A tripartite dsRNA genome characterizes phages in the Cystoviridae clade, a historically depauperate group, with 6 as its sole representative for >25 years after initial isolation. Eight other phages within this clade were recently isolated from bacteria-infested legumes (25), indicating that these viruses may be relatively common in many terrestrial habitats. Previous attempts to isolate additional Cystoviruses had failed, and it is unclear what has precipitated the more recent success. The high frequency of Cystoviruses within our environmental isolates hints that these phages may be a dominant predator in some bacterial communities.
Reassortment and Recombination. The most surprising result of our study is the complete lack of evidence for LD between some pairs of phage segments. LD is conspicuously absent between the S and L segments and the M and L segments, where we had the greatest power to detect it. Two factors decrease power in detecting LD: skewed allele frequencies (although this is mitigated by using randomization tests) and reduced genetic diversity. Thus, the two sets of segments in which we have the most power to detect LD are between the M and L, having the most genetic diversity; and between the S and L, having the least skewed allele frequencies. However, it is precisely these pairs that show no evidence of LD. These results thus imply that migration, coinfection, and selection all fail to limit reassortment between these segments. We consistently found LD (albeit at low levels) between the S and M segments; because all three LD tests bordered on significance, a lack of statistical power is likely to be important in the latter result. Overall, our study strongly suggests that the Cystoviridae experience frequent and widespread migration and regular coinfection of bacterial cells. Selection against reassortant phages seems to occur for distantly related S and M segments.
Several phages are identical for one segment and highly divergent for the other two (Fig. 1), indicating that the rate of reassortment is higher than the observed rate of substitution, even at 4-fold degenerate sites. If substitutions at 4-fold degenerate sites are neutral, we will observe them at the same rate as the per-base-pair mutation rate. We observed identical sequences for one segment and highly divergent sequences for a second segment; thus, reassortment has occurred before mutation at 4-fold degenerate sites within the first segment. Our sequence data encompassed a minimum of 7% of each segment, and, thus, we expect that the per-segment mutation rate does not exceed the per-segment rate of reassortment by >14 times (1/0.07).
Most virus studies concern strains pathogenic to humans and have concluded that reassortment occurs at low rates in virus populations. For example, reassortment in Influenza-A virus seems relatively uncommon (2-4) and depend on relatedness (6). Recent work has suggested that perhaps two or three reassortment events have occurred over a period of several years (4). Qualitative studies in Influenza-B virus have shown that, although reassortment is important for creating viral diversity (5, 9, 16), the rate does not approach that which we found in the Cystoviridae. High levels of LD are present between segments, even when examined across a relatively large time scale (25 years). Rotaviruses experience significant, although not high, rates of reassortment: Various measures of the frequency of reassortant viruses range between 2.7% (8) and 5.4% (14). Within the Bunyaviridae group, reassortment generally occurs infrequently (10) and depends on genetic similarity (7), whereas, in some taxa of pathogenic segmented viruses, reassortment has never been documented (15). Studies of disease-causing segmented viruses of plants have found low reassortment rates (13) and strong selection acting against reassortants (11, 12, 47). A drawback of these studies is that they give qualitative assessments of reassortment; however, the studies do suggest that it is not very common. It is thus surprising that our data indicate Cystovirus populations undergo genetic exchange at rates similar to those of obligately sexual populations. The result is even more surprising when one considers the amount of nucleotide divergence we observed between segments (>50% at 4-fold degenerate sites).
Laboratory assessments of intrasegment homologous recombination rates in the Cystoviridae (26, 46) are supported by our study. We found only marginally significant evidence for recombination; additionally, this evidence was inconsistent (Table 1). This test of intrasegment recombination (the decline of LD with increasing nucleotide distance) is extremely sensitive to small levels of recombination. Thus, although the result of the test is nonsignificant, its sensitivity suggests that recombination within segments is an extremely rare event. However, this analysis was done on a fairly small scale of hundreds of nucleotides; with sequence data spanning entire segments, significant levels of recombination may be detectable. It is important to note that the rate of recombination is extremely low relative to both the rate of mutation and the rate of reassortment (≈106-fold lower than both). Thus, homologous recombination is likely to play a relatively unimportant role in the evolution of this phage.
The primary limitation in the rate of homologous recombination is probably the nature of template replication in this phage family (26), although selection may also play a role in limiting recombination in various genomic regions. Specifically, some forms of epistasis will select against recombinants.
Genetic Exchange and the Viral Species Concept. Proper methods for assigning species boundaries in virus taxonomy are unclear, mostly because of inadequate understanding of what constitutes a species in naturally occurring viral populations. It would be informative to apply the biological species concept; viruses are capable of genetic exchange (2-16), and this method separates species according to barriers to genetic mixis. If viral species can be accurately delineated, effective population sizes can also be assessed. The population size of most natural viral populations is unknown, although this parameter may play a key role in processes such as disease emergence. It has been suggested that pathogens with higher mutation rates will produce more genetic variants and are, therefore, more likely to be generalists (48). This same prediction applies to larger population sizes, because a more appropriate measure of genetic variation is the population mutation rate (4Neμ in diploids). Thus, determination of viral species through the biological species concept and subsequent measurements of population size could provide an informative measure for the likelihood of emergent disease. It is likely that, in many viral groups, the rate of genetic mixis is a continuous trait, unlike the situation in most eukaryotic taxa, in which genetic exchange is, to a large extent, either present or absent, implying that, instead of distinct genetic clusters, such as those seen in eukaryotic and most prokaryotic taxa (49), loosely cohesive genetic clouds will be observed.
Migration. The geographic sampling was structured on a logarithmic scale, which should provide the most power for resolving structure at almost any scale. Notably, phages isolated from single clovers were not consistently more similar to each other than to phages isolated from sites across the country or from other previously isolated phages (Fig. 1). These data are thus consistent with frequent continent-wide migration in the Cystoviridae. To our knowledge, the only other study (50) addressing migration rates in phage populations also found continent-wide migration but in dsDNA (T7-like) marine phages. It is interesting that the only segment for which we find a significant association between geographical and genetic distance is the M segment. Limited migration cannot be the cause for this geographic structure, because neither the S nor L segments show evidence of regional structure. Nor is it likely that this structure has been introduced by a bias in the isolation process, because all of the samples should be subject to the same procedural bias in the laboratory. Rather, the result suggests that the M segment shows geographic structure because of selection. Notably, the M segment carries the genes responsible for host specificity (28); selection for local host adaptation may limit the amount of M segment migration.