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- Materials and methods
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Colour polymorphism is a widespread phenomenon in plant species affecting both vegetative and reproductive organs (e.g. Rafiński 1979; Smith 1986; Jorgensen & Andersson 2005; Whitney 2005; Rausher 2008). The co-occurrence of different colour morphs has ecological and evolutionary interest and can be due to a variety of evolutionary processes, from direct or indirect selection mediated by pollinators, frugivores, herbivores, pathogens or abiotic environmental effects, to random genetic drift (Epling & Dobzhansky 1942; Wright 1943; Rausher 2008).
Flower colour is a very conspicuous trait that is supposed to act as a selective target for pollinators. Directional selection by pollinators combined with random genetic drift should lead to the loss of floral colour polymorphism (Waser & Price 1981; Levin & Brack 1995; Campbell, Waser & Melendez-Ackerman 1997; Jones & Reithel 2001) but spatio-temporal fluctuations of pollinator spectrum could lead to divergent selection and maintenance of colour polymorphism (Subramanian & Rausher 2000; Schemske & Bierzychudek 2001). However, there are only relatively few direct demonstrations of pollinators exerting selection on flower colour variation, and recent studies have emphasized the need to critically reconsider the role attributed to pollinators in modelling floral characters (e.g. Clegg & Durbin 2000; Warren & Mackenzie 2001; Brown 2002; Strauss & Whittall 2006; Rausher 2008). It has been proposed that the evolution or predominance of a particular floral colour is mainly due to differences in the fitness associated with other correlated features (e.g. Levin & Brack 1995; Armbruster et al. 1997; Armbruster 2002; Frey 2004). Anthocyanins are omnipresent in angiosperms and probably evolved in early land plants long before the evolution of flowers. These pigments may have arisen in vegetative tissues in response to increased ultraviolet light, drought stress and herbivore pressures and were then subsequently co-opted by flowers to attract pollinators (Hanley, Lamont & Armbruster 2009; Whittal & Carlson 2009). In many cases, these pigments still maintain their original stress-related functions while also attracting pollinators (Winkel-Shirley 2002; Dick et al. 2011). The most common and studied flower colour polymorphism is the loss of anthocyanins by loss-of-function (LOF) mutations that cause pigmented flowers to become white. Unpigmented morphs often have lower tolerance to drought and heat than pigmented morphs (Schemske & Bierzychudek 2001, 2007; Strauss & Whittall 2006), and morphs with differential tolerance to abiotic conditions may be maintained by fluctuating environmental conditions or by geographical segregation (Dick et al. 2011). Although less frequent, the blue-purple/red-orange colour polymorphism is also common. This polymorphism can occur without any alteration of the responsible pigments involved, for example through vacuolar pH changes (Grotewold 2006). However, most frequently, the transition from blue-purple to red-orange is due to the production of less hydroxylated anthocyanins as a consequence of the inactivation of branches in the anthocyanin pathway (Rausher 2008). The biosynthetic pathway of anthocyanins comprises several branches differing in the B-ring hydroxylation level of their intermediate flavonoids and their final anthocyanins (Tanaka, Sasaki & Ohmiya 2008). As far as we are aware, environmentally related differences in fitness between morphs differing in such blue-red anthocyanin compositions have not been reported.
Scarlet pimpernel (Lysimachia arvensis (L.) U. Manns and Anderb., formerly Anagallis arvensis L.; Manns & Anderberg 2009) is a widely distributed annual herb, that was originally described as a red-flowered plant from Central Europe, but which also presents blue-flowered forms (Fig. 1). The two morphs of L. arvensis differ in anthocyanin composition, with malvidine and pelargonidine being mainly responsible for the blue and red coloration, respectively (Wiering & de Vlaming in Harborne 1968; Ishikura 1981). A search of L. arvensis in various Floras (e.g. Ferguson 1972) for the Mediterranean and European areas revealed that the red morph is most frequent in North and Central Europe, whereas in the Mediterranean region the blue morph is the most common, although some populations with both floral morphs occur in both areas. The climate of the Mediterranean region clearly differs from the Oceanic climate of North and Central Europe, being typically sunnier, hotter and dryer. Moreover, in an Oceanic climate, plants are rarely subjected to water stress since rainfall is distributed regularly throughout the year, while in the Mediterranean, besides an extended summer drought, there are shorter water stress periods in the rainy season due to unpredictability of rains. In L. arvensis, the colour polymorphism could be maintained by environmental heterogeneity if each morph performed better in different environments. In fact, in mixed Mediterranean populations, we have also observed that red plants show a tendency to inhabit wetter or shadier microsites (river banks, irrigated orchards or under tree canopy) than blue plants.
Figure 1. The blue and red morphs of Lysimachia arvensis and their frequencies throughout the western Mediterranean and south of Europe.
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There are no studies on pollinator discrimination between morphs in the scarlet pimpernel, but their role as selective agents might be minimized because the flowers show nastic movements and self-pollinate automatically (Gibbs & Talavera 2001). In this study, we have surveyed floral-morph proportions and environmental traits in 51 populations of scarlet pimpernel along a wide geographical range to test the hypothesis that geographical and local variations in flower colour are related to environmental variation. Moreover, to explore the existence of morph-by-environment interactions, we compared fitness components of blue and red morphs by growing plants under treatments simulating differences in water and light availability. In this way, we explored the hypothesis that flower colour polymorphism may be maintained by selection related to environmental heterogeneity.
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- Materials and methods
- Supporting Information
Colour polymorphism in plants has been traditionally considered to be a result of divergent direct selection driven by biotic agents. Polymorphisms of fruits and seeds are thought to be maintained by dispersers or by predators, and those of flowers by pollinators, although direct evidence is scarce (Whitney 2005; Rausher 2008; Porter 2013). Recently, the possibility of indirect selection by biotic or abiotic factors has become increasingly relevant as diverse cases are being reported. Polymorphisms of flowers, fruits and seeds can be caused by indirect selection due to biotic (Irwin et al. 2003; Whitney & Stanton 2004; Strauss & Whittall 2006) or abiotic agents (Willson & O'Dowd 1989; Traveset & Willson 1998; Schemske & Bierzychudek 2001; Whitney & Lister 2004). As we will show below, our results are consistent with the possibility that the flower colour polymorphism in L. arvensis is maintained by indirect selection driven by abiotic factors.
We found significant negative associations between blue morph frequency and latitude of populations, and between similarity in blue morph frequency and geographical distance of population pairs. This means that a geographical pattern of flower colour exists in L. arvensis, and it seems to be related to climatic features, which suggests that flower colour is not a neutral trait (Mayr 1965). The correlations found between blue morph frequency and the environmental variables studied indicate that blue plants are more frequent in dryer, hotter Mediterranean localities while red plants predominate in more temperate Oceanic areas. This could reflect a differential adequacy of morphs to environmental conditions that is also supported by the fact that red plants in southern mixed populations frequently occupied the wettest or shadiest places (M. Arista & P. L. Ortiz). The existence of a relationship between environmental gradient and geographical pattern does not prove a selective response to climatic traits and alternative explanations should be considered (Pope et al. 2013). The spectrum of pollinators of L. arvensis appears to differ in different localities; populations in the south of Spain receive very few visitors, mainly solitary bees (Gibbs & Talavera 2001; P. L. Ortiz et al. unpub. data), but those in Germany receive visits from B. terrestris (Raine & Chittka 2007). We have also found significant differences in flower size between colour morphs with red flowers bigger than blue ones without any morph-by-environment interactions. Flower colour preferences are variable for some pollinators (Chittka, Ings & Raine 2004), but they usually prefer large flowers to small ones (Galen & Newport 1987; Galen 1989; Vaughton & Ramsey 1998; Arista & Ortiz 2007). Pollinators could be the selection agents responsible for the geographical pattern observed if they covaried with environmental conditions. However, L. arvensis could be relatively independent from pollinator activity because its flowers self-pollinate when petals close after the first day of anthesis. Even so, pollinator attendance could be important if xenogamous seeds had higher fitness than autogamous seeds. Given that we have not studied either pollinator attendance or differences in fitness between autogamous and xenogamous seeds, we cannot disregard the fact that pollinators play a role in maintaining flower colour polymorphism.
If flower colour itself is not the target of selection, pleiotropy could be the primary mechanism favouring one morph over the other in different environments. Differences in colour have been associated with differences in seedling survival, flower and seed production, plant biomass, and herbivore and pathogen resistance (Koes, Quattrocchio & Mol 1994; Koes, Verweij & Quattrocchio 2005; Jonson, Berhow & Dowd 2008). In our experimental study, germination, seedling mass, seedling survival, and flower and ovule production all showed different morph-by-environment interactions. The blue morph showed lower germination in the shade and higher seedling mass in the sun treatment, while the red morph showed lower survival in the dry–sun combination, more flowers in the sun–wet combination and more ovules at sun or wet treatments. Since some treatment effects on the components of plant performance may counteract each other, they are poor predictors of the overall effect when analysed separately. Only by considering overall fitness, instead of each trait separately, enables us to assess how each colour morph is affected by the treatments (García & Ehrlén 2002). Overall male and female fitness of blue morph was markedly higher in dry conditions, and this suggests a better tolerance to more xeric environments. However, our experimental study failed to find a clear pattern of adequacy of red morph to more mesic environments as only in wet-sun but not in wet-shade conditions was overall female fitness higher (male fitness was also higher but not significantly). In fact, the wet–shade combination seems to be the less favourable for L. arvensis as both morphs showed their lowest fitness. Thus, the Mediterranean environment seems to be more suitable for the blue morph, while the red morph seems to perform better in wet and sunny places, such as those where it usually occurs in central Europe. But, it is possible that other environmental factors not considered here could also be responsible for the geographic pattern found in our survey.
Diverse studies have shown that flower colour correlates with anthocyanin content in vegetative tissues (Strauss & Whittall 2006) and that pigmented individuals often tolerate stressful conditions like drought and heat better than anthocyanin-less morphs (Warren & Mackenzie 2001; Strauss & Whittall 2006; Rausher 2008). However, the flower colour polymorphism of L. arvensis clearly differs from such a situation as both morphs are pigmented. It has been reported that high-hydroxylated flavonoids (cyanidin and malvidin branches) are more effective than low-hydroxylated ones (pelargonidin branch) in protecting against stress due to excess light and drought (Daniel et al. 1999; Winkel-Shirley 2002; Tattini et al. 2004; Tanaka, Sasaki & Ohmiya 2008; Agati & Tattini 2010). It is tempting to hypothesize that flavonoids of the blue morph confer a better fitness in more xeric environments than those of the red morph but this hypothesis needs to be appropriately tested.
Although most of the traits studied were affected by the experimental treatments, onset of flowering was markedly earlier in the blue morph in relation to the red morph without any morph-by-environment interactions. This difference between morphs, regardless of growing conditions, is one of our most notable results and suggests that this trait is linked to flower colour and is genetically determined. This pattern of flowering can be also observed in natural mixed populations (pers. obs.). Flowering phenology is a trait that is usually influenced by biotic and abiotic interactions, and so it is difficult to evaluate selection because different agents affect different fitness components (Ehrlén & Münzbergová 2009). Notwithstanding, in Mediterranean habitats, the flowering of annual species takes place early in spring when soil water is available. In these conditions, an earlier flowering could confer reproductive advantage ensuring fruit ripening when water still remains in the soil. In contrast, in northern regions, low temperatures instead of water availability limit the flowering season (Rathcke & Lacey 1985), and there a later flowering could be advantageous, avoiding the risks of reduced fitness from frost (Inouye 2008). The divergent flowering patterns of the colour morphs could be selectively advantageous in different environments, and could contribute to maintain colour polymorphism via indirect selection, which would be consistent with the geographical and local distribution patterns of colour morphs.
Taking into account all our results, we found in L. arvensis many monomorphic populations that were spatially isolated, and some mixed populations with observational and experimental evidence of divergence in flowering times between morphs. It is obvious that long-term spatial segregation can generate reproductive isolation and trigger speciation (Mayr 1965; Coyne 1992; Doebeli & Dieckmann 2003). However, even in absence of spatial barriers, differences in flowering time between morphs could cause assortative mating, leading to a decrease in gene flow between them and eventually to allochronic speciation (Fox 2003; Weis et al. 2005; Savolainen et al. 2006; Gavrilets & Vose 2007). Moreover, autonomous autogamy may also contribute to reduce gene flow between morphs. Theoretical models suggest that incipient allochronic speciation could be a common phenomenon in populations of limited size (Devaux & Lande 2008) and so, this may be occurring in the scarlet pimpernel.
In conclusion, a geographical pattern of flower colour distribution is found in L. arvensis, and although we lack definitive evidence, the mechanism of selection on flower colour seems to be related to differences in the fitness of both morphs in alternate environments. These fitness differences could explain the geographical pattern of flower colour and the maintenance of colour polymorphism in L. arvensis. However, it is difficult to extrapolate results from glasshouse to natural populations, and a reciprocal field transplant study could be very useful to test local adaptation. Thus, further research is needed to identify the precise mechanisms of selection and to explore alternative selective factors given that morphs differ in traits that affect pollinator attendance. The marked difference in flowering time between colour morphs leaves open the potential for assortative mating and speciation in L. arvensis.