The Genetic Basis of Thermal Reaction Norm Evolution in Lab and Natural Phage Populations

Two major goals of laboratory evolution experiments are to integrate from genotype to phenotype to fitness, and to understand the genetic basis of adaptation in natural populations. Here we demonstrate that both goals are possible by re-examining the outcome of a previous laboratory evolution experiment in which the bacteriophage G4 was adapted to high temperatures. We quantified the evolutionary changes in the thermal reaction norms—the curves that describe the effect of temperature on the growth rate of the phages—and decomposed the changes into modes of biological interest. Our analysis indicated that changes in optimal temperature accounted for almost half of the evolutionary changes in thermal reaction norm shape, and made the largest contribution toward adaptation at high temperatures. Genome sequencing allowed us to associate reaction norm shape changes with particular nucleotide mutations, and several of the identified mutations were found to be polymorphic in natural populations. Growth rate measures of natural phage that differed at a site that contributed substantially to adaptation in the lab indicated that this mutation also underlies thermal reaction norm shape variation in nature. In combination, our results suggest that laboratory evolution experiments may successfully predict the genetic bases of evolutionary responses to temperature in nature. The implications of this work for viral evolution arise from the fact that shifts in the thermal optimum are characterized by tradeoffs in performance between high and low temperatures. Optimum shifts, if characteristic of viral adaptation to novel temperatures, would ensure the success of vaccine development strategies that adapt viruses to low temperatures in an attempt to reduce virulence at higher (body) temperatures

Growth Rates of Phage Isolated from Nature at High Temperatures

<p>(A) Phylogenetic relationships of G4-like phages recently isolated from environmental samples, modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040201#pbio-0040201-b024" target="_blank">24</a>]. The genotypes of two pairs of sister taxa that differ at a locus (gene H: A47V) determined to have a large effect on reaction norm shape are indicated with blue (alanine) and red (valine). (B and C) Thermal reaction norms of the two phage pairs. Data are means ± s.e.m. determined from four or five replicate measures at each temperature.</p

TMV Decomposes Reaction Norm Shape Variation into Three Modes of Variation

<p>Vertical shifts produce changes in average performance, horizontal shifts produce changes in the optimal temperature, and generalist-specialist variation produces changes in niche width. An identical template polynomial is shown in each chart as a black dashed reaction norm. The transitions from blue to red curves illustrate hypothetical contributions of the three modes of variation to adaptation to high temperature.</p

Modes of Variation among the Reaction Norms of Evolving Genotypes

<p>TMV was used to decompose the variation among the reaction norms described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040201#pbio-0040201-g003" target="_blank">Figure 3</a> into three modes of variation. Each uppermost graph depicts the values of the fitted parameters from the model and the bottom graphs illustrate the impact of each parameter, if considered alone, on the shape of the reaction norm of G4_0. lnW is the natural log of growth rate.</p

Correlated Responses to Adaptation at High Temperature

<p>(A) Reaction norms estimated using TMV explain 71.79% of the sums of squares in the data. Data are mean growth rates ± s.e.m. for each phage genotype determined from three or four replicate measures at each temperature. (B) Correlated responses to selection at 41.5 °C (transfers 1–50; open circles) and to selection at 44 °C (transfers 51–100; closed circles). Data are the differences in mean ln(growth rate) ± s.e.d. between G4₅₀ and G4₀, and between G4₁₀₀ and G4₅₀, respectively.</p