In phylogenetics, the unrooted model of phylogeny and the strict molecular clock model are two extremes of a continuum. Despite their dominance in phylogenetic inference, it is evident that both are biologically unrealistic and that the real evolutionary process lies between these two extremes. Fortunately, intermediate models employing relaxed molecular clocks have been described. These models open the gate to a new field of “relaxed phylogenetics.” Here we introduce a new approach to performing relaxed phylogenetic analysis. We describe how it can be used to estimate phylogenies and divergence times in the face of uncertainty in evolutionary rates and calibration times. Our approach also provides a means for measuring the clocklikeness of datasets and comparing this measure between different genes and phylogenies. We find no significant rate autocorrelation among branches in three large datasets, suggesting that autocorrelated models are not necessarily suitable for these data. In addition, we place these datasets on the continuum of clocklikeness between a strict molecular clock and the alternative unrooted extreme. Finally, we present analyses of 102 bacterial, 106 yeast, 61 plant, 99 metazoan, and 500 primate alignments. From these we conclude that our method is phylogenetically more accurate and precise than the traditional unrooted model while adding the ability to infer a timescale to evolution.

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Relaxed Phylogenetics and Dating with Confidence

Mosquito-borne flaviviruses provide some of the most important examples of emerging and resurging diseases of global significance. Here, we describe three of them: the resurgence of dengue in tropical and subtropical areas of the world, and the spread and establishment of Japanese encephalitis and West Nile viruses in new habitats and environments. These three examples also illustrate the complexity of the various factors that contribute to their emergence, resurgence and spread. Whereas some of these factors are natural, such as bird migration, most are due to human activities, such as changes in land use, water impoundments and transportation, which result in changed epidemiological patterns. The three examples also show the ease with which mosquito-borne viruses can spread to and colonize new areas, and the need for continued international surveillance and improved public health infrastructure to meet future emerging disease threats.

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Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses

Dengue is a spectrum of disease caused by four serotypes of the most prevalent arthropod-borne virus affecting humans today, and its incidence has increased dramatically in the past 50 years. Due in part to population growth and uncontrolled urbanization in tropical and subtropical countries, breeding sites for the mosquitoes that transmit dengue virus have proliferated, and successful vector control has proven problematic. Dengue viruses have evolved rapidly as they have spread worldwide, and genotypes associated with increased virulence have expanded from South and Southeast Asia into the Pacific and the Americas. This review explores the human, mosquito, and viral factors that contribute to the global spread and persistence of dengue, as well as the interaction between the three spheres, in the context of ecological and climate changes. What is known, as well as gaps in knowledge, is emphasized in light of future prospects for control and prevention of this pandemic disease.

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Global spread and persistence of dengue.

The Virus Pathogen Database and Analysis Resource (ViPR, www.ViPRbrc.org) is an integrated repository of data and analysis tools for multiple virus families, supported by the National Institute of Allergy and Infectious Diseases (NIAID) Bioinformatics Resource Centers (BRC) program. ViPR contains information for human pathogenic viruses belonging to the Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Flaviviridae, Filoviridae, Hepeviridae, Herpesviridae, Paramyxoviridae, Picornaviridae, Poxviridae, Reoviridae, Rhabdoviridae and Togaviridae families, with plans to support additional virus families in the future. ViPR captures various types of information, including sequence records, gene and protein annotations, 3D protein structures, immune epitope locations, clinical and surveillance metadata and novel data derived from comparative genomics analysis. Analytical and visualization tools for metadata-driven statistical sequence analysis, multiple sequence alignment, phylogenetic tree construction, BLAST comparison and sequence variation determination are also provided. Data filtering and analysis workflows can be combined and the results saved in personal ‘Workbenches’ for future use. ViPR tools and data are available without charge as a service to the virology research community to help facilitate the development of diagnostics, prophylactics and therapeutics for priority pathogens and other viruses.

ViPR: an open bioinformatics database and analysis resource for virology research

BACKGROUND The extent of urban areas is increasing around the world, and most humans now live in cities. Urbanization results in dramatic environmental change, including increased temperatures, more impervious surface cover, altered hydrology, and elevated pollution. Urban areas also host more non-native species and reduced abundance and diversity of many native species. These environmental changes brought by global urbanization are creating novel ecosystems with unknown consequences for the evolution of life. Here, we consider how early human settlements led to the evolution of human commensals, including some of the most notorious pests and disease vectors. We also comprehensively review how contemporary urbanization affects the evolution of species that coinhabit cities. ADVANCES A recent surge of research shows that urbanization affects both nonadaptive and adaptive evolution. Some of the clearest results of urban evolution show that cities elevate the strength of random genetic drift (stochastic changes in allele frequencies) and restrict gene flow (the movement of alleles between populations due to dispersal and mating). Populations of native species in cities often represent either relicts that predate urbanization or populations that established after a city formed. Both scenarios frequently result in a loss of genetic diversity within populations and increased differentiation between populations. Fragmentation and urban infrastructure also create barriers to dispersal, and consequently, gene flow is often reduced among city populations, which further contributes to genetic differentiation between populations. The influence of urbanization on mutation and adaptive evolution are less clear. A small number of studies suggest that industrial pollution can elevate mutation rates, but the pervasiveness of this effect is unknown. A better studied phenomenon are the effects of urbanization on evolution by natural selection. A growing number of studies show that plant and animal populations experience divergent selection between urban and nonurban environments. This divergent selection has led to adaptive evolution in life history, morphology, physiology, behavior, and reproductive traits. These adaptations typically evolve in response to pesticide use, pollution, local climate, or the physical structure of cities. Despite these important results, the genetic basis of adaptive evolution is known from only a few cases. Most studies also examine only a few populations in one city, and experimental validation is rare. OUTLOOK The study of evolution in urban areas provides insights into both fundamental and applied problems in biology. The thousands of cities throughout the world share some features while differing in other aspects related to their age, historical context, governmental policies, and local climate. Thus, the phenomenon of global urbanization represents an unintended but highly replicated global study of experimental evolution. We can harness this global urban experiment to understand the repeatability and pace of evolution in response to human activity. Among the most important unresolved questions is, how often do native and exotic species adapt to the particular environmental challenges found in cities? Such adaptations could be the difference as to whether a species persists or vanishes from urban areas. In this way, the study of urban evolution can help us understand how evolution in populations may contribute to conservation of rare species, and how populations can be managed to facilitate the establishment of resilient and sustainable urban ecosystems. In a similar way, understanding evolution in urban areas can lead to improved human health. For example, human pests frequently adapt to pesticides and evade control efforts because of our limited understanding of the size of populations and movement of individuals. Applied evolutionary studies could lead to more effective mitigation of pests and disease agents. The study of urban evolution has rapidly become an important frontier in biology, with implications for healthy and sustainable human populations in urban ecosystems.

Evolution of life in urban environments.

In the last four decades the incidence of dengue fever has increased 30-fold worldwide, and over half the world's population is now threatened with infection from one or more of four co-circulating viral serotypes (DEN-1 through DEN-4). To determine the role of viral molecular evolution in emergent disease dynamics, we sequenced 40% of the genome of 82 DEN-4 isolates collected from Puerto Rico over the 20 years since the onset of endemic dengue on the island. Isolates were derived from years with varying levels of DEN-4 prevalence. Over our sampling period there were marked evolutionary shifts in DEN-4 viral populations circulating in Puerto Rico; viral lineages were temporally clustered and the most common genotype at a particular sampling time often arose from a previously rare lineage. Expressed changes in structural genes did not appear to drive this lineage turnover, even though these regions include primary determinants of viral antigenic properties. Instead, recent dengue evolution can be attributed in part to positive selection on the nonstructural gene 2A (NS2A), whose functions may include replication efficiency and antigenicity. During the latest and most severe DEN-4 epidemic in Puerto Rico, in 1998, viruses were distinguished by three amino acid changes in NS2A that were fixed far faster than expected by drift alone. Our study therefore demonstrates viral genetic turnover within a focal population and the potential importance of adaptive evolution in viral epidemic expansion.

Selection-driven evolution of emergent dengue virus.

Dengue virus is a circumtropical, mosquito-borne flavivirus that infects 50-100 million people each year and is expanding in both range and prevalence. Of the four co-circulating viral serotypes (DENV-1 to DENV-4) that cause mild to severe febrile disease, DENV-2 has been implicated in the onset of dengue haemorrhagic fever (DHF) in the Americas in the early 1980s. To identify patterns of genetic change since DENV-2's reintroduction into the region, molecular evolution in DENV-2 from Puerto Rico (PR) and surrounding countries was examined over a 20 year period of fluctuating disease incidence. Structural genes (over 20 % of the viral genome), which affect viral packaging, host-cell entry and immune response, were sequenced for 91 DENV-2 isolates derived from both low- and high-prevalence years. Phylogenetic analyses indicated that DENV-2 outbreaks in PR have been caused by viruses assigned to subtype IIIb, originally from Asia. Variation amongst DENV-2 viruses in PR has since largely arisen in situ, except for a lineage-replacement event in 1994 that appears to have non-PR New World origins. Although most structural genes have remained relatively conserved since the 1980s, strong evidence was found for positive selection acting on a number of amino acid sites in the envelope gene, which have also been important in defining phylogenetic structure. Some of these changes are exhibited by the multiple lineages present in 1994, during the largest Puerto Rican outbreak of dengue, suggesting that they may have altered disease dynamics, although their functional significance will require further investigation.

Maritza Chirivella

Papers

Selection-driven evolution of emergent dengue virus.

Molecular evolution of dengue 2 virus in Puerto Rico: positive selection in the viral envelope accompanies clade reintroduction

Insights into the molecular evolution of Dengue virus type 4 in Puerto Rico over two decades of emergence