Signatures of natural selection in tree topology shape of serially sampled viral phylogenies.

Tree shape metrics can be computed fast for trees of any size, which makes them promising alternatives to intensive statistical methods and parameter-rich evolutionary models in the era of massive data availability. Previous studies have demonstrated their effectiveness in unveiling important parameters in viral evolutionary dynamics, although the impact of natural selection on the shape of tree topologies has not been thoroughly investigated. We carried out a forward-time and individual-based simulation to investigate whether tree shape metrics of several kinds could predict the selection regime employed to generate the data. To examine the impact of the genetic diversity of the founder viral population, simulations were run under two opposing starting configurations of the genetic diversity of the infecting viral population. We found that four evolutionary regimes, namely, negative, positive, and frequency-dependent selection, as well as neutral evolution, were successfully distinguished by tree topology shape metrics. Two metrics from the Laplacian spectral density profile (principal eigenvalue and peakedness) and the number of cherries were the most informative for indicating selection type. The genetic diversity of the founder population had an impact on differentiating evolutionary scenarios. Tree imbalance, which has been frequently associated with the action of natural selection on intrahost viral diversity, was also characteristic of neutrally evolving serially sampled data. Metrics calculated from empirical analysis of HIV datasets indicated that most tree topologies exhibited shapes closer to the frequency-dependent selection or neutral evolution regimes.

Assessing the relative performance of fast molecular dating methods for phylogenomic data.

Advances in genome sequencing techniques produced a significant growth of phylogenomic datasets. This massive amount of data represents a computational challenge for molecular dating with Bayesian approaches. Rapid molecular dating methods have been proposed over the last few decades to overcome these issues. However, a comparative evaluation of their relative performance on empirical data sets is lacking. We analyzed 23 empirical phylogenomic datasets to investigate the performance of two commonly employed fast dating methodologies: penalized likelihood (PL), implemented in treePL, and the relative rate framework (RRF), implemented in RelTime. They were compared to Bayesian analyses using the closest possible substitution models and calibration settings. We found that RRF was computationally faster and generally provided node age estimates statistically equivalent to Bayesian divergence times. PL time estimates consistently exhibited low levels of uncertainty. Overall, to approximate Bayesian approaches, RelTime is an efficient method with significantly lower computational demand, being more than 100 times faster than treePL. Thus, to alleviate the computational burden of Bayesian divergence time inference in the era of massive genomic data, molecular dating can be facilitated using the RRF, allowing evolutionary hypotheses to be tested more quickly and efficiently.

The performance of outgroup-free rooting under evolutionary radiations.

Tree rooting implies a temporal dimension to phylogenies. Only after defining the position of the root node is that the ancestral-descendant relationship between branches can be fully deduced. Rooting has been usually carried out by employing evolutionarily close outgroup lineages, which is a drawback when these lineages are unavailable or unknown. Alternatively, outgroup-free rooting methods were proposed, which rely on the constancy of evolutionary rates to varying degrees. In this work we analyzed the performance of two of these methods, the midpoint rooting (MPR) and the minimal ancestor deviation (MAD), in rooting topologies evolved under challenging scenarios of fast evolutionary radiations derived from empirical data, characterized by short internal branches near the crown node. Considering all branch length combinations investigated, both methods exhibited average success rates below 50%, although MAD slightly outperformed MPR. Moreover, tree balance significantly impacted the relative performance of the methods. We found that, in four-taxa unrooted trees, the outcome of whether both methodologies will correctly root the tree can be roughly predicted by two simple dimensionless metrics: the coefficient of variation of the external branch lengths, and the ratio between the internal branch length to the total sum of branch lengths, which were employed to devise a general linear model that allowed calculating the probability of correct placing the root node for any four-taxa tree. We predicted that the performance of both outgroup-free rooting methods on loci representing the placental mammal radiation ranged between 50% and 75%.

Challenges in estimating virus divergence times in short epidemic timescales with special reference to the evolution of SARS-CoV-2 pandemic.

The estimation of evolutionary parameters provides essential information for designing public health policies. In short time intervals, however, nucleotide substitutions are ineffective to record all complexities of virus population dynamics. In this sense, the current SARS-CoV-2 pandemic poses a challenge for evolutionary analysis. We used computer simulation to evolve populations in scenarios of varying temporal intervals to evaluate the impact of the age of an epidemic on estimates of time and geography. Before estimating virus timescales, the shape of tree topologies can be used as a proxy to assess the effectiveness of the virus phylogeny in providing accurate estimates of evolutionary parameters. In short timescales, estimates have larger uncertainty. We compared the predictions from simulations with empirical data. The tree shape of SARS-CoV-2 was closer to shorter timescales scenarios, which yielded parametric estimates with larger uncertainty, suggesting that estimates from these datasets should be evaluated cautiously. To increase the accuracy of the estimates of virus transmission times between populations, the uncertainties associated with the age estimates of both the crown and stem nodes should be communicated. We place the age of the common ancestor of the current SARS-CoV-2 pandemic in late September 2019, corroborating an earlier emergence of the virus.

Phylogenetic analysis of the ATP-binding cassette proteins suggests a new ABC protein subfamily J in Aedes aegypti (Diptera: Culicidae).

BACKGROUND: We performed an in-depth analysis of the ABC gene family in Aedes aegypti (Diptera: Culicidae), which is an important vector species of arthropod-borne viral infections such as chikungunya, dengue, and Zika. Despite its importance, previous studies of the Arthropod ABC family have not focused on this species. Reports of insecticide resistance among pests and vectors indicate that some of these ATP-dependent efflux pumps are involved in compound traffic and multidrug resistance phenotypes. RESULTS: We identified 53 classic complete ABC proteins annotated in the A. aegypti genome. A phylogenetic analysis of Aedes aegypti ABC proteins was carried out to assign the novel proteins to the ABC subfamilies. We also determined 9 full-length sequences of DNA repair (MutS, RAD50) and structural maintenance of chromosome (SMC) proteins that contain the ABC signature. CONCLUSIONS: After inclusion of the putative ABC proteins into the evolutionary tree of the gene family, we classified A. aegypti ABC proteins into the established subfamilies (A to H), but the phylogenetic positioning of MutS, RAD50 and SMC proteins among ABC subfamilies-as well as the highly supported grouping of RAD50 and SMC-prompted us to name a new J subfamily of A. aegypti ABC proteins.

Survey for positively selected coding regions in the genome of the hematophagous tsetse fly Glossina morsitans identifies candidate genes associated with feeding habits and embryonic development.

Tsetse flies are responsible for the transmission of Trypanossoma sp. to vertebrate animals in Africa causing huge health issues and economic loss. The availability of the genome sequence of Glossina morsitans enabled the discovery of several genes related to medically important phenotypes and novel physiological features. However, a genome-wide scan for coding regions that underwent positive selection is still missing, which is surprising given the evolution of traits associated with the hematophagy in this lineage. In this study, we employed an experimental design that controlled for the rate of false positives and we performed a scan of 3,318 G. morsitans genes. We found 145 genes with significant historical signal of positive selection. These genes were categorized into 18 functional classes after careful manual annotation. Based on their attributed functions, we identified candidate genes related with feeding habits and embryonic development. When our results were contrasted with gene expression data, we confirmed that most genes that underwent adaptive molecular evolution were frequently expressed in organs associated with key physiological evolutionary innovations in the G. morsitans lineage, namely, the salivary gland, the midgut, fat body tissue, and in the spermatophore.

The range of sampling times affects Zika virus evolutionary rates and divergence times.

The rate of evolution of viral genomes is a fundamental parameter for understanding the origin and spread of epidemics. For instance, molecular dating is one of the many practical outcomes of evolutionary rate estimation. In this sense, the rate of evolution of ZIKV merits attention, because it has been shown to be higher than the average rate reported for other flaviviruses. It has been hypothesized that the higher rate of ZIKV evolution is due to a bias related to the analysis of sequences collected within a short time range, which would increase the chance of sampling slightly deleterious nucleotide polymorphisms. To investigate this hypothesis, we assembled datasets with different ranges of sampling times and also decomposed the ZIKV evolutionary rate into synonymous and non-synonymous rates. Our results demonstrated that the rate of ZIKV evolution is time dependent and that the observed increase in short-term rates is largely accounted for by a higher non-synonymous rate, suggesting the presence of slightly deleterious variants not yet eliminated by purifying selection. On the other hand, we show that synonymous rates were less impacted by the range of sampling times, generating timescales with reduced uncertainty. We conclude that, for inferring the ZIKV timescale and reconstructing the history of epidemics, synonymous changes are the most appropriate substitution type to be examined. We were able to obtain ZIKV divergence times that were time independent and exhibited greater precision than previous estimates. This observation should also hold for other serially sampled fast-evolving pathogens with evidence of time dependence of evolutionary rates.

The Estimated Pacemaker for Great Apes Supports the Hominoid Slowdown Hypothesis.

The recent surge of genomic data has prompted the investigation of substitution rate variation across the genome, as well as among lineages. Evolutionary trees inferred from distinct genomic regions may display branch lengths that differ between loci by simple proportionality constants, indicating that rate variation follows a pacemaker model, which may be attributed to lineage effects. Analyses of genes from diverse biological clades produced contrasting results, supporting either this model or alternative scenarios where multiple pacemakers exist. So far, an evaluation of the pacemaker hypothesis for all great apes has never been carried out. In this work, we tested whether the evolutionary rates of hominids conform to pacemakers, which were inferred accounting for gene tree/species tree discordance. For higher precision, substitution rates in branches were estimated with a calibration-free approach, the relative rate framework. A predominant evolutionary trend in great apes was evidenced by the recovery of a large pacemaker, encompassing most hominid genomic regions. In addition, the majority of genes followed a pace of evolution that was closely related to the strict molecular clock. However, slight rate decreases were recovered in the internal branches leading to humans, corroborating the hominoid slowdown hypothesis. Our findings suggest that in great apes, life history traits were the major drivers of substitution rate variation across the genome.

A multigene timescale and diversification dynamics of Ciliophora evolution.

Ciliophora is one of the most diverse lineages of unicellular eukaryotes. Nevertheless, a robust timescale including all main lineages and employing properly identified ciliate fossils as primary calibrations is lacking. Here, we inferred a time-calibrated multigene phylogeny of Ciliophora evolution, and we used this timetree to investigate the rates and patterns of lineage diversification through time. We implemented a two-step analytical approach that favored both gene and taxon sampling, reducing the uncertainty of time estimates and yielding narrower credibility intervals on the ribosomal-derived chronogram. We estimate the origin of Ciliophora at 1143 Ma, which is substantially younger than previously proposed ages, and the huge diversity explosion occurred during the Paleozoic. Among the current groups recognized as classes, Spirotrichea diverged earlier, its origin was dated at ca. 850 Ma, and Protocruziea was the younger class, with crown age estimated at 56 Ma. Macroevolutionary analysis detected a significant rate shift in diversification dynamics in the spirotrichean clade Hypotrichia + Oligotrichia + Choreotrichia, which had accelerated speciation rate ca. 570 Ma, during the Ediacaran-Cambrian transition. For all crown lineages investigated, speciation rates declined through time, whereas extinction rates remained low and relatively constant throughout the evolutionary history of ciliates.

Data partitioning and correction for ascertainment bias reduce the uncertainty of placental mammal divergence times inferred from the morphological clock.

Bayesian estimates of divergence times based on the molecular clock yield uncertainty of parameter estimates measured by the width of posterior distributions of node ages. For the relaxed molecular clock, previous works have reported that some of the uncertainty inherent to the variation of rates among lineages may be reduced by partitioning data. Here we test this effect for the purely morphological clock, using placental mammals as a case study. We applied the uncorrelated lognormal relaxed clock to morphological data of 40 extant mammalian taxa and 4,533 characters, taken from the largest published matrix of discrete phenotypic characters. The morphologically derived timescale was compared to divergence times inferred from molecular and combined data. We show that partitioning data into anatomical units significantly reduced the uncertainty of divergence time estimates for morphological data. For the first time, we demonstrate that ascertainment bias has an impact on the precision of morphological clock estimates. While analyses including molecular data suggested most divergences between placental orders occurred near the K-Pg boundary, the partitioned morphological clock recovered older interordinal splits and some younger intraordinal ones, including significantly later dates for the radiation of bats and rodents, which accord to the short-fuse hypothesis.

Large ancestral effective population size explains the difficult phylogenetic placement of owl monkeys.

The phylogenetic position of owl monkeys, grouped in the genus Aotus, has been a controversial issue for understanding Neotropical primate evolution. Explanations of the difficult phylogenetic assignment of owl monkeys have been elusive, frequently relying on insufficient data (stochastic error) or scenarios of rapid speciation (adaptive radiation) events. Using a coalescent-based approach, we explored the population-level mechanisms likely explaining these topological discrepancies. We examined the topological variance of 2,192 orthologous genes shared between representatives of the three major Cebidae lineages and the outgroup. By employing a methodological framework that allows for reticulated tree topologies, our analysis explicitly tested for non-dichotomous evolutionary processes impacting the finding of the position of owl monkeys in the cebid phylogeny. Our findings indicated that Aotus is a sister lineage of the callitrichines. Most gene trees (>50%) failed to recover the species tree topology, although the distribution of gene trees mismatching the true species topology followed the standard expectation of the multispecies coalescent without reticulation. We showed that the large effective population size of the common ancestor of Aotus and callitrichines was the most likely factor responsible for generating phylogenetic uncertainty. On the other hand, fast speciation scenarios or introgression played minor roles. We propose that the difficult phylogenetic placement of Aotus is explained by population-level processes associated with the large ancestral effective size. These results shed light on the biogeography of the early cebid diversification in the Miocene, highlighting the relevance of evaluating phylogenetic relationships employing population-aware approaches.

Newly Discovered Occurrences and Gene Tree of the Extracellular Globins and Linker Chains from the Giant Hexagonal Bilayer Hemoglobin in Metazoans.

Multicellular organisms depend on oxygen-carrying proteins to transport oxygen throughout the body; therefore, proteins such as hemoglobins (Hbs), hemocyanins, and hemerythrins are essential for maintenance of tissues and cellular respiration. Vertebrate Hbs are among the most extensively studied proteins; however, much less is known about invertebrate Hbs. Recent studies of hemocyanins and hemerythrins have demonstrated that they have much wider distributions than previously thought, suggesting that oxygen-binding protein diversity is underestimated across metazoans. Hexagonal bilayer hemoglobin (HBL-Hb), a blood pigment found exclusively in annelids, is a polymer comprised up to 144 extracellular globins and 36 linker chains. To further understand the evolutionary history of this protein complex, we explored the diversity of linkers and extracellular globins from HBL-Hbs using in silico approaches on 319 metazoan and one choanoflagellate transcriptomes. We found 559 extracellular globin and 414 linker genes transcribed in 171 species from ten animal phyla with new records in Echinodermata, Hemichordata, Brachiopoda, Mollusca, Nemertea, Bryozoa, Phoronida, Platyhelminthes, and Priapulida. Contrary to previous suggestions that linkers and extracellular globins emerged in the annelid ancestor, our findings indicate that they have putatively emerged before the protostome-deuterostome split. For the first time, we unveiled the comprehensive evolutionary history of metazoan HBL-Hb components, which consists of multiple episodes of gene gains and losses. Moreover, because our study design surveyed linkers and extracellular globins independently, we were able to cross-validate our results, significantly reducing the rate of false positives. We confirmed that the distribution of HBL-Hb components has until now been underestimated among animals.

Appropriate Assignment of Fossil Calibration Information Minimizes the Difference between Phylogenetic and Pedigree Mutation Rates in Humans.

Studies that measured mutation rates in human populations using pedigrees have reported values that differ significantly from rates estimated from the phylogenetic comparison of humans and chimpanzees. Consequently, exchanges between mutation rate values across different timescales lead to conflicting divergence time estimates. It has been argued that this variation of mutation rate estimates across hominoid evolution is in part caused by incorrect assignment of calibration information to the mean coalescent time among loci, instead of the true genetic isolation (speciation) time between humans and chimpanzees. In this study, we investigated the feasibility of estimating the human pedigree mutation rate using phylogenetic data from the genomes of great apes. We found that, when calibration information was correctly assigned to the human⁻chimpanzee speciation time (and not to the coalescent time), estimates of phylogenetic mutation rates were statistically equivalent to the estimates previously reported using studies of human pedigrees. We conclude that, within the range of biologically realistic ancestral generation times, part of the difference between whole-genome phylogenetic and pedigree mutation rates is due to inappropriate assignment of fossil calibration information to the mean coalescent time instead of the speciation time. Although our results focus on the human⁻chimpanzee divergence, our findings are general, and relevant to the inference of the timescale of the tree of life.

Incomplete lineage sorting impacts the inference of macroevolutionary regimes from molecular phylogenies when concatenation is employed: An analysis based on Cetacea.

Interest in methods that estimate speciation and extinction rates from molecular phylogenies has increased over the last decade. The application of such methods requires reliable estimates of tree topology and node ages, which are frequently obtained using standard phylogenetic inference combining concatenated loci and molecular dating. However, this practice disregards population-level processes that generate gene tree/species tree discordance. We evaluated the impact of employing concatenation and coalescent-based phylogeny inference in recovering the correct macroevolutionary regime using simulated data based on the well-established diversification rate shift of delphinids in Cetacea. We found that under scenarios of strong incomplete lineage sorting, macroevolutionary analysis of phylogenies inferred by concatenating loci failed to recover the delphinid diversification shift, while the coalescent-based tree consistently retrieved the correct rate regime. We suggest that ignoring microevolutionary processes reduces the power of methods that estimate macroevolutionary regimes from molecular data.

Multispecies coalescent analysis confirms standing phylogenetic instability in Hexapoda.

The multispecies coalescent (MSC) has been increasingly used in phylogenomic analyses due to the accommodation of gene tree topological heterogeneity by taking into account population-level processes, such as incomplete lineage sorting. In this sense, the phylogeny of insect species, which are characterized by their large effective population sizes, is suitable for a coalescent-based analysis. Furthermore, studies so far recovered short internal branches at early divergences of the insect tree of life, indicating fast evolutionary radiations that increase the probability of incomplete lineage sorting in deep time. Here, we investigated the performance of the MSC for a phylogenomic data set of hexapods compiled by Misof et al. (2014, Science 346:763). Our analysis recovered the monophyly of most insect orders, and major phylogenetic relationships were in agreement with current insect systematics. We identified, however, some evolutionary associations that were consistently problematic. Most noticeable, Hexapod monophyly was disrupted by the sister group relationship between the remiped crustacean and Insecta. Additionally, the interordinal relationships within Polyneoptera and Neuropteroidea were found to be phylogenetically unstable. We show that these controversial phylogenetic arrangements were also poorly supported by previous analyses, and therefore, we evaluated their robustness to stochastic errors from sampling sites and terminals, confirming standing problems in hexapod phylogeny in the genomics age.

Comparative evaluation of maximum parsimony and Bayesian phylogenetic reconstruction using empirical morphological data.

The use of discrete morphological data in Bayesian phylogenetics has increased significantly over the last years with the proposal of total evidence analysis and the treatment of fossils as terminal taxa in Bayesian molecular dating. Both approaches rely on the assumption that probabilistic Markov models reasonably accommodate all the complexity of morphological evolution of discrete traits. The performance of such morphological models used in Bayesian phylogenetics has been thoroughly investigated, but conclusions so far were based mostly on simulated data. In this study, we have surveyed MorphoBank and obtained a large number of morphological matrices to evaluate Bayesian phylogenetic inference (BI) under Lewis' Mk model in comparison with the maximum parsimony (MP) algorithm. We found that trees estimated by both methods frequently differed and that BI generated a larger amount of polytomic tree topologies. The number of trees contained in the 95% Bayesian credibility interval was significantly greater than the number of equally parsimonious trees. We also investigated which factors mostly influenced the topological difference between maximum parsimony and Bayesian tree topologies and found that the number of terminals in morphological matrices was the variable with the highest association with the topological distance between trees inferred by BI and MP. Surprisingly, we show that differences between both approaches were not influenced by increasing sample size. Our results, which were based on a large set of empirical matrices, corroborate recent findings that BI is less precise than MP.

Conservation analysis and decomposition of residue correlation networks in the phospholipase A2 superfamily (PLA2s): Insights into the structure-function relationships of snake venom toxins.

Phospholipases A2 (PLA2s) comprise a superfamily of glycerophospholipids hydrolyzing enzymes present in many organisms in nature, whose catalytic activity was majorly unveiled by analysis of snake venoms. The latter have pharmaceutical and biotechnological interests and can be divided into different functional sub-classes. Our goal was to identify important residues and their relation to the functional and class-specific characteristics in the PLA2s family with special emphasis on snake venom PLA2s (svPLA2s). We identified such residues by conservation analysis and decomposition of residue coevolution networks (DRCN), annotated the results based on the available literature on PLA2s, structural analysis and molecular dynamics simulations, and related the results to the phylogenetic distribution of these proteins. A filtered alignment of PLA2s revealed 14 highly conserved positions and 3 sets of coevolved residues, which were annotated according to their structural or functional role. These residues are mostly involved in ligand binding and catalysis, calcium-binding, the formation of disulfide bridges and a hydrophobic cluster close to the binding site. An independent validation of the inference of structure-function relationships from our co-evolution analysis on the svPLA2s family was obtained by the analysis of the pattern of selection acting on the Viperidae and Elapidae lineages. Additionally, a molecular dynamics simulation on the Lys49 PLA2 from Agkistrodon contortrix laticinctus was carried out to further investigate the correlation of the Lys49-Glu69 pair. Our results suggest this configuration can result in a novel conformation where the binding cavity collapses due to the approximation of two loops caused by a strong salt bridge between Glu69 and Arg34. Finally, phylogenetic analysis indicated a correlation between the presence of residues in the coevolved sets found in this analysis and the clade localization. The results provide a guide for important positions in the family of PLA2s, and potential new objects of investigation.

Impact of long-term chromosomal shuffling on the multispecies coalescent analysis of two anthropoid primate lineages.

Multispecies coalescent (MSC) theory assumes that gene trees inferred from individual loci are independent trials of the MSC process. As genes might be physically close in syntenic associations spanning along chromosome regions, these assumptions might be flawed in evolutionary lineages with substantial karyotypic shuffling. Neotropical primates (NP) represent an ideal case for assessing the performance of MSC methods in such scenarios because chromosome diploid number varies significantly in this lineage. To this end, we investigated the effect of sequence length on the theoretical expectations of MSC model, as well as the results of coalescent-based tree inference methods. This was carried out by comparing NP with hominids, a lineage in which chromosome macrostructure has been stable for at least 15 million years. We found that departure from the MSC model in Neotropical primates decreased with smaller sequence fragments, where sites sharing the same evolutionary history were more frequently found than in longer fragments. This scenario probably resulted from extensive karyotypic rearrangement occurring during the radiation of NP, contrary to the comparatively stable chromosome evolution in hominids.

Discovery of Novel Hemocyanin-Like Genes in Metazoans.

Among animals, two major groups of oxygen-binding proteins are found: proteins that use iron to bind oxygen (hemoglobins and hemerythrins) and two non-homologous hemocyanins that use copper. Although arthropod and mollusc hemocyanins bind oxygen in the same manner, they are distinct in their molecular structures. In order to better understand the range of natural variation in hemocyanins, we searched for them in a diverse array of metazoan transcriptomes by using bioinformatics tools to examine hemocyanin evolutionary history and to consequently revive the discussion about whether all metazoan hemocyanins shared a common origin with frequent losses or whether they originated separately after the divergence of Lophotrochozoa and Ecdysozoa. We confirm that the distribution of hemocyanin-like genes is more widespread than previously reported, including five putative novel mollusc hemocyanin genes in two annelid species from Chaetopteridae. For arthropod hemocyanins, 16 putative novel genes were retained, and the presence of arthropod hemocyanins in 11 annelid species represents a novel observation. Interestingly, Annelida is the lineage that presents the greatest repertoire of oxygen transport proteins reported to date, possessing all the main superfamily proteins, which could be explained partially by the immense variability of lifestyles and habitats. Work presented here contradicts the canonical view that hemocyanins are restricted to molluscs and arthropods, suggesting that the occurrence of copper-based blood pigments in metazoans has been underestimated. Our results also support the idea of the presence of oxygen carrier hemocyanins being widespread across metazoans with an evolutionary history characterized by frequent losses.