Analysis of selection in protein-coding sequences accounting for common biases.

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.

The influence of heterogeneous codon frequencies along sequences on the estimation of molecular adaptation.

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.

Analyzing the functional divergence of Slo1 and Slo3 channel subfamilies.

Slo1 and Slo3 encode close paralogues of the Slo potassium (K+) channels family. Despite their evolutionary relatedness, Slo1 and Slo3 channels show marked functional differences and evolutionary dynamics. Whereas Slo1 is a highly conserved and widely expressed channel, Slo3 is a rapidly evolving channel restricted to sperm. However, the molecular mechanisms behind the structural-functional differences of Slo1 and Slo3 channels are unknown. In this study, we explored the functional divergence of Slo1 and Slo3 subfamilies in vertebrates and examined the structure-function relationships of our predictions using experimental data. We found that ∼25% of sites between Slo1 and Slo3 underwent altered functional constraints, affecting some residues with important roles in Slo1 channel gating. Because functional divergence was principally generated by accelerated evolution of Slo3 after gene duplication, we explored selective forces behind Slo3 diversification. We observed that Slo3 subjected was principally subjected to relaxation of purifying selection, but we also identified several sites evolving under positive selection in the cytosolic domain of this channel . Concerning Slo1, this channel presented strong purifying selection. Whether residues evolving under different selection in Slo1 and Slo3 are responsible for functional differences observed between these channels, as well as among Slo3 orthologs, remains to be established.

A cytoplasmic Slo3 isoform is expressed in somatic tissues.

Slo3 is a pH-sensitive and weakly voltage-sensitive potassium channel that is essential for male fertility in mouse and whose expression is regarded as sperm-specific. These properties have proposed Slo3 as a candidate target for male contraceptive drugs. Nonetheless, the tissue distribution of Slo3 expression has not been rigorously studied yet. Applying computational and RT-PCR approaches, we identified expression of two short Slo3 isoforms in somatic mouse tissues such as brain, kidney and eye. These isoforms, which seem to result of transcription starting sites between exons 20 and 21, have an identical open reading frame, both encoding the terminal 381 amino acids of the cytosolic Slo3 domain. We corroborated the expression of these isoforms in mouse brain and testis by Western-blot. The complete isoform encoding the Slo3 ion channel was uniquely detected in testis, both at transcript and protein level. Although the functional role of the cytosolic Slo3 isoforms remains to be established, we propose that they may have a functional effect by modulating Slo channels trafficking and/or activity. This study confirms that expression of full-length Slo3 is sperm-specific but warns against developing contraceptive drugs targeting the C-terminal tail of Slo3 channels.

Comparative Sperm Proteomics in Mouse Species with Divergent Mating Systems.

Sexual selection is the pervasive force underlying the dramatic divergence of sperm form and function. Although it has been demonstrated that testis gene expression evolves rapidly, exploration of the proteomic basis of sperm diversity is in its infancy. We have employed a whole-cell proteomics approach to characterize sperm divergence among closely related Mus species that experience different sperm competition regimes and exhibit pronounced variation in sperm energetics, motility and fertilization capacity. Interspecific comparisons revealed significant abundance differences amongst proteins involved in fertilization capacity, including those that govern sperm-zona pellucida interactions, axoneme components and metabolic proteins. Ancestral reconstruction of relative testis size suggests that the reduction of zona pellucida binding proteins and heavy-chain dyneins was associated with a relaxation in sperm competition in the M. musculus lineage. Additionally, the decreased reliance on ATP derived from glycolysis in high sperm competition species was reflected in abundance decreases in glycolytic proteins of the principle piece in M. spretus and M. spicilegus. Comparison of protein abundance and stage-specific testis expression revealed a significant correlation during spermatid development when dynamic morphological changes occur. Proteins underlying sperm diversification were also more likely to be subject to translational repression, suggesting that sperm composition is influenced by the evolution of translation control mechanisms. The identification of functionally coherent classes of proteins relating to sperm competition highlights the utility of evolutionary proteomic analyses and reveals that both intensified and relaxed sperm competition can have a pronounced impact on the molecular composition of the male gamete.

Positive Selection in the Evolution of Mammalian CRISPs.

Cysteine-RIch Secretory Proteins (CRISPs) constitute a versatile family, with functions in reptilian venom and mammalian reproduction. Mammals generally express three CRISPs, four in mice, and all are highly expressed in male reproductive tissues, either testis or accessory organs. Because reproductive proteins often evolve adaptively in response to post-copulatory sexual selection, we hypothesized that mammalian CRISPs, with important roles in male reproduction, could have undergone positive selection promoting their divergence. We explored the molecular adaptation of mammalian CRISPs applying phylogenetic methods. Our analyses revealed the evidence of positive selection in all mammalian CRISPs. The intensity of positive selection was heterogeneous among CRISP members, being stronger in CRISP3 than in CRISP1 and CRISP2, and also across functional domains, having stronger impact on Pathogenesis-Related 1 (PR-1) in CRISP2 and on Ion Channel Regulator (ICR) in CRISP1 and CRISP3. In addition, we discovered a new CRISP in some rodent species, suggesting that the acquisition of new CRISP components could contribute to male reproductive success or to acquire new physiological roles. Signatures of positive selection were not focused on any particular mammalian group, suggesting that adaptive evolution is a recurrent pattern in mammalian CRISPs. Our findings support a model of CRISP family diversification driven by episodes of duplication and posterior neofunctionalization, and provide potential adaptive changes responsible for interspecific differences in CRISPs activity.

Sexual selection and the adaptive evolution of PKDREJ protein in primates and rodents.

PKDREJ is a testis-specific protein thought to be located on the sperm surface. Functional studies in the mouse revealed that loss of PKDREJ has effects on sperm transport and the ability to undergo an induced acrosome reaction. Thus, PKDREJ has been considered a potential target of post-copulatory sexual selection in the form of sperm competition. Proteins involved in reproductive processes often show accelerated evolution. In many cases, this rapid divergence is promoted by positive selection which may be driven, at least in part, by post-copulatory sexual selection. We analysed the evolution of the PKDREJ protein in primates and rodents and assessed whether PKDREJ divergence is associated with testes mass relative to body mass, which is a reliable proxy of sperm competition levels. Evidence of an association between the evolutionary rate of the PKDREJ gene and testes mass relative to body mass was not found in primates. Among rodents, evidence of positive selection was detected in the Pkdrej gene in the family Cricetidae but not in Muridae. We then assessed whether Pkdrej divergence is associated with episodes of sperm competition in these families. We detected a positive significant correlation between the evolutionary rates of Pkdrej and testes mass relative to body mass in cricetids. These findings constitute the first evidence of post-copulatory sexual selection influencing the evolution of a protein that participates in the mechanisms regulating sperm transport and the acrosome reaction, strongly suggesting that positive selection may act on these fertilization steps, leading to advantages in situations of sperm competition.

Structural evolution of CatSper1 in rodents is influenced by sperm competition, with effects on sperm swimming velocity.

BACKGROUND: Competition between spermatozoa from rival males for success in fertilization (i.e., sperm competition) is an important selective force driving the evolution of male reproductive traits and promoting positive selection in genes related to reproductive function. Positive selection has been identified in reproductive proteins showing rapid divergence at nucleotide level. Other mutations, such as insertions and deletions (indels), also occur in protein-coding sequences. These structural changes, which exist in reproductive genes and result in length variation in coded proteins, could also be subjected to positive selection and be under the influence of sperm competition. Catsper1 is one such reproductive gene coding for a germ-line specific voltage-gated calcium channel essential for sperm motility and fertilization. Positive selection appears to promote fixation of indels in the N-terminal region of CatSper1 in mammalian species. However, it is not known which selective forces underlie these changes and their implications for sperm function. RESULTS: We tested if length variation in the N-terminal region of CatSper1 is influenced by sperm competition intensity in a group of closely related rodent species of the subfamily Murinae. Our results revealed a negative correlation between sequence length of CatSper1 and relative testes mass, a very good proxy of sperm competition levels. Since CatSper1 is important for sperm flagellar motility, we examined if length variation in the N-terminus of CatSper1 is linked to changes in sperm swimming velocity. We found a negative correlation between CatSper1 length and several sperm velocity parameters. CONCLUSIONS: Altogether, our results suggest that sperm competition selects for a shortening of the intracellular region of CatSper1 which, in turn, enhances sperm swimming velocity, an essential and adaptive trait for fertilization success.

Coevolution of positively selected IZUMO1 and CD9 in rodents: evidence of interaction between gamete fusion proteins?

Proteins involved in sexual reproduction are known to evolve rapidly, often as the result of positive Darwinian selection, although the selective forces driving such adaptive changes are poorly understood. A process of coevolution between proteins in male and female gametes may promote rapid divergence of fertilization proteins. In the mouse, only two proteins have been shown so far to be essential for sperm-egg fusion, IZUMO1 in the sperm cell and CD9 in the egg. The role of these proteins has not been fully elucidated, and it has been suggested that they may act as fusogens, interacting in trans with proteins on the other cell, or regulators of fusogens through cis interactions. Here we analyze the evolution of IZUMO1 and CD9 in a group of rodent species. To assess possible protein interactions between IZUMO1 and CD9, we examined potential coevolution based on analyses of correlated evolutionary rates. We found evidence that both proteins evolve adaptively, with a more intense signal of positive selection in IZUMO1. In addition, our findings suggest that these proteins may have some form of interaction, although they have not been regarded as fusogens interacting directly with each other. The adaptive divergence of IZUMO1 and CD9 could influence reproductive compatibility, and, thus, these proteins may participate in the establishment of specific sperm-egg recognition systems. Further studies are required to uncover the role of IZUMO1 and CD9 during gamete fusion in order to understand the molecular basis of their coevolution, as other selective forces could also lead to general signatures of coevolution.

Evolution of protamine genes and changes in sperm head phenotype in rodents.

Little is known about the genetic basis of evolutionary changes in sperm phenotype. Postcopulatory sexual selection is associated with differences in protamine gene sequences and promoters and is a powerful force acting on sperm form and function, although links between protamine evolution and sperm phenotype are scarce. Protamines are involved in sperm chromatin condensation, and protamine deficiency negatively affects sperm morphology and male fertility, thus suggesting that they are important for sperm design and function. We examined changes in protamine genes and sperm phenotype in rodents to understand the role of sexual selection on protamine evolution and sperm design. We performed a genotype-phenotype association study using root-to-tip dN/dS (nonsynonymous/synonymous substitutions rate ratio) to account for evolutionary rates and phylogenetic generalized least squares analyses to compare genetic and morphometric data. Evolutionary rates of protamine 1 and the protamine 2 domain cleaved off during chromatin condensation correlated with head size and elongation. Protamine 1 exhibited restricted positive selection on some functional sites, which seemed sufficient to preserve its role in head design. The cleaved-protamine 2, whose relaxation is halted by sexual selection, seems to ensure small, elongated heads that would make sperm more competitive. No association existed between mature-protamine 2 and head phenotype, suggesting little involvement during chromatin condensation and a likely role maintaining the condensed state. Our results suggest that evolutionary changes in protamines could be related to complex developmental modifications in the sperm head. This represents an important step toward understanding the role of changes in gene coding sequences in the divergence of germ cell phenotype.

Evolutionary history of dimethylsulfoniopropionate (DMSP) demethylation enzyme DmdA in marine bacteria.

Dimethylsulfoniopropionate (DMSP), an osmolyte produced by oceanic phytoplankton and bacteria, is primarily degraded by bacteria belonging to the Roseobacter lineage and other marine Alphaproteobacteria via DMSP-dependent demethylase A protein (DmdA). To date, the evolutionary history of DmdA gene family is unclear. Some studies indicate a common ancestry between DmdA and GcvT gene families and a co-evolution between Roseobacter and the DMSP-producing-phytoplankton around 250 million years ago (Mya). In this work, we analyzed the evolution of DmdA under three possible evolutionary scenarios: (1) a recent common ancestor of DmdA and GcvT, (2) a coevolution between Roseobacter and the DMSP-producing-phytoplankton, and (3) an enzymatic adaptation for utilizing DMSP in marine bacteria prior to Roseobacter origin. Our analyses indicate that DmdA is a new gene family originated from GcvT genes by duplication and functional divergence driven by positive selection before a coevolution between Roseobacter and phytoplankton. Our data suggest that Roseobacter acquired dmdA by horizontal gene transfer prior to an environment with higher DMSP. Here, we propose that the ancestor that carried the DMSP demethylation pathway genes evolved in the Archean, and was exposed to a higher concentration of DMSP in a sulfur-rich atmosphere and anoxic ocean, compared to recent Roseobacter eco-orthologs (orthologs performing the same function under different conditions), which should be adapted to lower concentrations of DMSP.

Selective Pressures on Human Cancer Genes along the Evolution of Mammals.

Cancer is a disease driven by both somatic mutations that increase survival and proliferation of cell lineages and the evolution of genes associated with cancer risk in populations. Several genes associated with cancer in humans, hereafter cancer genes, show evidence of germline positive selection among species. Taking advantage of a large collection of mammalian genomes, we systematically looked for signatures of germline positive selection in 430 cancer genes available in COSMIC. We identified 40 cancer genes with a robust signal of positive selection in mammals. We found evidence for fewer selective constraints-higher number of non-synonymous substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site (dN/dS)-and higher incidence of positive selection-more positively selected sites-in cancer genes bearing germline and recessive mutations that predispose to cancer. This finding suggests a potential association between relaxed selection, positive selection, and risk of hereditary cancer. On the other hand, we did not find significant differences in terms of tissue or gene type. Human cancer genes under germline positive selection in mammals are significantly enriched in the processes of DNA repair, with high presence of Fanconi anaemia/Breast Cancer A (FA/BRCA) pathway components and T cell proliferation genes. We also show that the inferred positively selected sites in the two genes with the strongest signal of positive selection, i.e., BRCA2 and PTPRC, are in regions of functional relevance, which could be relevant to cancer susceptibility.

Premammalian origin of the sperm-specific Slo3 channel.

Slo3 is a sperm-specific potassium (K+) channel essential for male fertility. Slo3 channels have so far been considered to be specific to mammals. Through exploratory genomics, we identified the Slo3 gene in the genome of terrestrial (birds and reptiles) and aquatic (fish) vertebrates. In the case of fish, Slo3 has undergone several episodes of gene loss. Transcriptomic analysis showed that vertebrate Slo3 transcript orthologues are predominantly expressed in testis, in concordance with the mammalian Slo3. We conclude that the Slo3 gene arose during the radiation of early vertebrates, much earlier than previously thought. Our findings add to the growing evidence indicating that the phylogenetic profiles of sperm-specific channels are intermittent throughout metazoan evolution, which probably reflects the adaptation of sperm to different ionic milieus and fertilization environments.

Proteins involved in motility and sperm-egg interaction evolve more rapidly in mouse spermatozoa.

Proteomic studies of spermatozoa have identified a large catalog of integral sperm proteins. Rapid evolution of these proteins may underlie adaptive changes of sperm traits involved in different events leading to fertilization, although the selective forces underlying such rapid evolution are not well understood. A variety of selective forces may differentially affect several steps ending in fertilization, thus resulting in a compartmentalized adaptation of sperm proteins. Here we analyzed the evolution of genes associated to various events in the sperm's life, from sperm formation to sperm-egg interaction. Evolutionary analyses were performed on gene sequences from 17 mouse strains whose genomes have been sequenced. Four of these are derived from wild Mus musculus, M. domesticus, M. castaneus and M. spretus. We found a higher proportion of genes exhibiting a signature of positive selection among those related to sperm motility and sperm-egg interaction. Furthermore, sperm proteins involved in sperm-egg interaction exhibited accelerated evolution in comparison to those involved in other events. Thus, we identified a large set of candidate proteins for future comparative analyses of genotype-phenotype associations in spermatozoa of species subjected to different sexual selection pressures. Adaptive evolution of proteins involved in motility could be driven by sperm competition, since this selective force is known to increase the proportion of motile sperm and their swimming velocity. On the other hand, sperm proteins involved in gamete interaction could be coevolving with their egg partners through episodes of sexual selection or sexual conflict resulting in species-specific sperm-egg interactions and barriers preventing interspecies fertilization.

Sexual selection halts the relaxation of protamine 2 among rodents.

Sexual selection has been proposed as the driving force promoting the rapid evolutionary changes observed in some reproductive genes including protamines. We test this hypothesis in a group of rodents which show marked differences in the intensity of sexual selection. Levels of sperm competition were not associated with the evolutionary rates of protamine 1 but, contrary to expectations, were negatively related to the evolutionary rate of cleaved- and mature-protamine 2. Since both domains were found to be under relaxation, our findings reveal an unforeseen role of sexual selection: to halt the degree of degeneration that proteins within families may experience due to functional redundancy. The degree of relaxation of protamine 2 in this group of rodents is such that in some species it has become dysfunctional and it is not expressed in mature spermatozoa. In contrast, protamine 1 is functionally conserved but shows directed positive selection on specific sites which are functionally relevant such as DNA-anchoring domains and phosphorylation sites. We conclude that in rodents protamine 2 is under relaxation and that sexual selection removes deleterious mutations among species with high levels of sperm competition to maintain the protein functional and the spermatozoa competitive.