Microbes are the most numerous entities in the biosphere, and viruses that infect bacteria (bacteriophages) constitute the majority of these organisms (1). Various mechanisms exist for genetic exchange (sex) between individual microbes, but the importance of these processes in structuring virus populations remains unclear. Several studies have estimated that the rate of genetic exchange in virus populations is relatively low (2–16). However, only a few medically or agriculturally important viruses have been examined, few of which represent random population samples. These biases prevent the drawing of general conclusions. Bacteriophages are an excellent choice for studying the rate and significance of genetic exchange in virus populations. It is increasingly evident that phages often exert significant control over the population sizes of their hosts and, thus, influence large-scale ecological and biogeochemical processes attributed to bacteria (17, 18). Furthermore, it has long been proposed that sex may be generally favored, because it promotes linkage equilibrium, which (in addition to de novo mutations) can provide the genetic variability that is the raw material for natural selection (19–23). Therefore, from a theoretical standpoint, it is crucial to gauge the rate of genetic exchange in phages and to determine whether sexual processes act to structure biological populations that comprise a major portion of our global ecosystem.
Bacteriophages from the Cystoviridae clade were first isolated in 1971 from bean straw infested with Pseudomonas syringae pathovar phaseolicola (Pp) (24). Cystoviridae are characterized by a tripartite dsRNA genome, and the three RNA segments per phage particle are referred to as L, M, and S (large, medium, and small, respectively). Additional phages from this clade were not isolated until 25 years later from the leaves of several agricultural species (25). Experimental manipulations have shown that intrasegment recombination in Cystoviruses is infrequent, occurring at a rate of ≈10-7 per segment per generation (26), but reassortment (exchange of segments between viruses) readily occurs when host cells are multiply infected (27). These characteristics are biologically similar to those of segmented RNA viruses of medical and agricultural importance, such as Influenza-A. Reassortment between very distantly related phages in the Cystoviridae clade has been documented in vitro (28). However, the frequency of reassortment among Cystoviruses in the wild is unknown.
Here, we report the results of a biogeography study involving Cystoviridae isolates from different regions across the United States. We use nucleotide sequence data derived from newly and previously isolated strains (25, 29) to characterize migration rates and linkage disequilibrium (LD); LD is a measure of the nonrandomness of the association between alleles at different sites. We test for a correlation between genetic and geographic distance between segments and for LD within and between segments. The rarity of intrasegment recombination documented in laboratory studies of the phage suggests that LD within segments will be high. In contrast, frequent genetic reassortment among segments would cause low LD between segments. We test for LD using three methods: a Mantel test of genetic association, a test using standard metrics of LD (r2 and D′), and a third method based on measures of phylogenetic congruence. In contrast to previous studies of viral populations, we find no consistent evidence for LD between most segment pairs, despite the fact that some segments have >50% nucleotide divergence at 4-fold degenerate sites. This extraordinary rate of genetic exchange between highly unrelated individuals is unprecedented in any taxa and implies that sex within this group of viruses occurs frequently and, in some cases, without limitation by selection.