Sex is the dominant mode of reproduction among euka-ryotic life, but why sex prevails over asexual reproduction is among the most important unresolved and contested questions in biology (Bell, 1982; Barton & Charlesworth, 1998; Salathéet al., 2008b; Otto, 2009; Becks & Agrawal, 2010; Lively, 2010). Despite being less common, asexual reproduction is widespread across the tree of life and is associated with many ecological and evolutionary advantages over sexual reproduction. For example, asexual individuals can avoid ecological and genetic costs of sex by reproducing faster (‘cost of males’, Maynard Smith, 1978), and they also exhibit greater relatedness to offspring (‘cost of meiosis’, Williams, 1975; Lively & Lloyd, 1990). In contrast, the prevalence of sex is attributed to evolutionary advantages associated with recombination and segregation of alleles, which are greatly suppressed or completely absent in asexual individuals. For example, evolutionary theory predicts that recombination and segregation enable more effective purging of deleterious mutations (Muller, 1964; Lynch & Gabriel, 1990) and promote beneficial (adaptive) allele combinations (Muller, 1932; Agrawal, 2006; Otto, 2009). There has been some empirical work in animal systems, showing that asexual lineages have the propensity to accumulate more deleterious mutations than related sexual lineages (Paland & Lynch, 2006; Johnson & Howard, 2007; Neiman et al., 2010), but we are unaware of any studies that examine whether asexual lineages also experience slower rates of adaptive molecular evolution (positive selection) than related sexual lineages.
Natural enemies of plants are ubiquitous in nature and can have major negative fitness consequences for host species. Under such circumstances, natural enemies are expected to impose selection on host species to evolve adaptive defence traits that help them avoid attack and/or minimize negative fitness consequences of attack (Ehrlich & Raven, 1964; Zangerl & Berenbaum, 2005; Stenberg et al., 2006). Understanding the effects of recombination and segregation on the molecular evolution of host defence genes could provide key insights into whether sexually reproducing hosts have an advantage over asexually reproducing hosts when responding to natural enemies and whether sex promotes adaptive molecular evolution. For example, if plants adapt to natural enemies by altering proteins that recognize or attack natural enemies, then sexual lineages should exhibit stronger signatures of positive selection on defence proteins than asexual lineages. Such adaptive changes might not only involve structural changes to proteins (e.g. amino acid replacements in coding regions) but might also rely on changes in regulatory regions or a combination of these factors (Doebley, 1993; Purugganan, 1998; Hoekstra & Coyne, 2007).
Here, we first show that pathogens can potentially exert selection on Onagraceae species and then we test whether a loss of sexual recombination and segregation in plants negatively impacts adaptive molecular evolution in a plant gene involved in defence against pathogens. We focus on a class I basic chitinase (chiB) because plant chitinases are well studied and have a clear function in plants. Specifically, plant chitinases are disease resistance enzymes that are present in virtually all plants, and they aid in both the recognition and attack of fungal pathogens (Collinge et al., 1993; Kasprzewska, 2003; Ferreira et al., 2007). ChiB also offers an excellent model to test whether recombination and segregation (two fundamental components of sex) promote adaptive molecular changes within a plant defence gene because previous work on plants, without regard to variation in sexual reproduction, showed that specific amino acid sites within chiB are under positive selection (Bishop et al., 2000; Tiffin, 2004). We test our hypotheses by examining species belonging to the evening primrose family (Onagraceae). This family is an ideal plant system for testing the effects of sex on plant defences because it contains many sexual and functionally asexual species, where a breakdown in sex arises because of a genetic system called permanent translocation heterozygosity (PTH). PTH species retain fertilization but experience a near-complete suppression of both recombination and segregation such that all seeds produced by a maternal plant are genetically identical (Stebbins, 1950; Cleland, 1972; Rauwolf et al., 2008, 2011). Furthermore, there have been many (> 20) independent transitions between sexual and functionally asexual (PTH) reproduction in the Onagraceae (Holsinger & Ellstrand, 1984; Johnson et al., 2009, 2011), which allows for a naturally replicated and robust test of the effects of sex on plant defence evolution.
Based on evolutionary theory, we predicted that the repeated losses of recombination and segregation in the Onagraceae cause decreased rates of adaptive molecular evolution in chiB. To test this prediction, we applied maximum-likelihood comparative phylogenetic approaches to infer rates of structural molecular evolution of the chiB enzyme across 16 sexual and 16 asexual evening primrose (Onagraceae) plant lineages. We further compared chiB gene expression across a subset of these lineages to determine whether a loss of sex is also associated with changes in regulatory evolution.