J Mol Evol (2014) 78:101–108 DOI 10.1007/s00239-013-9604-x ORIGINAL ARTICLE The Effects of Stabilizing and Directional Selection on Phenotypic and Genotypic Variation in a Population of RNA Enzymes Eric J. Hayden • Sinisa Bratulic • Iwo Koenig • Evandro Ferrada • Andreas Wagner Received: 26 September 2013 / Accepted: 23 November 2013 / Published online: 6 December 2013 Ó Springer Science+Business Media New York 2013 Abstract The distribution of variation in a quantitative Introduction trait and its underlying distribution of genotypic diversity can both be shaped by stabilizing and directional selection. A population of organisms or molecules with some quanti- Understanding either distribution is important, because it tative phenotype P, where natural selection favors an determines a population’s response to natural selection. increase or decrease in P, and where the population mean of P Unfortunately, existing theory makes conflicting predictions is consequently changing over time, is said to be under about how selection shapes these distributions, and very little directional selection. This mode of selection typically occurs pertinent experimental evidence exists. Here we study a when a population experiences a new environment and many simple genetic system, an evolving RNA enzyme (ribozyme) individuals find themselves poorly adapted. Over time, such in which a combination of high throughput genotyping and a population would adapt to its environment, and enter a measurement of a biochemical phenotype allow us to address regime of stabilizing selection, during which the population this question. We show that directional selection, compared mean phenotype P no longer changes. Because of its to stabilizing selection, increases the genotypic diversity of importance for understanding Darwinian evolution, the an evolving ribozyme population. In contrast, it leaves the effects of directional and stabilizing selection on phenotypes variance in the phenotypic trait unchanged. have received considerable attention by theorists. One simple question with no straightforward answer Keywords Ribozymes Á Experimental evolution Á regards the effects of directional and stabilizing selection Genotype to phenotype Á Directional selection Á on the phenotypic variance of a population. Because Stabilizing selection Á Sequence space directional selection can favor extreme phenotypes, one might think that it would cause an increase not only in the mean, but also in the phenotypic variance of the popula- E. J. Hayden (&) Department of Biological Science, Boise State University, Boise, tion. However, this is not necessarily the case. Whether it ID, USA does depends on several factors, including the shape— e-mail: [email protected] convex or concave—of the fitness function (Layzer 1978), the distribution of trait values (Bu¨rger 1991; Hansen 1992), S. Bratulic Á A. Wagner Institute of Evolutionary Biology and Environmental Studies, whether a trait optimum fluctuates with time, the presence University of Zurich, Zurich, Switzerland and kind of genotype-by-environment interactions (Her- misson and Wagner 2004), the existence of epistatic S. Bratulic Á A. Wagner interactions among loci (Hansen 2006), and the extent of The Swiss Institute of Bioinformatics, Lausanne, Switzerland phenotypic canalization before the onset of directional I. Koenig selection (de Visser et al. 2003; Hermisson and Wagner Department of Biochemistry, University of Zurich, Zurich, 2004). The change in phenotypic variance caused by Switzerland periods of stabilizing selection is no easier to predict. It E. Ferrada Á A. Wagner depends upon several attributes of genetic architecture, The Santa Fe Institute, Santa Fe, NM, USA which include epistasis (Hermisson et al. 2003), the 123 102 J Mol Evol (2014) 78:101–108 structure of the fitness landscape (Barton and Turelli 1987), individual sequence occupies a discrete point in this space. the number of loci involved in a trait (Turelli and Barton Two sequences have a distance that is defined as the number 1990;Bu¨rger 1991), and the extent of canalization (Wagner of nucleotide changes necessary to change one sequence into et al. 1997). Overall, existing theory does not make any the other. The genotypes of a given population can be viewed simple and general predictions for the change in pheno- as a cloud in genotype space. Here, we describe the effects on typic variance over time. Unfortunately, there is also very this cloud of genotypes as it experiences stabilizing selection little experimental data that speak to this question, with the that is followed by a period of directional selection. exception of the observation that stressful environments We previously conducted evolution experiments where can cause a release of previously hidden phenotypic vari- we exposed the Azoarcus group I ribozyme to two different ation with a genetic basis (Waddington 1959; Scharloo chemical environments (Hayden et al. 2011). The ribozyme 1991; Rutherford and Lindquist 1998; True and Lindquist has the ability to cleave an exogenous RNA substrate 2000; Gibson and Dworkin 2004; Flatt 2005; Hayden et al. because this activity is a component of its catalytic role in 2011). nature as a self-splicing intron (Tanner and Cech 1996). In Directional and stabilizing selection can not only affect the first environment we supplied a ribozyme population the distribution of phenotypes, but also the distribution of with a ‘‘native’’ RNA oligonucleotide. Cleavage of this oli- genotypes, and especially their diversity. Next to no gonucleotide results in the ligation of a portion of the sub- experimental data are available about these effects, and strate to the 30-end of the ribozyme, and we can exploit this mainly for two reasons. First, it has not been feasible until sequence modification to selectively amplify catalytically recently to genotype many individuals in an evolving successful molecules (Beaudry and Joyce 1992; Lehman and population. Second, many phenotypic traits are complex Joyce 1993). Molecules with higher activity are more likely and have unknown genetic components. Next-generation to be replicated. We previously observed that in this chem- sequencing technologies are beginning to allow us to ical environment, the average activity of the population did overcome the first problem (Kvitek and Sherlock 2011; not change significantly over eight rounds of mutation and Ensminger et al. 2012; Traverse et al. 2013; Herron and selection on this native substrate. Further, we did not detect Doebeli 2013; Jime´nez et al. 2013), especially for the rel- any mutations that were increasing in frequency over time, atively simple phenotypic traits of molecules, where the which would indicate a selective advantage for ribozymes second problem is not an issue. For such molecular traits, containing them. We concluded that the population was one can obtain complete genotypic information of many well-adapted to this native substrate. Herein, we refer to the individuals in an evolving population (Hietpas et al. 2011; population from the eighth round of this selection as our Hayden et al. 2011). The purpose of this paper is to study Stabilizing Selection population (SS). the evolution of both phenotypic and genotypic variation in The second chemical environment is one to which the an especially transparent molecular system, a population of ancestral wild-type Azoarcus ribozyme is poorly adapted. evolving RNA enzymes. Specifically, we introduced a chemical modification The phenotypic trait in our study system is the catalytic (phosphorothioate) into the RNA backbone of the substrate, activity of the RNA enzyme (ribozyme) derived from the and exposed a ribozyme population to this modified sub- Azoarcus group I self-splicing intron (Tanner and Cech strate. We mutagenized the members of this SS population, 1996). Ribozymes are nucleic acid sequences with a catalytic and then exerted selection by amplifying molecules that function. Thus, phenotype and genotype are linked to a could cleave this new substrate, performing an additional single molecule in this system. Populations of ribozyme eight ‘‘generations’’ of mutation and selection in this way. genotypes can be enzymatically replicated in the lab. Under this second selection pressure, the population adapted Genotypic and phenotypic diversity is introduced through rapidly to the new substrate. The average activity of the nucleotide mutations made during replication. Selection can population after eight generations was significantly higher be applied to exploit the differences in catalytic activity than it was prior to this selection. This demonstrates direc- caused by these mutations, resulting in Darwinian evolution tional selection exerted by the introduction of the new sub- (Joyce 2004). An important feature of our study system is strate. We will refer to the population from the eighth round that the phenotypes are encoded in very small genotypes of this second selection as the directional selection (DS) (\200 bp). For such genotypes, high throughput sequencing population. These experimentally derived populations technology can be used to analyze many genotypes in facilitate a comparison between the distribution of pheno- evolving RNA populations (Pitt and Ferre-D’Amare 2010; types and genotypes resulting from an equal number of Hayden et al. 2011). In other words, they allow us to study rounds (eight) of each type of selection. Our previous activity empirically how such populations spread through the vast measurements were based on