frameworks for the computer simulations of protein-coding data and estimation of molecular adaptation are SGWE and PAML, respectively. Both are publicly available and referenced in the study. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SUMMARY: SPLATCHE3 simulates genetic data under a variety of spatially explicit evolutionary scenarios, extending previous versions of the framework. The new capabilities include long-distance migration, spatially and temporally heterogeneous short-scale migrations, alternative hybridization models, simulation of serial samples of genetic data and a large variety of DNA mutation models. These implementations have been applied independently to various studies, but grouped together in the current version. AVAILABILITY AND IMPLEMENTATION: SPLATCHE3 is written in C++ and is freely available for non-commercial use from the website http://www.splatche.com/splatche3. It includes console versions for Linux, MacOs and Windows and a user-friendly GUI for Windows, as well as detailed documentation and ready-to-use examples.

MOTIVATION: Models of molecular evolution aim at describing the evolutionary processes at the molecular level. However, current models rarely incorporate information from protein structure. Conversely, structure-based models of protein evolution have not been commonly applied to simulate sequence evolution in a phylogenetic framework, and they often ignore relevant evolutionary processes such as recombination. A simulation evolutionary framework that integrates substitution models that account for protein structure stability should be able to generate more realistic in silico evolved proteins for a variety of purposes. RESULTS: We developed a method to simulate protein evolution that combines models of protein folding stability, such that the fitness depends on the stability of the native state both with respect to unfolding and misfolding, with phylogenetic histories that can be either specified by the user or simulated with the coalescent under complex evolutionary scenarios, including recombination, demographics and migration. We have implemented this framework in a computer program called ProteinEvolver. Remarkably, comparing these models with empirical amino acid replacement models, we found that the former produce amino acid distributions closer to distributions observed in real protein families, and proteins that are predicted to be more stable. Therefore, we conclude that evolutionary models that consider protein stability and realistic evolutionary histories constitute a better approximation of the real evolutionary process.

This study is intended for evolutionary biologists interested in strategies for the simulation of molecular data under diverse evolutionary scenarios. It begins with a brief background on simulation approaches and describes some of the most important simulators developed to date. Then, several practical examples for simulating particular scenarios are presented, and finally, details are given on a variety of relevant applications of simulated data. Overall, this study provides a practical guide for applying simulation techniques to real world problems in molecular evolution.

BACKGROUND: Typical evolutionary events like recombination, hybridization or gene transfer make necessary the use of phylogenetic networks to properly depict the evolution of DNA and protein sequences. Although several theoretical classes have been proposed to characterize these networks, they make stringent assumptions that will likely not be met by the evolutionary process. We have recently shown that the complexity of simulated networks is a function of the population recombination rate, and that at moderate and large recombination rates the resulting networks cannot be categorized. However, we do not know whether these results extend to networks estimated from real data. RESULTS: We introduce a web server for the categorization of explicit phylogenetic networks, including the most relevant theoretical classes developed so far. Using this tool, we analyzed statistical parsimony phylogenetic networks estimated from approximately 5,000 DNA alignments, obtained from the NCBI PopSet and Polymorphix databases. The level of characterization was correlated to nucleotide diversity, and a high proportion of the networks derived from these data sets could be formally characterized. CONCLUSIONS: We have developed a public web server, NetTest (freely available from the software section at http://darwin.uvigo.es), to formally characterize the complexity of phylogenetic networks. Using NetTest we found that most statistical parsimony networks estimated with the program TCS could be assigned to a known network class. The level of network characterization was correlated to nucleotide diversity and dependent upon the intra/interspecific levels, although no significant differences were detected among genes. More research on the properties of phylogenetic networks is clearly needed.

BACKGROUND: Coalescent simulations have proven very useful in many population genetics studies. In order to arrive to meaningful conclusions, it is important that these simulations resemble the process of molecular evolution as much as possible. To date, no single coalescent program is able to simulate codon sequences sampled from populations with recombination, migration and growth. RESULTS: We introduce a new coalescent program, called Recodon, which is able to simulate samples of coding DNA sequences under complex scenarios in which several evolutionary forces can interact simultaneously (namely, recombination, migration and demography). The basic codon model implemented is an extension to the general time-reversible model of nucleotide substitution with a proportion of invariable sites and among-site rate variation. In addition, the program implements non-reversible processes and mixtures of different codon models. CONCLUSION: Recodon is a flexible tool for the simulation of coding DNA sequences under realistic evolutionary models. These simulations can be used to build parameter distributions for testing evolutionary hypotheses using experimental data. Recodon is written in C, can run in parallel, and is freely available from http://darwin.uvigo.es/.