Our results reveal quantitative variations in the morphology and nectar characteristics of P. bourgaeana flowers at the intra-individual, inter-individual, and microgeographical levels. Thus, although flower characters are traditionally considered to have low variance, our results support recent reviews (Cresswell 1998; Herrera 2009), which indicate significant intra-specific variance in flower characters.
Variation within and among individuals
In general, our results indicate greater variation of nectar characters than morphological characters, both among and within individuals. In particular, the inter-individual CVs of nectar characters (43%) doubled those for morphological characters (21%). This clearly agrees with a trend reported by Cresswell (1998) across a wide range of taxa; in that study, the mean variation of nectar characters (54%) was about twofold greater than that of corolla morphological characters (22%). Cresswell (1998) concluded that the lower variability of morphological characters was due to selective pressure to preserve the “mechanical fit” of flowers and pollinators. However, in case of P. bourgaeana flowers, “mechanical fit” between flowers and their pollinators seems unlikely because they are not specialized.
Our results showed that intra-individual variation of flower morphological characters were only a minor part of total flower variation. This is in contrast to the conclusions of a recent review by Herrera (2009), who suggested that high intra-plant variation is common in continuous and nearly continuous morphological characters of flowers. In particular, Herrera found that for 97 animal-pollinated plants, the percentage of variance for floral characters was 5.8–100%, and was more than 50% in 27% of species. Our results for flowers of P. bourgaeana suggest that intra-individual flower morphological characters have relatively low variance.
The lower variance of morphological characters than nectar characters presumably results from plant–pollinator interactions. Pollinators tend to avoid individuals with high intra-individual variance in flower characters (Real 1981; Real and Rathcke 1988; Shafir et al. 1999; Biernaskie et al. 2002), and a higher CV is associated with a stronger aversion (Shafir 2000). Within-plant choice of flowers means that insects must invest time in assessing the phenotypic diversity of available flowers, thus reducing foraging efficiency (Herrera 2009). Flower morphological characters attract pollinators; hence, we can assume that the lower intra-individual variance would be associated with a greater chance of pollinator visitation.
A similar trend might be naively expected for nectar, because pollinators would be expected to choose plants with more consistent rewards. However, there is an important difference between variance in nectar and variance in flower morphology, because, in order to test nectar character variability, pollinators must actually approach and sample numerous flowers. While feeding on nectar, insects unintentionally pollinate flowers. Reducing the time that a pollinator spends on a given tree will promote transport of pollen to other trees, thereby providing increased outcrossing and reproductive success. Thus, a high variance of nectar characters forces pollinators to move to other plants (Biernaskie et al. 2002). On the other hand, within-tree variability in nectar can reduce the energy that the plant invests in “rewards”, a strategy known as the “blank-bonanza” pattern (Feinsinger 1978). In this model, flowers with a large amount of nectar (“bonanzas”) are dispersed among many flowers with little or no nectar (“blanks”) so that plants expend less energy per flower and obtain increased pollinators movement and pollen dispersal (Feinsinger 1978). Thus, it is possible that the high intra-individual variability of nectar characters that we observed in P. bourgaeana provides an adaptive advantage for individuals.
Our results indicate that differences among trees were the most important source of variation in flower morphological characters. Similar results have been reported previously for other species (Cresswell 1998; Kearns and Inouye 1993; Møler and Eriksson 1994). Inter-individual variation in flower morphological characters could be an effect of phenotypic plasticity, and related to differences in microhabitat conditions. Previous studies have shown that variability in environmental factors (e.g., water, light, temperature, nutrient level) can influence floral characters (Villarreal and Freeman 1990; Lambrecht and Dawson 2007; Delesalle and Mazer 1996; Vogler et al. 1999; Catley et al. 2002). In our study, the most probable differences in environmental conditions within populations seem to relate to water and nutrient levels. Therefore, further studies assessing the effect of resource variation on floral morphology and nectar are clearly needed.
The inter-individual variation of flower morphological characters that we found for P. bourgaeana could also result from genetic variation. It would suggest a potential to respond to selection (Vogler et al. 1999) which may be important for a fragmented metapopulation with low population density, as in our metapopulation in Doñana. Such fragmentation and low population density often have negative effects on population genetic diversity, and can even cause population decline (Jacquemyn et al. 2003; Arroyo-Rodriguez et al. 2007). If the individual variance among trees has a genetic basis, it would suggest that the metapopulation of P. bourgaeana in Doñana is maintaining its genetic diversity despite fragmentation. Importantly, genetic variation and phenotypic plasticity may both contribute to our observed results.
Our results also revealed that, for nectar characters, the magnitudes of intra- and inter-individual variations were comparable and high, a phenomenon that has been documented for many plant species (Feinsinger 1978; Zimmerman and Pyke 1986; Boose 1997). Nectar characters are highly plastic, and light, water, fertilization, temperature, CO2 concentration, and other factors can have a strong influence (Villarreal and Freeman 1990; Boose 1997; Orians et al. 2002). The intra-individual variance may be due to phenotypic plasticity in organ-level responses to spatial variation of microenvironmental conditions. Resource allocation is another important cause of variation (Obeso 2004; Weiner 2004). Previous studies have shown that nectar characters can change significantly over time (Mitchell 2004 review), so the day-to-day variation in nectar volume and overall variance in nectar volume may exceed the variation among individual plants (Real and Rathcke 1988). Daily high variability of nectar secretion has been reported for Pyrus cultivars (Farkas and Orosz-Kovács 2003). In some cases, variation in the quantity of nectar arises from differences in the number of nectaries (Herrera and Soriguer 1983).
Similar magnitudes of intra- and inter-individual variation in nectar characters could suggest that direct selection by pollinators is unlikely in this case, because inter-individual variation could be difficult for pollinators to assess (Boose 1997; Kawecki and Ebert 2004). However, the level of the variation can itself be under selection, because animals that interact with plants can respond to intra-individual variability and act as selective agents (Pleasants 1983; Boose 1997; Herrera 2009).
Differences among localities and their consistency between years
We found no differences among localities for most floral traits. We can suggest at least four reasons for this result. First, in Doñana, fragmentation of the P. bourgaeana population occurred within the past ~200 years. P. bourgaeana can live more than 100 years (authors, unpublished data), so the time since fragmentation has been too short for significant local selection. Second, it is possible that our localities did not differ sufficiently in either insect assemblages or selective pressures exerted by pollinators. Third, even if selection pressures by pollinators differ among localities, ‘trait remixing’ (sensu Thompson 2005b) among localities would be likely via pollen and/or seed dispersal. Indeed, our target population is actually a metapopulation and, by definition, there is gene flow among localities (i.e. subpopulations), which acts against local adaptation (Kawecki and Ebert 2004). And lastly, P. bourgaeana flowers show radial symmetry and are pollinated by unspecialized pollinators (Hymenoptera, Diptera and Coleoptera; Herrera 1988; authors, unpublished data). Local adaptation is considered unlikely to occur in cases of generalist interactions (Kawecki and Ebert 2004). In such systems, there is frequent temporal variation in the identity and abundance of the most important selective agents, causing strong fluctuations in selective regimes (Waser et al. 1996; Gómez and Zamora 2006; but see Gómez et al. 2008).
Phenotypic plasticity is a likely source of some variability observed among localities (Sultan 2000; Givnish 2002). Environmental heterogeneity favors the evolution of adaptive phenotypic plasticity and leads to adaptive phenotypic differentiation without underlying genetic differentiation (e.g., Williams and Conner 2001; Kawecki and Ebert 2004). On the other hand, at the microhabitat scale, phenotypic plasticity can cause trees of one locality growing in a unique microhabitat to differ in phenotypic response. If there was a high heterogeneity of microhabitats in our localities, this may explain why differences among localities are not apparent.
Moisture availability can be a limiting factor for plants in arid and semi-arid environments (e.g., those in Mediterranean regions; Thompson 2005a). For example, Lambrecht and Dawson (2007) reported that increasing moisture availability increases the area of flowers. Thus, part of the interannual differences in P. bourgaeana floral characters that we observed could be related to differences in water availability in 2008 and 2009. In fact, there was lower rainfall in 2008 than 2009, and we found that the number of flowers per inflorescence was greater in 2009 at all localities. There were also significant differences in the area of petals in 2008 and 2009, but this was not consistent among localities. It seems likely that, because our localities differed in the availability of ground water (water table ranges 8–20 m a.s.l.; Instituto Tecnologico Geominero Español 1992), they were not equally sensitive to changes in rainfall water.
Our results also have implications for Pyrus taxonomical discrimination. Thus, while Aldasoro et al. (1996) estimates of petal length for P. bourgaeana and P. communis ranged from 5.4 to 12.0 mm (mean 8.7 mm) and from 12.0 to 15.0 mm (mean 13.2 mm), respectively, in Doñana, we found that petal length for P. bourgaeana was longer than 12 mm in about 50% of cases. Because of such an overlap between both Pyrus species, discrimination based on floral traits (Aldasoro et al. 1996) should be considered with caution. Other traits (e.g., leaf traits) and, especially, genetic profiles should be considered in future assessments.
In conclusion, we found significant variations in flower morphological and nectar characters of P. bourgaeana at microgeographical, inter-individual, intra-individual, and intra-inflorescence levels. The magnitude of flower phenotypic variation was different at different levels. Therefore, we suggest that, when investigating the causes and consequences of variation, it is important to consider its scale-dependent nature. In particular, it is essential to consider intra- and inter-individual variance. For P. bourgaeana, inter-individual variation was the main source of variation of flower morphology, but nectar characters had significant variation at both intra- and inter-individual levels. We found only small differences among the five studied localities, and these were not consistent between years. Although considerable data are available on long-term quantitative variation of flower set (mast seeding studies), there are few long-term data on qualitative variation of flower characters. Even though our study suggests general constancy on floral traits between years, we also found interannual significant differences for a few traits during the relatively short time frame considered (2 years). Greater differences on floral traits would be most likely found on a longer time span. Therefore, short-term studies of floral characters should be viewed with caution.