Materials and methods

- Widespread genetic exchange among terrestrial bacteriophages

Sampling. Twenty-five phages were isolated from white clovers (Trifolium repens) on the campus of the University of California, San Diego (UCSD) and Chico, CA, and one from green bean (Phaseolus vulgaris) in Hamden, CT. General nomenclature is as follows: state of origin, initials of collector, and order of sample isolation. Clones taken from a single clover are identified with an alphabetical suffix that is serially assigned. At the UCSD location, five clones were isolated from each of three clovers (CA/KW064a-e, CA/KW065a-e, and CA/KW066a-e) and five other phages (CA/KW067-071) were isolated from individual clovers, all lying within a 1 m² quadrant. Four phages (CA/KW051, 052, 072, and 073) were isolated from clovers located ≈30 m, 50 m, 375 m, and 600 m from this quadrant. CA/OS001 was isolated in Chico, CA, (865 km from San Diego), and CT/KO001 was isolated in Hamden, CT (3,995 km from San Diego). All of the CA/KW clones were collected in August and September of 2002, the CA/OS clone was collected in July of 2003, and the CT/KO clone was collected in June of 2003.

Culture. Individual clovers collected in CA were placed in 10 ml of LB broth (Lennox medium, pH 7.5) inoculated with 10⁸Pp cells and grown overnight at 25°C. This culture was filter-sterilized through a 0.22-μm membrane, and a volume was plated on a lawn of Pp. One to five plaques were isolated from each plate. During culture, maximum phage densities after overnight culture were measured at 10⁵ per ml, and bacterial densities were maintained at >10⁷ per ml; thus, the likelihood of reassortment during isolation was low (probability of coinfection for each individual isolate -2). Phage isolation from plant material collected in CT differed, in that the initial medium was not inoculated with Pp.

Extraction, PCR, and Sequencing. Genomic extraction was performed as described in ref. 30 or by using QIAamp Viral RNA minikits (Qiagen, Valencia, CA). The dsRNA genome was reverse-transcribed by using random hexamers, and resultant cDNA was used as PCR template (primers available upon request). PCR products were purified by using the QIAquick PCR purification kit (Qiagen) or ExoSAP-IT (United States Biological, Swampscot, MA). Sequencing was performed by using the BigDye Terminator reaction v3.1 on an ABI 3100 (Applied Biosystems). Sequence reads were curated by eye by using the program sequencher. Isolate CA/KW066e could not be amplified and was not included in the analysis. All sequences used in this study have been deposited in GenBank (L segment, DQ273591-DQ273614; M segment, DQ273615-DQ273638; and S segment, DQ273639-DQ273662).

Population Structure Analysis. Genetic isolation by geographic distance for each segment was tested by a Mantel test (31) by using the program zt (32). Genetic distances were calculated by using the general time-reversible model (33) with γ distributed rate variation among sites, as implemented in the program tree-puzzle (34). Geographic coordinates were measured on the UCSD campus by GPS or downloaded from the U.S. Geological Survey website. Distances were measured by using a campus map or calculated by using the javascript program available at www.wcrl.ars.usda.gov/cec/java/lat-long.htm. Permutation tests to assess significance of LD within segments were performed by using the program pairwise, part of the LDHat package (downloaded from www.stats.ox.ac.uk/~mcvean/LDhat/LDhat1.0/LDhat1.0.html). All other analyses of LD and other permutation tests were implemented in activeperl 5.8.4.810.

The two metrics of LD we used were r² and D′. r² is the squared correlation coefficient (35) between loci and is a simple measure indicating how often an allele found at one locus is associated with an allele at a second locus. Consider a locus with two alleles, A and a. If there is a second locus with alleles B and b, and the frequency of each allele is denoted as p_i and the frequency of each genotype as p_ij, then r² = (p_AB - p_Ap_B)²/p_Ap_Bp_ap_b.

D′ is a measure of LD relative to the maximum possible value under the observed allele frequencies (36). Considering the above set of biallelic loci, D′ = D/D_max, where D ≡ p_AB - p_Ap_B and D_max is the lesser of p_Ap_b or p_ap_B, if D is negative, or the lesser of p_Ap_B or p_ap_b, if D is positive. The sign of D′ is arbitrary, so we use ,D′, in all cases. r² is sensitive to variation in allele frequencies between loci, whereas D′ is less so (37).

For the analysis of LD between segments, r² or D′ was measured between all pairs of polymorphic loci (nucleotides) and an average value was obtained for all nucelotides between each pair of segments. One set of segments was then randomized, and the average r² or D′ was recalculated. This process was repeated 10,000 times to test whether the observed average LD statistic was significantly greater than expected for a random (unlinked) collection of segments.

Phylogenetic Analysis. Orthologous sequences were obtained from the National Center for Biotechnology Information for the L, M, and S segments of the Cystovirus phage 6 (38-40), and the M and S segments from the Cystovirus phages 7, 9, and 10 (25). No orthologous L-segment sequence is available for these latter three phages. Nucleotide sequences were aligned by using the program clustalx (41) and manually corrected by eye. The program mrbayes v3.0b4 (42) was used for phylogeny construction. Analyses were performed by using the general time-reversible model, with γ distributed rate variation across sites. MCMC chains were run for 200,000 generations, with the first 100,000 discarded as burn-in for construction of the 50% majority-rule consensus trees. Tree-to-tree distance metrics were calculated by using the program component v2.00a (43). Five-hundred random trees generated by using component or 500 random trees from the set of 5,000 most-recently visited trees of the MCMC chain were used for the phylogenetic comparisons. Two distance metrics were used: the number of identical and the number of different triplets between trees.