Darren J. Obbard

The evolution of RNAi as a defence against viruses and transposable elements.

RNA interference (RNAi) is an important defence against viruses and transposable elements (TEs). RNAi not only protects against viruses by degrading viral RNA, but hosts and viruses can also use RNAi to manipulate each others gene expression, and hosts can encode microRNAs that target viral sequences. In response, viruses have evolved a myriad of adaptations to suppress and evade RNAi. RNAi can also protect cells against TEs, both by degrading TE transcripts and by preventing TE expression through heterochromatin formation. The aim of our review is to summarize and evaluate the current data on the evolution of these RNAi defence mechanisms. To this end, we also extend a previous analysis of the evolution of genes of the RNAi pathways. Strikingly, we find that antiviral RNAi genes, anti-TE RNAi genes and viral suppressors of RNAi all evolve rapidly, suggestive of an evolutionary arms race between hosts and parasites. Over longer time scales, key RNAi genes are repeatedly duplicated or lost across the metazoan phylogeny, with important implications for RNAi as an immune defence.

Quantifying Adaptive Evolution in the Drosophila Immune System

It is estimated that a large proportion of amino acid substitutions in Drosophila have been fixed by natural selection, and as organisms are faced with an ever-changing array of pathogens and parasites to which they must adapt, we have investigated the role of parasite-mediated selection as a likely cause. To quantify the effect, and to identify which genes and pathways are most likely to be involved in the host–parasite arms race, we have re-sequenced population samples of 136 immunity and 287 position-matched non-immunity genes in two species of Drosophila. Using these data, and a new extension of the McDonald-Kreitman approach, we estimate that natural selection fixes advantageous amino acid changes in immunity genes at nearly double the rate of other genes. We find the rate of adaptive evolution in immunity genes is also more variable than other genes, with a small subset of immune genes evolving under intense selection. These genes, which are likely to represent hotspots of host–parasite coevolution, tend to share similar functions or belong to the same pathways, such as the antiviral RNAi pathway and the IMD signalling pathway. These patterns appear to be general features of immune system evolution in both species, as rates of adaptive evolution are correlated between the D. melanogaster and D. simulans lineages. In summary, our data provide quantitative estimates of the elevated rate of adaptive evolution in immune system genes relative to the rest of the genome, and they suggest that adaptation to parasites is an important force driving molecular evolution.

The Discovery, Distribution, and Evolution of Viruses Associated with Drosophila melanogaster

Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs, we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2,000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont—which is known to be protective against virus infections in Drosophila—we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets, we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host–virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research.

Simple allelic-phenotype diversity and differentiation statistics for allopolyploids

The analysis of genetic diversity within and between populations is a routine task in the study of diploid organisms. However, population genetic studies of polyploid organisms have been hampered by difficulties associated with scoring and interpreting molecular data. This occurs because the presence of multiple alleles at each locus often precludes the measurement of genotype or allele frequencies. In allopolyploids, the problem is compounded because genetically distinct isoloci frequently share alleles. As a result, analysis of genetic diversity patterns in allopolyploids has tended to rely on the interpretation of phenotype frequencies, which loses information available from allele composition. Here, we propose the use of a simple allelic-phenotype diversity statistic (H′) that measures diversity as the average number of alleles by which pairs of individuals differ. This statistic can be extended to a population differentiation measure (F′ST), which is analogous to FST. We illustrate the behaviour of these statistics using coalescent computer simulations that show that F′ST behaves in a qualitatively similar way to FST, thus providing a useful way to quantify population differentiation in allopolyploid species.

HYBRIDIZATION, POLYPLOIDY, AND THE EVOLUTION OF SEXUAL SYSTEMS IN MERCURIALIS (EUPHORBIACEAE)

Abstract Hybridization and polyploidy are widely believed to be important sources of evolutionary novelty in plant evolution. Both can lead to novel gene combinations and/or novel patterns of gene expression, which in turn provide the variation on which natural selection can act. Here, we use nuclear and plastid gene trees, in conjunction with morphological data and genome size measurements, to show that both processes have been important in shaping the evolution of the angiosperm genus Mercurialis, particularly a clade of annual lineages that shows exceptional variation in the sexual system. Our results indicate that hexaploid populations of M. annua, in which the rare sexual system androdioecy is common (the occurrence of males and hermaphrodites) is of allopolyploid origin involving hybridization between an autotetraploid lineage of M. annua and the related diploid species M. huetii. We discuss the possibility that androdioecy may have evolved as a result of hybridization between dioecious M. huetii and monoecious tetraploid M. annua, an event that brought together the genes for specialist males with those for hermaphrodites.

Host Phylogeny Determines Viral Persistence and Replication in Novel Hosts

Pathogens switching to new hosts can result in the emergence of new infectious diseases, and determining which species are likely to be sources of such host shifts is essential to understanding disease threats to both humans and wildlife. However, the factors that determine whether a pathogen can infect a novel host are poorly understood. We have examined the ability of three host-specific RNA-viruses (Drosophila sigma viruses from the family Rhabdoviridae) to persist and replicate in 51 different species of Drosophilidae. Using a novel analytical approach we found that the host phylogeny could explain most of the variation in viral replication and persistence between different host species. This effect is partly driven by viruses reaching a higher titre in those novel hosts most closely related to the original host. However, there is also a strong effect of host phylogeny that is independent of the distance from the original host, with viral titres being similar in groups of related hosts. Most of this effect could be explained by variation in general susceptibility to all three sigma viruses, as there is a strong phylogenetic correlation in the titres of the three viruses. These results suggest that the source of new emerging diseases may often be predictable from the host phylogeny, but that the effect may be more complex than simply causing most host shifts to occur between closely related hosts.

Sexual Systems and Population Genetic Structure in an Annual Plant: Testing the Metapopulation Model

The need for reproductive assurance during dispersal, along with the pressure of local mate competition, means that the importance of frequent or repeated colonization is implicit in the literature on sexual system evolution. However, there have been few empirical tests of the association between colonization history and sexual system in plants, and none within a single species. Here we use patterns of genetic diversity to provide such a test in the Mercurialis annua species complex, which spans the range of systems from self‐compatible monoecy through androdioecy to dioecy. This variation has been hypothesized to result from differing patterns of metapopulation turnover and recolonization. Because monoecy should be favored during colonization, androdioecy and dioecy will be maintained only in regions with low rates of local extinction and recolonization, and these differences should also be reflected in patterns of neutral genetic diversity. We show that monoecious populations of M. annua display lower within‐population genetic diversity than androdioecious populations and higher genetic differentiation than dioecious and androdioecious populations, as predicted by metapopulation models. In contrast, regional diversity in M. annua appears to be primarily a product of postglacial range expansion from two refugia in the eastern and western Mediterranean Basin.

The recent spread of a vertically transmitted virus through populations of Drosophila melanogaster

The sigma virus is a vertically transmitted pathogen that commonly infects natural populations of Drosophila melanogaster. This virus is the only known host‐specific pathogen of D. melanogaster, and so offers a unique opportunity to study the genetics of Drosophila–viral interactions in a natural system. To elucidate the population genetic processes that operate in sigma virus populations, we collected D. melanogaster from 10 populations across three continents. We found that the sigma virus had a prevalence of 0–15% in these populations. Compared to other RNA viruses, we found that levels of viral genetic diversity are very low across Europe and North America. Based on laboratory measurements of the viral substitution rate, we estimate that most European and North American viral isolates shared a common ancestor approximately 200 years ago. We suggest two explanations for this: the first is that D. melanogaster has recently acquired the sigma virus; the second is that a single viral type has recently swept through D. melanogaster populations. Furthermore, in contrast to Drosophila populations, we find that the sigma viral populations are highly structured. This is surprising for a vertically transmitted pathogen that has a similar migration rate to its host. We suggest that the low structure in the viral populations can be explained by the smaller effective population size of the virus.

Induction and suppression of tick cell antiviral RNAi responses by tick-borne flaviviruses

Arboviruses are transmitted by distantly related arthropod vectors such as mosquitoes (class Insecta) and ticks (class Arachnida). RNA interference (RNAi) is the major antiviral mechanism in arthropods against arboviruses. Unlike in mosquitoes, tick antiviral RNAi is not understood, although this information is important to compare arbovirus/host interactions in different classes of arbovirus vectos. Using an Ixodes scapularis-derived cell line, key Argonaute proteins involved in RNAi and the response against tick-borne Langat virus (Flaviviridae) replication were identified and phylogenetic relationships characterized. Analysis of small RNAs in infected cells showed the production of virus-derived small interfering RNAs (viRNAs), which are key molecules of the antiviral RNAi response. Importantly, viRNAs were longer (22 nucleotides) than those from other arbovirus vectors and mapped at highest frequency to the termini of the viral genome, as opposed to mosquito-borne flaviviruses. Moreover, tick-borne flaviviruses expressed subgenomic flavivirus RNAs that interfere with tick RNAi. Our results characterize the antiviral RNAi response in tick cells including phylogenetic analysis of genes encoding antiviral proteins, and viral interference with this pathway. This shows important differences in antiviral RNAi between the two major classes of arbovirus vectors, and our data broadens our understanding of arthropod antiviral RNAi.

Sigma viruses from three species of Drosophila form a major new clade in the rhabdovirus phylogeny

The sigma virus (DMelSV), which is a natural pathogen of Drosophila melanogaster, is the only Drosophila-specific rhabdovirus that has been described. We have discovered two new rhabdoviruses, D. obscura and D. affinis, which we have named DObsSV and DAffSV, respectively. We sequenced the complete genomes of DObsSV and DMelSV, and the L gene from DAffSV. Combining these data with sequences from a wide range of other rhabdoviruses, we found that the three sigma viruses form a distinct clade which is a sister group to the Dimarhabdovirus supergroup, and the high levels of divergence between these viruses suggest that they deserve to be recognized as a new genus. Furthermore, our analysis produced the most robustly supported phylogeny of the Rhabdoviridae to date, allowing us to reconstruct the major transitions that have occurred during the evolution of the family. Our data suggest that the bias towards research into plants and vertebrates means that much of the diversity of rhabdoviruses has been missed, and rhabdoviruses may be common pathogens of insects.