Evolutionary genomics of host-use in bifurcating demes of RNA virus phi-6

Abstract

Background: Viruses are exceedingly diverse in their evolved strategies to manipulate hosts for viral replication. However, despite these differences, most virus populations will occasionally experience two commonly-encountered challenges: growth in variable host environments, and growth under fluctuating population sizes. We used the segmented RNA bacteriophage ϕ6 as a model for studying the evolutionary genomics of virus adaptation in the face of host switches and parametrically varying population sizes. To do so, we created a bifurcating deme structure that reflected lineage splitting in natural populations, allowing us to test whether phylogenetic algorithms could accurately resolve this 'known phylogeny'. The resulting tree yielded 32 clones at the tips and internal nodes; these strains were fully sequenced and measured for phenotypic changes in selected traits (fitness on original and novel hosts).

Results: We observed that RNA segment size was negatively correlated with the extent of molecular change in the imposed treatments; molecular substitutions tended to cluster on the Small and Medium RNA chromosomes of the virus, and not on the Large segment. Our study yielded a very large molecular and phenotypic dataset, fostering possible inferences on genotype-phenotype associations. Using further experimental evolution, we confirmed an inference on the unanticipated role of an allelic switch in a viral assembly protein, which governed viral performance across host environments.

Conclusions: Our study demonstrated that varying complexities can be simultaneously incorporated into experimental evolution, to examine the combined effects of population size, and adaptation in novel environments. The imposed bifurcating structure revealed that some methods for phylogenetic reconstruction failed to resolve the true phylogeny, owing to a paucity of molecular substitutions separating the RNA viruses that evolved in our study.

Figure 1

Design for experimental evolution of phage ϕ6 populations propagated via bifurcating demes, under various host-use challenges and population sizes. Labeled root, nodes and tips of the tree indicate isolated clones, subjected to genome sequencing

Figure 2

Molecular changes observed in the experimental clade more often experiencing infection on P. pseudoalcaligenes ERA (“ERA clade”). Changes in coding regions list the affected protein amino-acid substitution, RNA segment (L, M or S) and gene; changes in non-coding (n.c.) regions list the base position and segment. Mutations are relative to those observed in the immediate predecessor clone

Figure 3

Molecular changes observed in the experimental clade more often experiencing infection on P. phaseolicola (“PP clade”). Changes in coding regions list the affected protein amino-acid substitution, RNA segment (L, M or S) and gene; changes in non-coding (n.c.) regions list the base position and segment. Mutations are relative to those observed in the immediate predecessor clone

Figure 4

Molecular changes, in light of segment location, treatment regime and protein function

Figure 5

Phenotypic evolution through time, in light of the imposed experimental design. Values are mean log fitness of sequenced clones on (A) P. phaseolicola (PP) and on (B) P. pseudoalcaligenes (ERA). See Table 1 for numerical values and statistics

Figure 6

Inferred topology using only aligned sequences from tips (clones F1 thru F16) of the tree. Maximum parsimony, maximum likelihood, and Bayesian methods all yielded the same inferred tree (see text for details). MrBayes provided estimates of branch lengths. Numbers at nodes indicate marginal posterior probabilities, and bootstrap percentages in parentheses