Narcissus triandrus exhibits unusual floral morphology and style-morph frequencies for a tristylous species. Because of the absence of heteromorphic incompatibility, floral morphology plays an important role in governing pollen dispersal, female fertility and patterns of outcrossed mating. Our theoretical results indicate that variation in female fertility has the potential to play a significant role in morph-ratio evolution, particularly when the strength of frequency-dependent selection varies among the morphs because of asymmetrical mating patterns. Our empirical studies of natural populations of N. triandrus revealed that this component of female reproductive success was associated with sexual system, flower size and morph frequencies within populations. We discuss the potential mechanisms responsible for these associations and their consequence for the evolution of sexual systems.
The majority of studies that have investigated seed production in tristylous species have not detected morph-specific differences in female fertility (Dulberger, 1970; Barrett, 1977; Price & Barrett, 1982; Barrett et al., 1989). For example, Oxalis alpina resembles N. triandrus in several respects, with low frequencies of the M-morph characteristic of tristylous populations and dimorphic populations missing the M-morph. However, reduced female fertility of the M-morph was not evident based on extensive surveys of natural populations (Weller, 1981, 1986). A few cases are reported of morph-specific differences in female fertility in tristylous species. For example, in Decodon verticillatus (Eckert & Barrett, 1995) and Lythrum salicaria (Ågren, 1996; Ågren & Ericson, 1996), lower seed set of the L-morph is reported in some L-biased populations, perhaps reflecting frequency-dependent effects. In N. triandrus we found significant differences in proportional seed set in seven of the nine trimorphic populations (Fig. 5) and two of the six dimorphic populations (Fig. 4) that we surveyed. Hence, unlike most other tristylous species, female fertility differences among the morphs appear to be a common feature of N. triandrus populations.
Female fertility and variation in M-morph frequency
Narcissus triandrus populations display wide variation in frequency of the M-morph (0–0.63; Barrett et al., 1997, 2004). A central goal of our study was to identify if differences in female fertility among morphs could contribute to this pattern. The results of our pollen-transfer models revealed that a reduction in female fertility of the M-morph via either ovule discounting or pollen limitation could result in a decline and loss of this morph from populations. When mating asymmetries occur in the manner predicted for N. triandrus, even slight reductions in M-morph fertility resulted in very low-equilibrium M-morph frequencies and the loss of this morph from populations (Fig. 2h). Thus a positive correlation between M-morph frequency and the fertility of the M-morph is predicted if population morph ratios are in equilibrium. Alternatively, a negative correlation is predicted if populations are not at equilibrium and morph ratios are evolving towards an isoplethic equilibrium by frequency-dependent selection (Ågren & Ericson, 1996). Negative correlations between fitness components and morph ratios have been demonstrated in other plant species with polymorphic traits under frequency-dependent selection (McCauley & Brock, 1998; Gigord et al., 2001; Thompson et al., 2003; Olson et al., 2005).
Our empirical results support the prediction that a female fertility disadvantage of the M-morph of N. triandrus may play a role in its loss from populations. In the three populations with the lowest M-morph frequency, the M-morph had significantly lower proportional seed set than the other two morphs (Fig. 5). Moreover, as predicted, M-morph frequency was positively correlated with both the proportional and relative seed set of the M-morph (Fig. 6a). However, as pollination is often a stochastic process and N. triandrus is a perennial plant, evaluation of this pattern across multiple years would be important in order to support this hypothesis. Differences among N. triandrus populations in local environmental conditions that influence the female fertility of the M-morph probably contribute to the variation in its frequency. Identifying the proximate ecological mechanisms represents a major challenge.
Female fertility in plant populations can be influenced by both pollen quality (e.g. self vs outcross pollen) and the amount of pollen transferred to stigmas (reviewed by Wilcock & Neiland, 2002). In N. triandrus, prior self-pollination reduces outcrossed seed set by up to 74%, indicating that there could be significant fertility costs associated with self-pollination and ovule discounting (Barrett et al., 1997). Elsewhere, it has been demonstrated that the M-morph in tristylous species experiences significantly higher selfing rates than the L- and S-morphs (Kohn & Barrett, 1992), presumably because of its particular morphology. Mid-level stigmas are sandwiched between two stamen levels, and this arrangement is likely to promote higher levels of self-pollination (Charlesworth, 1979). Our theoretical work indicates that morph-specific differences in ovule discounting can influence morph ratios, and it is possible that such effects could play a role in explaining the positive association between M-morph frequency and fertility that we observed (Fig. 6a). In dimorphic species of Narcissus, ovule discounting has also been implicated as a factor involved in morph-frequency variation (Barrett et al., 1996; Cesaro et al., 2004).
Modifications to sex-organ position in tristylous populations have the potential to influence patterns of outcrossed siring success and therefore morph ratios (Weller, 1986; Barrett et al., 2004). Our pollen-transfer model also demonstrated that, in pollen-limited conditions, such morphological changes affect female fertility because of differences in pollen receipt among morphs. In these situations, morph frequencies will be determined by both negative frequency-dependent selection and selection via female fertility. We found that pollen receipt of the M-morph declined as the upper-level stamens of the L-morph corresponded more closely in position to stigmas of the L- rather than the M-morph. With pollen limitation (represented by decreasing α), this resulted in reduced fertility of the M-morph compared with the other morphs, and the frequency of the M-morph declined rapidly as Q increased (Fig. 3b,c). Once the M-morph is lost from populations, the L-morph will go to fixation when assortative mating in this morph reaches 1/2 (Baker et al., 2000b). Therefore, under pollen-limited conditions, the anomalous positioning of the upper-level stamens of the L-morph of N. triandrus could cause reductions in the fertility of the M-morph. This effect, combined with ovule discounting resulting from self-pollination typical of pollen-limited situations, may be sufficient to cause repeated loss of the M-morph from tristylous populations.
Pollen limitation has been identified as an important ecological factor that can influence the evolution of polymorphic sexual systems (Charlesworth & Charlesworth, 1979b, 1979c; Washitani et al., 1994; McCauley & Taylor, 1997). Our model demonstrates that, if pollen limitation occurs in tristylous populations, it could lead to the evolution of dimorphic populations. Dimorphic populations of N. triandrus are of lower density and smaller size than tristylous populations (Hodgins & Barrett, 2006). These demographic characteristics often make populations more prone to pollen limitation (Ågren, 1996; Groom, 1998; Ward & Johnson, 2005). However, levels of pollen limitation in N. triandrus were not significantly different between dimorphic and trimorphic populations, and no dimorphic populations exhibited pollen limitation. Proportional seed set was actually 13% higher in dimorphic compared with trimorphic populations. Evolutionary changes to floral morphology, such as an increase in flower size (Hodgins & Barrett, 2006), may have occurred in dimorphic populations, facilitating more proficient pollen transfer and mitigating pollen limitation.
There were no significant differences among morphs in pollen limitation, even in populations with significantly lower proportional seed set of the M-morph. This suggests that pollen quantity was not responsible for the fertility differences we observed. However, the two populations with the lowest frequency of M-morph were pollen-limited and exhibited low M-morph fertility. More extensive studies of morph-specific differences in pollen limitation in trimorphic populations having low frequencies of the M-morph are necessary to determine whether morph-specific differences in pollen limitation cause the extensive variation in frequency of the M-morph.
The L-morph bias and stability of the S-morph
There was no evidence that differences in female reproductive success could account for the L-biased morph ratios that characterize populations of N. triandrus. Unlike the M-morph, the frequency of the L-morph among populations was not correlated with either its proportional or relative seed set. This strongly suggests that significant assortative mating in the L-morph, facilitated by the elevated position of its upper-level stamens, is largely responsible for its predominance in both trimorphic and dimorphic populations. High levels of assortative mating by the L-morph are also implicated as the major cause of L-biased morph ratios in Narcissus species with stigma-height dimorphism (Barrett et al., 1996; Baker et al., 2000a, 2000b; Arroyo et al., 2002).
Our theoretical results indicate that, with assortative mating in the L-morph, both M- and L-morphs can easily be lost from populations when seed set is reduced in these morphs (Fig. 2g,h). The fact that no N. triandrus populations lack the L-morph or show a deficiency of this morph indicates that, unlike the M-morph, the L-morph does not commonly experience a female fertility disadvantage in populations. In N. triandrus, the pendulous flowers and long-level stigma located above the upper-level stamens probably restrict opportunities for self-pollination and ovule discounting. Moreover, as all three morphs possess long-level stamens targeting stigmas of the L-morph (Fig. 1), the relative fertility of this morph is unlikely to suffer in environments with reduced pollinator service.
In tristylous populations of N. triandrus, the S-morph often has the lowest representation (average frequency 0.22) and exhibits strikingly less variation in frequency among populations compared with the L- and M-morphs (Barrett et al., 2004). Our models demonstrate that the frequency of the S-morph is not influenced by changes in pollen transfer between the L- and M-morphs (increased Q), but remains at a stable equilibrium frequency of 1/3 (Fig. 2f,i). Ovule discounting in the S-morph can contribute to a reduction in its frequency to below 1/3 (Fig. 2c,f,i). In contrast to the L- and M-morphs, mating asymmetries resulting from assortative mating in the L-morph do not hasten decline in frequency of the S-morph with reduced female fertility. This is because of the S-morph's equal interdependence on both L- and M-morphs for pollen import and export. Therefore the mating patterns of the L- and M-morphs do not influence the frequency of the S-morph, because these morphs are interchangeable as maternal parents of seeds sired by the S-morph. Because of the distinct combination of stamen levels in the S-morph, in comparison with the shared stamen positions of the L and M-morphs (Fig. 1), the S-morph should experience more intense frequency-dependent selection. This probably explains the remarkable stability in frequency of the S-morph among populations of N. triandrus.
Floral morphology, female fertility and geographical variation in morph frequencies
Geographical variation in the floral morphology of N. triandrus is correlated with morph-frequency variation (Barrett et al., 2004). In large-flowered populations in the north-western parts of the Iberian Peninsula, the M-morph is either at low frequency or absent. In contrast, in small-flowered populations further south, the M-morph occurs at higher frequencies. This flower-size variation is associated with contrasting allometric relations among sex organ positions, particularly mid-level organs, with consequences for pollen transfer. Our data revealed a negative correlation between flower size and proportional seed set of the M-morph (Fig. 6b). Significantly, this association was not evident in the other morphs. The mechanism(s) causing this reduction in fertility of the M-morph in N. triandrus are unclear. Geographical changes in the behaviour and size of the major flower visitors (Anthophora vs Bombus), and their effectiveness in promoting cross- vs self-pollination when interacting with the different floral morphologies of the style morphs, may be involved.
The ability to predict the frequency of the M-morph in N. triandrus populations based on the flower size and female fertility of this morph suggests that local ecological conditions play an important role in governing morph-ratio evolution. The association between heterostyly and a self-incompatibility system that permits assortative mating results in complex mating asymmetries not evident in heterostylous species with heteromorphic incompatibility. In N. triandrus, interactions between floral morphology and pollinators visiting flowers largely govern the character of frequency-dependent selection on morph ratios. These interactions are dependent on regional context, particularly climatic gradients, because N. triandrus is widely distributed in the Iberian Peninsula. Accordingly, morph-frequency variation exhibits predictable geographical patterns not often observed in polymorphic species. This variation provides a valuable spatial template for future studies of the causes and consequences of floral evolution for morph-ratio dynamics in heterostylous plants.