Comparative Analysis of the Complete Genome Sequence of the California MSW Strain of Myxoma Virus Reveals Potential Host Adaptations

Myxomatosis is a rapidly lethal disease of European rabbits that is caused by myxoma virus (MYXV). The introduction of a South American strain of MYXV into the European rabbit population of Australia is the classic case of host-pathogen coevolution following cross-species transmission. The most virulent strains of MYXV for European rabbits are the Californian viruses, found in the Pacific states of the United States and the Baja Peninsula, Mexico. The natural host of Californian MYXV is the brush rabbit, Sylvilagus bachmani. We determined the complete sequence of the MSW strain of Californian MYXV and performed a comparative analysis with other MYXV genomes. The MSW genome is larger than that of the South American Lausanne (type) strain of MYXV due to an expansion of the terminal inverted repeats (TIRs) of the genome, with duplication of the M156R, M154L, M153R, M152R, and M151R genes and part of the M150R gene from the right-hand (RH) end of the genome at the left-hand (LH) TIR. Despite the extreme virulence of MSW, no novel genes were identified; five genes were disrupted by multiple indels or mutations to the ATG start codon, including two genes, M008.1L/R and M152R, with major virulence functions in European rabbits, and a sixth gene, M000.5L/R, was absent. The loss of these gene functions suggests that S. bachmani is a relatively recent host for MYXV and that duplication of virulence genes in the TIRs, gene loss, or sequence variation in other genes can compensate for the loss of M008.1L/R and M152R in infections of European rabbits.     

Experimental Evolution of a Bacteriophage Virus Reveals the Trajectory of Adaptation across a Fecundity/Longevity Trade-Off

Life history theory attempts to account for how organisms lead their lives, balancing the conflicting demands of reproduction and survival. Here, we track the genomic and phenotypic evolution of the bacteriophage virus T7 across a postulated fecundity/longevity constraint. We adapted T7 to a challenging survival environment (6M urea). Our evolved strain displayed a significant improvement in propagule survival, coupled with a significant loss of fecundity (reduced growth rate on host cells). However, the increased resistance to urea did not generalise to increased resistance against temperature stress, highlighting that propagule durability is environment dependent. Previous comparative studies predicted that changes in propagule resistance would be mediated by changes in capsid proteins or gene deletions. In contrast, we found that point mutations in internal core protein genes (6.7 and 16) were responsible for the increased urea resistance of our evolved strain. Prior to the emergence of the 6.7 and 16 mutations, a distinct set of 5-point mutations peaked at over 20% prevalence before attenuating, suggestive of negative epistatic interactions during adaptation. Our results illustrate that parasites can adapt to specific transmission environments, and that this adaptation can impose costs on the subsequent ability to exploit host cells, potentially constraining durable parasites to lower virulence.     

The Mitochondrial Genome of Soybean Reveals Complex Genome Structures and Gene Evolution at Intercellular and Phylogenetic Levels

Determining mitochondrial genomes is important for elucidating vital activities of seed plants. Mitochondrial genomes are specific to each plant species because of their variable size, complex structures and patterns of gene losses and gains during evolution. This complexity has made research on the soybean mitochondrial genome difficult compared with its nuclear and chloroplast genomes. The present study helps to solve a 30-year mystery regarding the most complex mitochondrial genome structure, showing that pairwise rearrangements among the many large repeats may produce an enriched molecular pool of 760 circles in seed plants. The soybean mitochondrial genome harbors 58 genes of known function in addition to 52 predicted open reading frames of unknown function. The genome contains sequences of multiple identifiable origins, including 6.8 kb and 7.1 kb DNA fragments that have been transferred from the nuclear and chloroplast genomes, respectively, and some horizontal DNA transfers. The soybean mitochondrial genome has lost 16 genes, including nine protein-coding genes and seven tRNA genes; however, it has acquired five chloroplast-derived genes during evolution. Four tRNA genes, common among the three genomes, are derived from the chloroplast. Sizeable DNA transfers to the nucleus, with pericentromeric regions as hotspots, are observed, including DNA transfers of 125.0 kb and 151.6 kb identified unambiguously from the soybean mitochondrial and chloroplast genomes, respectively. The soybean nuclear genome has acquired five genes from its mitochondrial genome. These results provide biological insights into the mitochondrial genome of seed plants, and are especially helpful for deciphering vital activities in soybean.     

A Nonparametric Test Reveals Selection for Rapid Flowering in the Arabidopsis Genome

The detection of footprints of natural selection in genetic polymorphism data is fundamental to understanding the genetic basis of adaptation, and has important implications for human health. The standard approach has been to reject neutrality in favor of selection if the pattern of variation at a candidate locus was significantly different from the predictions of the standard neutral model. The problem is that the standard neutral model assumes more than just neutrality, and it is almost always possible to explain the data using an alternative neutral model with more complex demography. Today's wealth of genomic polymorphism data, however, makes it possible to dispense with models altogether by simply comparing the pattern observed at a candidate locus to the genomic pattern, and rejecting neutrality if the pattern is extreme. Here, we utilize this approach on a truly genomic scale, comparing a candidate locus to thousands of alleles throughout the Arabidopsis thaliana genome. We demonstrate that selection has acted to increase the frequency of early-flowering alleles at the vernalization requirement locus FRIGIDA. Selection seems to have occurred during the last several thousand years, possibly in response to the spread of agriculture. We introduce a novel test statistic based on haplotype sharing that embraces the problem of population structure, and so should be widely applicable.     

Rapid Evolution of Culture-Impaired Bacteria during Adaptation to Biofilm Growth

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Single-Molecule FRET Reveals Three Conformations for the TLS Domain of Brome Mosaic Virus Genome

Metabolite-dependent conformational switching in RNA riboswitches is now widely accepted as a critical regulatory mechanism for gene expression in bacterial systems. More recently, similar gene regulation mechanisms have been found to be important for viral systems as well. One of the most abundant and best-studied systems is the tRNA-like structure (TLS) domain, which has been found to occur in many plant viruses spread across numerous genera. In this work, folding dynamics for the TLS domain of Brome Mosaic Virus have been investigated using single-molecule fluorescence resonance energy transfer techniques. In particular, burst fluorescence methods are exploited to observe metal-ion ([Mⁿ⁺])-induced folding in freely diffusing RNA constructs resembling the minimal TLS element of brome mosaic virus RNA3. The results of these experiments reveal a complex equilibrium of at least three distinct populations. A stepwise, or consecutive, thermodynamic model for TLS folding is developed, which is in good agreement with the [Mⁿ⁺]-dependent evolution of conformational populations and existing structural information in the literature. Specifically, this folding pathway explains the metal-ion dependent formation of a functional TLS domain from unfolded RNAs via two consecutive steps: 1) hybridization of a long-range stem interaction, followed by 2) formation of a 3′-terminal pseudoknot. These two conformational transitions are well described by stepwise dissociation constants for [Mg²⁺] (K₁ = 328 ± 30 μM and K₂ = 1092 ± 183 μM) and [Na⁺] (K₁ = 74 ± 6 mM and K₂ = 243 ± 52 mM)-induced folding. The proposed thermodynamic model is further supported by inhibition studies of the long-range stem interaction using a complementary DNA oligomer, which effectively shifts the dynamic equilibrium toward the unfolded conformation. Implications of this multistep conformational folding mechanism are discussed with regard to regulation of virus replication.     

Genome Comparison of a Nonpathogenic Myxoma Virus Field Strain with Its Ancestor,the Virulent Lausanne Strain

One of the best-studied examples of host-virus coevolution is the release of myxoma virus (MV) for biological control of European rabbits in Australia and Europe. To investigate the genetic basis of MV adaptation to its new host, we sequenced the genome of 6918, an attenuated Spanish field strain, and compared it with that of Lausanne, the strain originally released in Europe in 1952. Although isolated 43 years apart, the genomes were highly conserved (99.95% identical). Only 32 of the 159 MV predicted proteins revealed amino acid changes. Four genes (M009L, M036L, M135R, and M148R) in 6918 were disrupted by frameshift mutations.Myxoma virus (MV), the causative agent of myxomatosis, belongs to the Leporipoxvirus genus of the Poxviridae family (9). Two distinct types of MV have been identified: South American MV, which circulates in Sylvilagus brasiliensis, and Californian MV, which circulates in Sylvilagus bachmani. Each virus is highly adapted to its host, causing a benign cutaneous fibroma at the site of inoculation. Both types of MV infect the European rabbit (Oryctolagus cuniculus), causing myxomatosis. The Californian strain MSW is more virulent for European rabbits than South American strains such as SLS or Lausanne (54). Another leporipoxvirus, Shope fibroma virus (SFV), is found in eastern North America in Sylvilagus floridanus. SFV protects European rabbits against myxomatosis (24), and it is routinely used as a vaccine.One of the best-studied examples of host-virus coevolution is the use of MV for biological control of European rabbits (22, 23, 29). It is particularly unusual because the precise time the virus was released is known, and the original viruses are available for comparison with current strains. MV (the SLS strain) was deliberately released in Australia in 1950 and soon after (1952) in France (the Lausanne strain), whence it rapidly spread across Europe, and it has become endemic since then. For almost 60 years, a complex coevolution of host and virus has occurred, characterized by the emergence of attenuated viral strains and rabbits selected for resistance to MV (11, 12, 30).The MV Lausanne strain and SFV have been completely sequenced (13, 61). MV encodes 171 genes, versus 165 encoded by SFV. The genetic information is highly conserved between the two viruses. Recently, preliminary sequencing of the MSW strain indicated that the major genomic differences with the Lausanne strain localize at the left terminal end of the MSW genome (31). In MSW, the terminal inverted repeats (TIRs) are extended, causing the duplication of five complete open reading frames (ORFs), which are present as a single copy near the right TIR in the Lausanne strain (9). To date, little molecular analysis concerning the adaptation of MV to its new host has been performed. Studies involving Australian field strains found small differences with reference to the SLS and Lausanne strains (49, 50), suggesting that adaptation (and the concomitant attenuation) of MV is not associated with major genetic changes such as large deletions. This finding is in contrast to what has been reported for attenuated poxviruses obtained by extensive cell culture passaging, which usually present substantial genomic deletions or rearrangements (5, 25, 36, 47, 48).Strain 6918 is a naturally attenuated MV isolated in Spain in 1995 (7). It is therefore a descendant of the Lausanne strain recovered after 43 years of continuous evolution in the field. It has been used for the development of a “transmissible vaccine” intended to protect wild-rabbit populations against both myxomatosis and rabbit hemorrhagic disease virus (RHDV) in Spain, where the European rabbit plays a key role in the Mediterranean ecosystems (18). For this purpose, a recombinant virus, 6918VP60-T2, was constructed by inserting the capsid gene of RHDV into the genome of strain 6918 (4, 6, 7, 56, 57). The genomes of 6918 and 6918VP60-T2 have been sequenced. Here we report the results of our comparison of the genomic sequences of Lausanne and 6918. To our knowledge, this is the first comparative genomic analysis involving two poxvirus field strains linked by a clearly recorded lineage, one being fully virulent and the other virtually nonpathogenic. The results provide relevant insights into the mechanisms of MV attenuation that occurred as a consequence of the adaptation of the virus to its new host.     

The Solanum commersonii Genome Sequence Provides Insights into Adaptation to Stress Conditions and Genome Evolution of Wild Potato Relatives

Here, we report the draft genome sequence of Solanum commersonii, which consists of ∼830 megabases with an N50 of 44,303 bp anchored to 12 chromosomes, using the potato (Solanum tuberosum) genome sequence as a reference. Compared with potato, S. commersonii shows a striking reduction in heterozygosity (1.5% versus 53 to 59%), and differences in genome sizes were mainly due to variations in intergenic sequence length. Gene annotation by ab initio prediction supported by RNA-seq data produced a catalog of 1703 predicted microRNAs, 18,882 long noncoding RNAs of which 20% are shown to target cold-responsive genes, and 39,290 protein-coding genes with a significant repertoire of nonredundant nucleotide binding site-encoding genes and 126 cold-related genes that are lacking in S. tuberosum. Phylogenetic analyses indicate that domesticated potato and S. commersonii lineages diverged ∼2.3 million years ago. Three duplication periods corresponding to genome enrichment for particular gene families related to response to salt stress, water transport, growth, and defense response were discovered. The draft genome sequence of S. commersonii substantially increases our understanding of the domesticated germplasm, facilitating translation of acquired knowledge into advances in crop stability in light of global climate and environmental changes.     

Highly Sensitive Real-Time In Vivo Imaging of an Influenza Reporter Virus Reveals Dynamics of Replication and Spread

The continual public health threat posed by the emergence of novel influenza viruses necessitates the ability to rapidly monitor infection and spread in experimental systems. To analyze real-time infection dynamics, we have created a replication-competent influenza reporter virus suitable for in vivo imaging. The reporter virus encodes the small and bright NanoLuc luciferase whose activity serves as an extremely sensitive readout of viral infection. This virus stably maintains the reporter construct and replicates in culture and in mice with near-native properties. Bioluminescent imaging of the reporter virus permits serial observations of viral load and dissemination in infected animals, even following clearance of a sublethal challenge. We further show that the reporter virus recapitulates known restrictions due to host range and antiviral treatment, suggesting that this technology can be applied to studying emerging influenza viruses and the impact of antiviral interventions on infections in vivo. These results describe a generalizable method to quickly determine the replication and pathogenicity potential of diverse influenza strains in animals.     

Widespread Adaptive Evolution in the Human Immunodeficiency Virus Type 1 Genome

We investigated variable selective pressures among amino acid sites in HIV-1 genes. Selective pressure at the amino acid level was measured by using the nonsynonymous/synonymous substitution rate ratio ( = d_N/d_S). To identify amino acid sites under positive selection with > 1, we applied maximum likelihood models that allow variable ratios among sites to analyze genomic sequences of 26 HIV-1 lineages including subtypes A, B, and C. Likelihood ratio tests detected sites under positive selection in each of the major genes in the genome: env, gag, pol, vif, and vpr. Positive selection was also detected in nef, tat, and vpu, although those genes are very small. The majority of positive selection sites is located in gp160. Positive selection was not detected if was estimated as an average across all sites, indicating the lack of power of the averaging approach. Candidate positive selection sites were mapped onto the available protein tertiary structures and immunogenic epitopes. We measured the physiochemical properties of amino acids and found that those at positive selection sites were more diverse than those at variable sites. Furthermore, amino acid residues at exposed positive selection sites were more physiochemically diverse than at buried positive selection sites. Our results demonstrate genomewide diversifying selection acting on the HIV-1.     

Rapid Evolution of Virus Sequences in Intrinsically Disordered Protein Regions

Nodamura Virus (NoV) is a nodavirus originally isolated from insects that can replicate in a wide variety of hosts, including mammals. Because of their simplicity and ability to replicate in many diverse hosts, NoV, and the Nodaviridae in general, provide a unique window into the evolution of viruses and host-virus interactions. Here we show that the C-terminus of the viral polymerase exhibits extreme structural and evolutionary flexibility. Indeed, fewer than 10 positively charged residues from the 110 amino acid-long C-terminal region of protein A are required to support RNA1 replication. Strikingly, this region can be replaced by completely unrelated protein sequences, yet still produce a functional replicase. Structure predictions, as well as evolutionary and mutational analyses, indicate that the C-terminal region is structurally disordered and evolves faster than the rest of the viral proteome. Thus, the function of an intrinsically unstructured protein region can be independent of most of its primary sequence, conferring both functional robustness and sequence plasticity on the protein. Our results provide an experimental explanation for rapid evolution of unstructured regions, which enables an effective exploration of the sequence space, and likely function space, available to the virus.     

Origin and Rapid Evolution of a Novel Murine Erythroleukemia Virus of the Spleen Focus-Forming Virus Family

The Friend spleen focus-forming virus (SFFV) env gene encodes a glycoprotein with apparent M_r of 55,000 that binds to erythropoietin receptors (EpoR) to stimulate erythroblastosis. A retroviral vector that does not encode any Env glycoprotein was packaged into retroviral particles and was coinjected into mice in the presence of a nonpathogenic helper virus. Although most mice remained healthy, one mouse developed splenomegaly and polycythemia at 67 days; the virus from this mouse reproducibly caused the same symptoms in secondary recipients by 2 to 3 weeks postinfection. This disease, which was characterized by extramedullary erythropoietin-independent erythropoiesis in the spleens and livers, was also reproduced in long-term bone marrow cultures. Viruses from the diseased primary mouse and from secondary recipients converted an erythropoietin-dependent cell line (BaF3/EpoR) into factor-independent derivatives but had no effect on the interleukin-3-dependent parental BaF3 cells. Most of these factor-independent cell clones contained a major Env-related glycoprotein with an M_r of 60,000. During further in vivo passaging, a virus that encodes an M_r-55,000 glycoprotein became predominant. Sequence analysis indicated that the ultimate virus is a new SFFV that encodes a glycoprotein of 410 amino acids with the hallmark features of classical gp55s. Our results suggest that SFFV-related viruses can form in mice by recombination of retroviruses with genomic and helper virus sequences and that these novel viruses then evolve to become increasingly pathogenic.The independently isolated Friend and Rauscher erythroleukemia viruses (18, 48) are complexes of a replication competent murine leukemia virus (MuLV) and a replication-defective spleen focus-forming virus (SFFV) (39, 42, 47). The SFFVs encode Env glycoproteins (gp55) that are inefficiently processed to form larger cell surface derivatives (gp55^p) (19). The gp55 binds to erythropoietin receptors (EpoR) to cause erythroblast proliferation and splenomegaly in susceptible mice. Evidence has suggested that the critical mitogenic interaction occurs exclusively on cell surfaces (7, 16).SFFVs are structurally closely related to mink cell focus-inducing viruses (MCFs) (2, 5, 10, 50), a class of replication-competent murine retroviruses that has a broad host range termed polytropic (15, 21). Although MCFs are not inherited as replication-competent intact proviruses, the mouse genome contains numerous dispersed polytropic env gene sequences (8, 17, 27). MCFs apparently readily form de novo by recombination when ecotropic host range MuLVs replicate in mice (14, 15, 26, 43). MCF env genes typically are hybrid recombinants that contain a 5′ polytropic-specific region and a 3′ ecotropic-specific portion (26). They encode a gPr90 Env glycoprotein that is cleaved by partial proteolysis to form the products gp70 surface (SU) glycoprotein plus p15E transmembrane (TM) protein (32, 39, 47). In addition, MCFs often differ from ecotropic MuLVs in their long terminal repeat (LTR) sequences (8, 20, 26, 28, 29, 45).Based on their sequences, SFFVs could have derived from MCFs by a 585-base deletion and by a single-base addition in the ecotropic-specific portion of the env gene (10). Evidence suggests that both the 585-bp deletion and the frameshift mutation probably contribute to SFFV pathogenesis (3, 49). Several pathogenic differences among SFFV strains have also been ascribed to amino acid sequence differences in the ecotropic-specific portion of the Env glycoproteins (9, 41).This report describes the origin and rapid stepwise evolution of a new SFFV. This new pathogenic virus initially formed in a mouse that had been injected with an ecotropic strain of MuLV in the presence of a retroviral vector that does not encode any Env glycoprotein. The mouse developed erythroleukemia, splenomegaly, and polycythemia after a long lag phase. At that time the spleen contained viruses with env genes that were able to activate EpoR. Serial passage of this initial virus isolate resulted in selection of a novel SFFV that encodes a gp55 glycoprotein of 410 amino acids. This experimental system provides a method for isolating new SFFVs and for mapping the stages in their genesis.     

Phylogenetic Analysis Reveals Rapid Evolutionary Dynamics in the Plant RNA Virus Genus Tobamovirus

Early studies on the evolutionary dynamics of plant RNA viruses suggested that they may evolve more slowly than their animal counterparts, sometimes dramatically so. However, these estimates were often based on an assumption of virus–host codivergence over time-scales of many millions of years that is difficult to verify. An important example are viruses of the genus Tobamovirus, where the assumption of host–virus codivergence over 100 million years has led to rate estimates in the range of ~1 × 10⁻⁸ nucleotide substitutions per site, per year. Such a low evolutionary rate is in apparent contradiction with the ability of some tobamoviruses to quickly overcome inbred genetic resistance. To resolve how rapidly molecular evolution proceeds in the tobomaviruses, we estimated rates of nucleotide substitution, times to common ancestry, and the extent of congruence between virus and host phylogenies. Using Bayesian coalescent methods applied to time-stamped sequences, we estimated mean evolutionary rates at the nucleotide and amino acid levels of between 1 × 10⁻⁵ and 1.3 × 10⁻³ substitutions per site, per year, and hence similar to those seen in a broad range of animal and plant RNA viruses. Under these rates, a conservative estimate for the time of origin of the sampled tobamoviruses is within the last 100,000 years, and hence a far more recently than proposed assuming codivergence. This is supported by our cophylogeny analysis which revealed significantly discordant evolutionary histories between the tobamoviruses and the plant families they infect.     

Novel Virus Discovery and Genome Reconstruction from Field RNA Samples Reveals Highly Divergent Viruses in Dipteran Hosts

We investigated whether small RNA (sRNA) sequenced from field-collected mosquitoes and chironomids (Diptera) can be used as a proxy signature of viral prevalence within a range of species and viral groups, using sRNAs sequenced from wild-caught specimens, to inform total RNA deep sequencing of samples of particular interest. Using this strategy, we sequenced from adult Anopheles maculipennis s.l. mosquitoes the apparently nearly complete genome of one previously undescribed virus related to chronic bee paralysis virus, and, from a pool of Ochlerotatus caspius and Oc. detritus mosquitoes, a nearly complete entomobirnavirus genome. We also reconstructed long sequences (1503-6557 nt) related to at least nine other viruses. Crucially, several of the sequences detected were reconstructed from host organisms highly divergent from those in which related viruses have been previously isolated or discovered. It is clear that viral transmission and maintenance cycles in nature are likely to be significantly more complex and taxonomically diverse than previously expected.     

Forward Genetics by Genome Sequencing Reveals That Rapid Cyanide Release Deters Insect Herbivory of Sorghum bicolor

Whole genome sequencing has allowed rapid progress in the application of forward genetics in model species. In this study, we demonstrated an application of next-generation sequencing for forward genetics in a complex crop genome. We sequenced an ethyl methanesulfonate-induced mutant of Sorghum bicolor defective in hydrogen cyanide release and identified the causal mutation. A workflow identified the causal polymorphism relative to the reference BTx623 genome by integrating data from single nucleotide polymorphism identification, prior information about candidate gene(s) implicated in cyanogenesis, mutation spectra, and polymorphisms likely to affect phenotypic changes. A point mutation resulting in a premature stop codon in the coding sequence of dhurrinase2, which encodes a protein involved in the dhurrin catabolic pathway, was responsible for the acyanogenic phenotype. Cyanogenic glucosides are not cyanogenic compounds but their cyanohydrins derivatives do release cyanide. The mutant accumulated the glucoside, dhurrin, but failed to efficiently release cyanide upon tissue disruption. Thus, we tested the effects of cyanide release on insect herbivory in a genetic background in which accumulation of cyanogenic glucoside is unchanged. Insect preference choice experiments and herbivory measurements demonstrate a deterrent effect of cyanide release capacity, even in the presence of wild-type levels of cyanogenic glucoside accumulation. Our gene cloning method substantiates the value of (1) a sequenced genome, (2) a strongly penetrant and easily measurable phenotype, and (3) a workflow to pinpoint a causal mutation in crop genomes and accelerate in the discovery of gene function in the postgenomic era.     

Long-Term Evolution of the Luteoviridae: Time Scale and Mode of Virus Speciation

Despite their importance as agents of emerging disease, the time scale and evolutionary processes that shape the appearance of new viral species are largely unknown. To address these issues, we analyzed intra- and interspecific evolutionary processes in the Luteoviridae family of plant RNA viruses. Using the coat protein gene of 12 members of the family, we determined their phylogenetic relationships, rates of nucleotide substitution, times to common ancestry, and patterns of speciation. An associated multigene analysis enabled us to infer the nature of selection pressures and the genomic distribution of recombination events. Although rates of evolutionary change and selection pressures varied among genes and species and were lower in some overlapping gene regions, all fell within the range of those seen in animal RNA viruses. Recombination breakpoints were commonly observed at gene boundaries but less so within genes. Our molecular clock analysis suggested that the origin of the currently circulating Luteoviridae species occurred within the last 4 millennia, with intraspecific genetic diversity arising within the last few hundred years. Speciation within the Luteoviridae may therefore be associated with the expansion of agricultural systems. Finally, our phylogenetic analysis suggested that viral speciation events tended to occur within the same plant host species and country of origin, as expected if speciation is largely sympatric, rather than allopatric, in nature.Although RNA viruses are the most common agents of emerging disease, key aspects of their evolution are still only partly understood. This is of both academic and practical importance, as virus evolution may compromise disease control strategies, including the rapid generation of genotypes that are able to evade host immune responses or of those that are resistant to antivirals or crop genetic resistance (20, 34, 47).Most of our knowledge of the rapidity of RNA virus evolution comes from the study of animal viruses, for which estimates of rates of nucleotide substitution normally fall within 1 order of magnitude of 1 × 10⁻³ nucleotide substitutions per site per year (subs/site/year) and largely reflect the background mutation rate (10, 13, 29, 37, 53). Equivalent studies on plant RNA viruses have reported more heterogeneous rates. Early studies suggested that some plant RNA viruses evolved more slowly than RNA viruses that infect animals. For example, estimates of the nucleotide substitution rate in the range of ∼1 × 10⁻⁶ to 1 × 10⁻⁸ subs/site/year have been obtained for Turnip yellow mosaic virus (4, 23) and some tobamoviruses (21, 25). In contrast, more recent estimates using Bayesian coalescent methods applied to sequences with known dates of sampling and allowing for rate variation among lineages have reported substitution rates in the same range as those of animal RNA viruses (22, 63) and therefore suggest relatively high rates of mutation, as expected, given the intrinsically error-prone nature of RNA replication (15, 65). As well as the differences in how these rates are estimated, a reasonable biological explanation for such a diversity of rate estimates is that they are increased in the short term due to the presence of mutational polymorphisms but lower in the long term because any such deleterious mutations would have then been removed by purifying selection (17, 22). In particular, severe population bottlenecks at transmission would allow deleterious mutations to rise to a high frequency due to strong genetic drift. Such effects make it dangerous to extrapolate long-term rates of evolutionary change from the analysis of intraspecific sequence data (31). Differences in the strength of adaptive evolution could also cause rate heterogeneity, including such processes as competition for susceptible individuals and the colonization of new host species (22, 65).Although there is a growing body of data on intraspecific evolutionary processes in plant RNA viruses, including rates of nucleotide substitution, there has been a general neglect of long-term evolutionary patterns, including the determinants of viral speciation. Exceptions are recent analyses of the Potyviridae and the Sobemovirus, which associated viral speciation with the development of agriculture (17, 24). Although RNA viruses reproduce asexually, it is informative to consider as analogies the two major forms of speciation used in studies of sexually reproducing eukaryotes: allopatric speciation, in which reproductive isolation follows geographic separation, and sympatric speciation, in which reproductive isolation occurs within an interbreeding population (67). In the context of RNA viruses, allopatric speciation can be thought of as the genetic diversification that occurs when viruses jump to new host species and thereafter evolve independently, as is commonly associated with the process of viral “emergence.” In contrast, sympatric speciation would occur when viruses diversify within a single host species, perhaps by exploiting different cell types (34). Despite the importance of these processes for our understanding of the macroevolution of RNA viruses, their respective roles are currently unknown.To better understand the nature of long-term evolutionary processes in plant RNA viruses, we undertook an extensive molecular evolutionary analysis of the family Luteoviridae, a heterogeneous family of plant viruses divided into three genera, Luteovirus, Polerovirus, and Enamovirus, containing five, nine, and one classified species, respectively, as well as a number of unclassified species (18). The Luteoviridae possess positive-sense single-stranded RNA genomes of 5,600 to 6,000 nucleotides (nt). These genomes can harbor five or six open reading frames (ORFs). 5′-proximal partially overlapping ORF1 and -2 encode proteins P1 and P2, which are involved in virus replication. Low-frequency −1 ribosomal frameshifting in the overlapping region results in the P1-P2 fusion RNA-dependent RNA polymerase protein (RdRp). ORF3 encodes the coat protein (CP) and completely contains ORF4, which is not found in Enamovirus and is needed for virus movement in the plant (the movement protein, MP). ORF5, which is necessary for aphid transmission (6, 27, 49) and is also involved in virus movement (57) and Luteoviridae phloem limitation (58), is translated through in-frame read-through of the ORF3 stop codon, existing as a read-through domain (RTD) fused to the CP. Members of the genus Polerovirus have an extra ORF0 in the 5′ end of the genome partially overlapping ORF1. Its translation product (P0) acts as a repressor of the RNA-silencing plant defense response (44, 59). Finally, some Luteovirus species have an additional ORF6 with an unknown function in the 3′ end of the genome (18, 48, 70). As a consequence of this particular genomic organization, approximately one-third of the Polerovirus genome, and a smaller fraction in the Luteovirus genome, is composed of overlapping regions.Due to their agronomic importance, gene sequence data, together with information on host range and geographical distribution, are available for a relatively large number of members of the family Luteoviridae. However, to date the only luteovirus for which rates of evolutionary change have been estimated is Barley yellow dwarf virus (BYDV). In this case, an analysis of substitution rates based on viral RNA extracted from herbarium specimens produced estimates of between 6.2 × 10⁻⁴ and 9.7 × 10⁻⁵ subs/site/year (43). Similarly, only one estimate of the point at which genetic diversity arose in the family Luteoviridae has been obtained, i.e., approximately 9,000 years ago, and therefore it is perhaps associated with the rise of agriculture (17). However, only a limited number of Luteoviridae species and sequences were included in this analysis. No studies have yet considered the mechanisms of speciation in the family Luteoviridae.The family Luteoviridae also represents a useful data set to study two other evolutionary phenomena: the pattern and determinants of recombination, which appears to be commonplace within the family Luteoviridae (26, 49, 51, 70, 71), and the differing evolutionary dynamics in genes with overlapping reading frames. There are contrasting hypotheses as to why overlapping reading frames are so commonly used in RNA viruses. According to one view, gene overlapping maximizes the genetic information in smaller genomes (1, 39). Alternatively, it has been suggested that gene overlap generates mutational robustness (i.e., the ability to preserve phenotypes despite the genomic mutational load) at the population level (2, 16, 42). Under the latter hypothesis, gene overlapping generates hypersensitivity to deleterious mutations, as these affect more than one gene. Although this hypersensitivity reduces the capacity of each individual to buffer mutation effects, it represents a selective advantage for wild-type genotypes, which then bolsters robustness at the population level (16, 42). As a consequence of this elevated burden of deleterious mutation, RNA viruses with larger proportions of their genomes present as overlapping reading frames are expected to exhibit lower rates of nucleotide substitution (41, 50). Such a rate reduction has been observed in many animal DNA and RNA viruses (for example, see references 35, 36, 55, 77, and 78), although only a few studies have considered plant RNA viruses in this context (28, 61).