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Flower diversification in animal-pollinated species is thought to result from the selective pressures exerted by pollinators (Herrera et al., 2006; Johnson, 2006). Flowers are thus envisioned as mechanical devices optimized through the action of natural selection to maximize pollen transfer between potential mates (Darwin, 1877; Stebbins, 1970). In the long term, this selective process is expected to result in a precise relationship between the flowers of animal-pollinated species and pollinator morphology. Because flowers are modular entities composed of several relatively independent organs (perianth, androecium and gynoecium), the reciprocal adjustment between flowers and pollinators should involve the evolution of particular allometric relationships among floral organs, that is, the combination of trait values maximizing the rates of pollen delivery and receipt (Nilsson, 1988; Alexandersson & Johnson, 2002). Natural selection is expected to favour the evolution of floral integration, in other words, high phenotypic covariances among traits associated with pollinator attraction and reward, pollen delivery and pollen receipt (Berg, 1960; Stebbins, 1970; Conner & Via, 1993; Armbruster et al., 1999; Herrera, 2001; Herrera et al., 2002; Armbruster et al., 2004; Badyaev, 2004; Anderson & Busch, 2006; but see Ordano et al., 2008).
The floral phenotype can be represented as a square matrix of phenotypic variances and covariances (Chernoff & Magwene, 1999; Steppan et al., 2002; Smith & Rausher, 2008). This matrix allows the calculation of both the magnitude and pattern of floral integration, the first describing the strength with which floral traits correlate with each other, and the second referring to the overall structure of the phenotypic correlations among traits (Ordano et al., 2008). Depending on its magnitude and pattern, floral integration may favour or constrain the response to the selective pressures exerted by pollinators or other agents (Campbell, 2009). Evolutionary changes in the floral phenotype may be constrained if the trajectory favoured by natural selection differs from the direction of the axis of maximum variation of the flower (i.e. when the direction of the eigenvector associated with the largest eigenvalue of the variance–covariance matrix of the flower differs from the direction of the vector of selection gradients) (Kirkpatrick & Lofsvold, 1992; Schluter, 1996; Merilä & Björklund, 2004). The higher the discrepancy between these two axes, the stronger the restrictions on adaptive evolution (Arnold, 1992; Schlichting & Pigliucci, 1998). Evolutionary transitions between reproductive systems involving adaptive changes in the functioning and morphology of flowers offer an ideal opportunity to examine the evolution of phenotypic integration.
The remarkable evolutionary lability exhibited by heterostylous reproductive systems (Ornduff, 1972; Weller, 1992; Domínguez et al. 1997) makes them interesting study subjects for addressing the evolution of floral integration. Literature on heterostyly shows several examples describing evolutionary transitions from a polymorphism with three floral morphs (tristyly) to one with only two floral morphs (distyly) (Ornduff, 1972; Charlesworth, 1979; Weller, 1986, 1992; Eckert & Barrett, 1994; Barrett et al., 2004; Weller et al., 2007). Heterostyly is characterized by the reciprocal positioning of anthers and stigmas between floral morphs (Lloyd & Webb, 1992; see Fig. 1a,d), and by the presence, in many species, of a heteromorphic incompatibility system preventing seed production after self- and same-morph pollinations. Theoretically, efficient pollen transfer in heterostylous species requires a precise relationship between flower and pollinator morphology, because pollen from different stamen levels adheres to specific parts of the pollinator’s body (Ganders, 1979; Barrett & Glover, 1985). Therefore, the precise location of sexual organs (anthers and stigmas) within and between flowers (i.e. the reciprocal placement of anthers and stigmas from different floral morphs; hereafter reciprocity) is the primary mechanism maintaining the evolutionary stability of heterostylous systems (Charlesworth, 1979). Such a mechanism is reinforced by a tubular or closed funnel-shaped perianth that constrains the way in which pollinators enter the flower (Ganders, 1979), thus increasing the likelihood of pollen transfer between same-level sexual organs (legitimate crosses, sensuDarwin, 1877). Hence, selection for features promoting legitimate pollen flow should produce high covariances among floral traits, and therefore relatively high levels of floral integration.
Figure 1. Hypothetical changes in the reciprocal placement of sexual organs among floral morphs during the evolution of distyly from tristyly in Oxalis alpina. Horizontal arrows indicate legitimate pollen transfer between mid-level anthers and mid stigmas in a hypothetical ancestral tristylous population (a). During the evolutionary transition towards distyly, modifications of incompatibility in short- and long-styled morphs result in a reduction in the frequency of the mid-styled morph. This, in turn, reduces the availability of mid styles and, consequently, the probability of legitimate pollen transfer from mid-level anthers (b, c). The arrow thickness represents the probability of successful pollen transfer from mid anthers to target stigmas. Although the shift from tristyly to distyly involves the loss of the mid-styled morph and, consequently, mid styles in the population, both short- and long-styled plants retain mid-level stamens. Thus, natural selection should increase the efficiency of pollen transfer between the mid-level stamens of the short- and long-styled morphs through changes in the length of the mid-level stamens, such that they match the positions of short and long stigmas. See vertical arrows above mid level anthers in (c). Consequently, the two stamen whorls of the short or long floral morph should converge to the length of the style in its reciprocal morph (d). A new phenotype in which each floral morph has all its stamens at a single level is expected to evolve, thus making pollen transfer more efficient.
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Altered patterns of pollen transfer and incompatibility modifications have been shown to account for the breakdown of tristyly through the loss of one floral morph (Charlesworth, 1979; Weller, 1986; Weller et al., 2007). This evolutionary transition is expected to shift the adaptive peak of a tristylous floral phenotype towards a new adaptive peak in which only two floral morphs coexist (distyly; see Fig. 1 for details). Given that the breakdown of tristyly generally involves the loss of the mid-styled morph (Ornduff, 1972; Charlesworth, 1979; Weller, 1986, 1992; Eckert & Barrett, 1994; Barrett et al., 2004; Weller et al., 2007), its gradual loss would reduce the availability of target stigmas for pollen from the mid anthers of short- and long-styled plants (Fig. 1b,c). Thus, natural selection should favour any modification of the floral phenotype of the short- and long-styled morphs that increases the rates of pollen transfer from their mid stamens towards their new target styles (Mulcahy, 1964; Lewis & Rao, 1971; Weller, 1992; Eckert & Mavraganis, 1996). Such modifications should produce a derived dimorphism in which both anther whorls of short- and long-styled plants would have the same length as the style of their complementary morph (long and short styles, respectively; see Fig. 1d). In other words, increased reciprocal placement between sexual organs of the short- and long-styled floral morphs is expected during the evolution of distyly from tristyly (i.e. during the loss of the mid-styled morph).
If flowers constitute integrated modules with correlated floral traits, mid stamens as well as other floral traits (e.g. other sexual organs or perianth traits) should respond to the selective pressures that promote an increased reciprocity between the long- and short-styled morphs. At some point during the evolution of a new distylous floral morphology, correlations among floral traits may constrain further evolutionary changes (Merilä & Björklund, 2004; Campbell, 2009). Thus, natural selection is expected to reduce temporarily the magnitude and modify the pattern of floral integration during the transition from tristyly to distyly. Although some studies have shown that the breakdown of tristyly generally involves modifications in the length of the mid stamens of both short- and long-styled morphs (Mulcahy, 1964; Ornduff, 1964; Lewis & Rao, 1971; Lewis, 1975; Weller, 1979), no studies have examined whether such changes involve an increase in the extent of reciprocity between formerly illegitimate sexual organs during the transition from tristyly to distyly, whether other floral traits are involved in this process, and how these modifications affect the magnitude and pattern of floral integration.
Within the Sonoran Desert Sky Islands (Arizona, New Mexico, USA; and Sonora, Mexico), the heterostylous species Oxalis alpina shows a marked variation in the frequency of floral morphs, ranging from isoplethic tristylous (equal frequency of the three morphs) to distylous populations in which the mid-styled morph is absent (Weller et al., 2007). The loss of the mid-styled morph in this species results from incompatibility modifications (defined as the relative loss of incompatibility between illegitimate crosses) in the short- and long-styled morphs (see details in Fig. 2; Weller et al., 2007). Accordingly, the presence of populations representing different steps in the evolutionary transition from tristyly to distyly offers a unique opportunity to explore changes in flower morphology associated with this evolutionary shift.
In this study, we first describe the floral variation among morphs and populations of O. alpina representing the evolutionary transition from tristyly to distyly. Second, we test whether the variation in flower morphology among these populations fits the hypothesized pattern of increased reciprocity between short- and long-styled plants associated with changes in the position of sexual organs within the flower. Third, we assess how these changes affect the magnitude and pattern of floral integration during this evolutionary transition.
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In this study, we have shown that the loss of the mid-styled morph in O. alpina is associated with modifications in the floral phenotypes that increase the morphological reciprocity between short- and long-styled plants. Such changes, in turn, seem to be moving the floral phenotypes towards a new distylous adaptive peak. Although this adjustment entails modifications in both the flower size and the relationships among sexual traits, no evidence of changes in the magnitude of floral integration was found. Our analyses further revealed that the pattern of floral integration (the structure of the phenotypic floral matrix) changed during the evolution of distyly from tristyly, probably reflecting modifications in the allometric, functional or pleiotropic relationships among floral traits.
Previous studies analysing the consequences of the breakdown of tristyly on floral morphology have reported changes in the length of the mid stamens (Mulcahy, 1964; Ornduff, 1964; Weller, 1979; Eckert & Barrett, 1994; Eckert & Mavraganis, 1996). Other important features, however, such as modifications in flower size (Barrett et al., 2004; Hodgins & Barrett, 2006, 2008), other sexual organs or the entire variance–covariance matrix have received much less attention (but see O’Neil & Schmitt, 1993). As predicted, the loss of the mid-styled morph in O. alpina resulted in modifications in the floral phenotype, increasing the reciprocity between formerly illegitimate sexual organs of the short- and long-styled floral morphs. This process involved both the within- and between-morph components of reciprocity and, as revealed by PCA, morphological adjustment entailed modifications in the length of both the stamens and styles, and in the flower size. Our results indicate that the selective pressures favouring a higher reciprocity between short- and long-styled plants affect the whole floral phenotype.
Even though the breakdown of tristyly involved changes in both the flower size and relative position of the sexual organs, the magnitude of floral integration was apparently independent of this evolutionary process. The average magnitude of floral integration among populations of O. alpina (20.37% ± 2.10) falls near the average observed for flowering plants (21.5% ± 1.72, Ordano et al., 2008). This result is unexpected because a high magnitude of floral integration was predicted on the basis of the selective value of reciprocal morphology between floral morphs in heterostylous species. The magnitude of floral integration varied greatly among populations, with a threefold difference between the population having the lowest and highest value. It is likely that the magnitude of floral integration in O. alpina has played a minor role during the evolution of a distylous floral phenotype.
Although other studies have tested the adaptive role of the magnitude of floral integration, the evidence of its putative adaptive value is controversial (Fornoni et al., 2009; Harder, 2009). For example, experimental modifications of the extent of floral integration in Lavandula latifolia had no significant effects on pollination success (Herrera, 2001). Ordano et al. (2008) estimated the intensity of selection acting on individual floral integration in three Rosaceae species, and showed that this attribute is selectively neutral. Studies based on the comparative method, or looking for differences in floral integration among populations with dissimilar pollinator faunas, have found no support for the adaptive value of floral integration (Herrera et al., 2002; Armbruster et al., 2004). Pérez-Barrales et al. (2007), but Pérez et al. (2007), in contrast, reported that phenotypic floral integration responded to pollinator-mediated selection in Narcissus papyraceus and Schizanthus species, respectively. Anderson & Busch (2006) found that outcrossing species of the family Brassicaceae had higher levels of floral integration than conspecific selfing species. Higher levels of phenotypic integration in floral relative to vegetative traits in Dalechampia scandens implicitly assume the importance of floral integration in pollination biology (Hansen et al., 2007). Thus, more empirical studies are necessary before we can definitively conclude that there is an adaptive role for the magnitude of floral integration (Fornoni et al., 2009).
In contrast with the magnitude of floral integration, we found a significant relationship between the pattern of floral integration and the extent of incompatibility modification, a result in agreement with other studies showing that the pattern of phenotypic integration is generally responsive to natural selection (Herrera et al., 2002; Ashman, 2003; Olsson, 2004). Our analyses also revealed that the pattern of covariation among floral traits changed during the breakdown of tristyly, and this was consistent with our adaptive hypothesis. Hence, it is likely that natural selection modified the strength of covariation among specific floral traits in order to reshape the flower of short- and long-styled plants according to the new selective regime imposed by the loss of the mid-styled morph. These adjustments resulted in an increment in the reciprocal morphology during the evolution of a derived distylous floral phenotype.
Our analyses assume that the floral morphology in populations of O. alpina has evolved independently. A preliminary phylogeographical study suggests that geographical aggregation of distylous populations of O. alpina in the northwestern Sky Islands (Fig. 3) resulted from several independent evolutionary events (J. Pérez-Alquicira, unpublished). Future studies should explore how the ancestry of these populations may have influenced the geographical variation in flower morphology in O. alpina.
In addition to the increase in reciprocity between short- and long-styled plants, the breakdown of tristyly involved a marked change in flower size. This was an unexpected result because flower size, at least in theory, evolves in response to the selective pressures exerted by the most common/efficient pollinators (Galen, 1989; Mitchell, 1994; Conner & Rush, 1996). The reduction in flower size in distylous populations suggests that the magnitude of the selective pressures favouring increased reciprocity may have negative consequences for the pollination of O. alpina. Given that the identity of the principal pollinators [two species of small bee (Heterosaurus; Andrenidae) contributing to 94.5–100% of the floral visits] does not change among the populations studied, the reduction in flower size and the increase in reciprocity cannot be explained by changes in pollinator species. The similarity in pollinator fauna of tristylous and distylous populations of O. alpina suggests that the fitness consequences of a reduction in flower size should be explored.
Our PCA indicates that more than half of the morphological floral variation is a result of differences in size, with an additional 15% accounted for by differences in the relative position of sexual organs. Although the shortest path to evolve a distylous floral phenotype in O. alpina would require modifications only in the relationships among sexual organs, it is the variation in flower size that corresponds to the axis of maximum variation. Because this axis is one of minimum resistance to morphological change, we suggest that the reduction in flower size is a byproduct of the selective pressures acting on the rearrangement of the sexual organs (Björklund, 1996; Merilä & Björklund, 2004). Results from our study support this interpretation because flower size was reduced by 0.78 SD (the difference between the means of the standardized scores (PC1) of tristylous and distylous populations), whereas the relative position of sexual organs (PC2) changed by only 0.40 and −0.33 SD (for short- and long-styled morphs, respectively), a twofold difference between these two components. These results suggest that, although natural selection is apparently driving the floral phenotype towards a distylous adaptive peak, the constraints imposed by the structure of the phenotypic matrix have forced the evolution of smaller flowers. This explanation relies on the assumption that the phenotypic variance–covariance matrix represents a fair estimate of the genetic floral matrix (Roff, 1997; Waitt & Levin, 1998; Steppan et al., 2002).
Taken together, results from this study indicate that the breakdown of tristyly in O. alpina is accompanied by major changes in the morphology of short- and long-styled plants. Most of the observed floral modifications were associated with flower size, suggesting that the evolutionary trajectory is driven chiefly by the axis of maximum phenotypic variation. Accordingly, the evolution of flower morphology in O. alpina seems to have been conditioned (but not prevented) by the structure of the phenotypic floral matrix. In spite of this restriction, reciprocal herkogamy between formerly illegitimate sexual organs of short- and long-styled plants increased during the evolution from tristyly to distyly. Thus, the available evidence suggests that natural selection operating on flower morphology is strong enough to overcome any potential constraint resulting from the correlations among traits.