Phylogeography of hepatitis B: The role of Portugal in the early dissemination of HBV worldwide

In Portugal, the genetic diversity, origin of HBV, and the Portuguese role in the dissemination of HBV worldwide were never investigated. In this work, we studied the epidemic history and transmission dynamics of HBV genotypes that are endemic in Portugal. HBV pol gene was sequenced from 130 patients followed in Lisbon. HBV genotype A (HBV/A) was the most prevalent (n=54, 41.5%), followed by D [HBV/D; (n=44, 33.8%)], and E [HBV/E; (n=32, 24.6%)]. Spatio-temporal evolutionary dynamics was reconstructed in BEAST using a Bayesian Markov Chain Monte Carlo method, with a GTR nucleotide substitution model, an uncorrelated lognormal relaxed molecular clock model, a Bayesian skyline plot, and a continuous diffusion model. HBV/D4 was the first subgenotype to be introduced in Portugal around 1857 (HPD 95% 1699-1931) followed by HBV/D3 and A2 a few decades later. HBV/E and HBV/A1 were introduced in Portugal later, almost simultaneously. Our results also indicate a very important role of Portugal in the exportation of HBV/D4 and A2 to Brazil and Cape Verde, respectively, at the beginning of the XX century. This work clarifies the epidemiological history of HBV in Portugal and shows that Portugal had an important role in the global spread of this virus.

Plagued by a cryptic clock: Insight and issues from the global phylogeny of Yersinia pestis

Plague has an enigmatic history as a zoonotic pathogen. This potentially devastating infectious disease will unexpectedly appear in human populations and disappear just as suddenly. As a result, a long-standing line of inquiry has been to estimate when and where plague appeared in the past. However, there have been significant disparities between phylogenetic studies of the causative bacterium, Yersinia pestis, regarding the timing and geographic origins of its reemergence. Here, we curate and contextualize an updated phylogeny of Y. pestis using 601 genome sequences sampled globally. We perform a detailed Bayesian evaluation of temporal signal in subsets of these data and demonstrate that a Y. pestis-wide molecular clock model is unstable. To resolve this, we devised a new approach in which each Y. pestis population was assessed independently. This enabled us to recover significant temporal signal in five populations, including the ancient pandemic lineages which we now estimate may have emerged decades, or even centuries, before a pandemic was historically documented from European sources. Despite this, we only obtain robust divergence dates from populations sampled over a period of at least 90 years, indicating that genetic evidence alone is insufficient for accurately reconstructing the timing and spread of short-term plague epidemics. Finally, we identify key historical data sets that can be used in future research, which will complement the strengths and mitigate the weaknesses of genomic data.

Frequent lineage-specific substitution rate changes support an episodic model for protein evolution

Since the inception of the molecular clock model for sequence evolution, the investigation of protein divergence has revolved around the question of a more or less constant change of amino acid sequences, with specific overall rates for each family. Although anomalies in clock-like divergence are well known, the assumption of a constant decay rate for a given protein family is usually taken as the null model for protein evolution. However, systematic tests of this null model at a genome-wide scale have lagged behind, despite the databases’ enormous growth. We focus here on divergence rate comparisons between very closely related lineages since this allows clear orthology assignments by synteny and reliable alignments, which are crucial for determining substitution rate changes. We generated a high-confidence dataset of syntenic orthologs from four ape species, including humans. We find that despite the appearance of an overall clock-like substitution pattern, several hundred protein families show lineage-specific acceleration and deceleration in divergence rates, or combinations of both in different lineages. Hence, our analysis uncovers a rather dynamic history of substitution rate changes, even between these closely related lineages, implying that one should expect that a large fraction of proteins will have had a history of episodic rate changes in deeper phylogenies. Furthermore, each of the lineages has a separate set of particularly fast diverging proteins. The genes with the highest percentage of branch-specific substitutions are ADCYAP1 in the human lineage (9.7%), CALU in chimpanzees (7.1%), SLC39A14 in the internal branch leading to humans and chimpanzees (4.1%), RNF128 in gorillas (9%), and S100Z in gibbons (15.2%). The mutational pattern in ADCYAP1 suggests a biased mutation process, possibly through asymmetric gene conversion effects. We conclude that a null model of constant change can be problematic for predicting the evolutionary trajectories of individual proteins.

Inferring Evolutionary Timescales without Independent Timing Information: An Assessment of “Universal” Insect Rates to Calibrate a Collembola (Hexapoda) Molecular Clock

Previous estimates of nucleotide substitution rates are routinely applied as secondary or “universal” molecular clock calibrations for estimating evolutionary timescales in groups that lack independent timing information. A major limitation of this approach is that rates can vary considerably among taxonomic groups, but the assumption of rate constancy is rarely evaluated prior to using secondary rate calibrations. Here I evaluate whether an insect mitochondrial DNA clock is appropriate for estimating timescales in Collembola—a group of insect-like arthropods characterized by high levels of cryptic diversity. Relative rates of substitution in cytochrome oxidase subunit 1 (COI) were inferred via Bayesian analysis across a topologically constrained Hexapod phylogeny using a relaxed molecular clock model. Rates for Collembola did not differ significantly from the average rate or from the rates estimated for most other groups (25 of 30), suggesting that (1) their apparent cryptic diversity cannot be explained by accelerated rates of molecular evolution and (2) clocks calibrated using “universal” insect rates may be appropriate for estimating evolutionary timescales in this group. However, of the 31 groups investigated, 10 had rates that deviated significantly from the average (6 higher, 4 lower), underscoring the need for caution and careful consideration when applying secondary insect rate calibrations. Lastly, this study exemplifies a relatively simple approach for evaluating rate constancy within a taxonomic group to determine whether the use of secondary rates are appropriate for molecular clock calibrations.

Frequent branch-specific changes of protein substitution rates in closely related lineages

Since its inception, the investigation of protein divergence has revolved around a more or less constant rate of sequence information decay that led to the formation of the molecular clock model for sequence evolution. We use here the classical approach of amino acid sequence comparisons to examine the overall divergence of proteins and the possibility of lineage-specific acceleration. By generating and analysing a high-confidence dataset of 13,160 syntenic orthologs from four ape species, including humans, we found that only less than 1% of the ortholog families are entirely in line with the clock model in each of their branches. The most common departure from the expected decay rate involves higher than expected substitutions on just one or two branches of the individual families. However, when taken as aggregate, even a small set of families conform well with the clock assumptions. We identified ADCYAP1 as the most divergent human protein-coding gene with 10% human-specific substitutions. Such lineage-specific highly accelerated genes were not limited to humans but appear as a general pattern that accompanies the formation of species. Our analysis uncovers a much more dynamic history of substitution rate changes in most protein families than usually assumed. Such fluctuations can result in bursts of rapid acceleration followed by periods of strong conservation that effectively cancel each other. Although this gives an impression of a long-term constant rate, the actual history of protein sequence evolution appears to be more complicated.

Additive Uncorrelated Relaxed Clock Models for the Dating of Genomic Epidemiology Phylogenies

Phylogenetic dating is one of the most powerful and commonly used methods of drawing epidemiological interpretations from pathogen genomic data. Building such trees requires considering a molecular clock model which represents the rate at which substitutions accumulate on genomes. When the molecular clock rate is constant throughout the tree then the clock is said to be strict, but this is often not an acceptable assumption. Alternatively, relaxed clock models consider variations in the clock rate, often based on a distribution of rates for each branch. However, we show here that the distributions of rates across branches in commonly used relaxed clock models are incompatible with the biological expectation that the sum of the numbers of substitutions on two neighboring branches should be distributed as the substitution number on a single branch of equivalent length. We call this expectation the additivity property. We further show how assumptions of commonly used relaxed clock models can lead to estimates of evolutionary rates and dates with low precision and biased confidence intervals. We therefore propose a new additive relaxed clock model where the additivity property is satisfied. We illustrate the use of our new additive relaxed clock model on a range of simulated and real data sets, and we show that using this new model leads to more accurate estimates of mean evolutionary rates and ancestral dates.

Great ape mutation spectra vary across the phylogeny and the genome due to distinct mutational processes that evolve at different rates

ABSTRACTRecent studies of hominoid variation have shown that mutation rates and spectra can evolve rapidly, contradicting the fixed molecular clock model. The relative mutation rates of three-base-pair motifs differ significantly among great ape species, suggesting the action of unknown modifiers of DNA replication fidelity. To illuminate the footprints of these hypothetical mutators, we measured mutation spectra of several functional compartments (such as late-replicating regions) that are likely targeted by localized mutational processes. Using genetic diversity from 88 great apes, we find that compartment-specific mutational signatures appear largely conserved between species. These signatures layer with species-specific signatures to create rich mutational portraits: for example, late-replicating regions in gorillas contain an identifiable mixture of a replication timing signature and a gorilla-specific signature. Our results suggest that cis-acting mutational modifiers are highly conserved between species and transacting modifiers are driving rapid mutation spectrum evolution.

Bayesian Estimation of Past Population Dynamics in BEAST 1.10 Using the Skygrid Coalescent Model

Inferring past population dynamics over time from heterochronous molecular sequence data is often achieved using the Bayesian Skygrid model, a nonparametric coalescent model that estimates the effective population size over time. Available in BEAST, a cross-platform program for Bayesian analysis of molecular sequences using Markov chain Monte Carlo, this coalescent model is often estimated in conjunction with a molecular clock model to produce time-stamped phylogenetic trees. We here provide a practical guide to using BEAST and its accompanying applications for the purpose of drawing inference under these models. We focus on best practices, potential pitfalls, and recommendations that can be generalized to other software packages for Bayesian inference. This protocol shows how to use TempEst, BEAUti, and BEAST 1.10 (http://beast.community/; last accessed July 29, 2019), LogCombiner as well as Tracer in a complete workflow.

Highly Resolved Phylogenetic Relationships within Order Acipenseriformes According to Novel Nuclear Markers

Order Acipenseriformes contains 27 extant species distributed across the northern hemisphere, including so-called “living fossil” species of garfish and sturgeons. Previous studies have focused on their mitochondrial genetics and have rarely used nuclear genetic data, leaving questions as to their phylogenetic relationships. This study aimed to utilize a bioinformatics approach to screen for candidate single-copy nuclear genes, using transcriptomic data from sturgeon species and genomic data from the spotted gar, Lepisosteus oculatus. We utilized nested polymerase chain reaction (PCR) and degenerate primers to identify nuclear protein-coding (NPC) gene markers to determine phylogenetic relationships among the Acipenseriformes. We identified 193 nuclear single-copy genes, selected from 1850 candidate genes with at least one exon larger than 700 bp. Forty-three of these genes were used for primer design and development of 30 NPC markers, which were sequenced for at least 14 Acipenseriformes species. Twenty-seven NPC markers were found completely in 16 species. Gene trees according to Bayesian inference (BI) and maximum likelihood (ML) were calculated based on the 30 NPC markers (20,946 bp total). Both gene and species trees produced very similar topologies. A molecular clock model estimated the divergence time between sturgeon and paddlefish at 204.1 Mya, approximately 10% later than previous estimates based on cytochrome b data (184.4 Mya). The successful development and application of NPC markers provides a new perspective and insight for the phylogenetic relationships of Acipenseriformes. Furthermore, the newly developed nuclear markers may be useful in further studies on the conservation, evolution, and genomic biology of this group.

Genome skimming provides well resolved plastid and nuclear phylogenies, showing patterns of deep reticulate evolution in the tropical carnivorous plant genus Nepenthes (Caryophyllales)

Nepenthes is a genus of carnivorous plants consisting of ~160 species that are distributed in the paleotropics. Molecular systematics has so far not been able to resolve evolutionary relationships of most species because of the limited genetic divergence in previous studies. In the present study, we used a genome-skimming approach to infer phylogenetic relationships on the basis of 81 plastid genes and the highly repetitive rRNA (external transcribed spacer (ETS)–26S) for 39 accessions representing 34 species from eight sections. Maximum-likelihood analysis and Bayesian inference were performed separately for the nuclear and the plastid datasets. Divergence-time estimations were conducted on the basis of a relaxed molecular-clock model, using secondary calibration points. The phylogenetic analyses of the nuclear and plastid datasets yielded well resolved and supported phylogenies. Incongruences between the two datasets were detected, suggesting multiple hybridisation events or incomplete lineage sorting in the deeper and more recent evolutionary history of the genus. The inclusion of several known and suspected hybrids in the phylogenetic analysis provided insights into their parentage. Divergence-time estimations placed the crown diversification of Nepenthes in the early Miocene, c. 20 million years ago. This study showed that genome skimming provides well resolved nuclear and plastid phylogenies that provide valuable insights into the complex evolutionary relationships of Nepenthes.