Direct responses to selection
The Brassica rapa‘populations’ that we studied possessed the genetic potential to respond to selection on pollen size. Truncation selection that allowed mating between only the ~11% of individuals with either the smallest or largest pollen caused mean pollen size to diverge 3–4.5 SDs during three generations. If pollen size affects mating success, then such heritable genetic variation would allow natural selection to increase the incidence of pollen sizes that promote pollination and/or fertilization. The extensive variation in pollen size among species (Wodehouse, 1935; Muller, 1979) and limited variation within species (e.g. Vonhof & Harder, 1995; Cresswell, 1998) suggests that pollen size commonly experiences selection. Indeed, pollen size is phenotypically less variable than most other floral characteristics (Cresswell, 1998), which in turn are less variable than vegetative traits (Briggs & Walters, 1997).
Our heritability estimates (Table 2) probably indicate the maximal responsiveness to selection on the size of B. rapa pollen grains. The laboratory conditions under which we grew our plants resulted in phenotypic coefficients of variation around 2%. This variation equals the smallest coefficient of variation observed for pollen diameter for 30 species from natural environments and is about 60% smaller than the median CV for these species (3.3%: see Vonhof & Harder, 1995; Cresswell, 1998). Based on our results and an expected coefficient of variation of 3.3%, eqn 1 predicts h2 ≈ 0.16 in natural conditions. Two points are evident from this result. First, although our laboratory experiment provided strong evidence of additive genetic variation for pollen size in B. rapa, it would be more difficult to identify this genetic variation in natural conditions. Secondly, in natural conditions pollen size would respond to selection of the intensity applied in our experiment about one-third as strongly as we observed in the laboratory. These results illustrate both the virtue of our laboratory approach for isolating additive genetic variation for pollen size and the care that must be exercised when considering the implications of laboratory estimates of heritability.
Studies of six species provide contrasting evidence for genetic variation in pollen size. The histories of the plants studied may contribute to this variation. In particular, the three species with demonstrated heritable variation in pollen size have histories of genetic manipulation by plant breeders or are widespread agricultural weeds (R. sativus, Mazer & Schick, 1991; Mazer, 1992; P. vulgaris, Montes-R & White, 1996; B. rapa, this study), both circumstances that facilitate gene flow and could increase genetic diversity. In contrast, genetic variation for pollen size was not detected for three wild species (S. marina, Delesalle & Mazer, 1995; M. guttatus, Fenster & Carr, 1997; C. rapunculoides, Vogler et al., 1999). However, this seeming dichotomy may also reflect the statistical power to isolate genetic variation. Selection experiments in controlled environments provide a powerful means of detecting genetic variation, in part because they involve many plants, and both studies that applied selection on pollen size observed significant responses (Montes-R & White, 1996; this study). The remaining studies evaluated genetic variation based on either the resemblance of offspring to parents or differences between families. The ability of these approaches to detect genetic variation varies inversely with sample size, so it is not surprising that the study that found significant variation (Mazer & Schick, 1991) also considered two to eight times more families (n=60) than those that did not (Delesalle & Mazer, 1995; n=15 families per population; Fenster & Carr, 1997; n=23 and 25; Vogler et al., 1999; n=7). Two studies of R. sativus provide a particularly interesting comparison, as Mazer & Schick (1991) considered 60 families and found additive genetic variation for pollen size, whereas Young et al. (1994) considered only 11 families and found no significant variation. Indeed, Young et al. (1994) estimated coefficients of additive genetic variation (3–4%) about twice as large as our estimates for B. rapa (Table 2), although they assessed this variation to be not statistically significant. Hence, it is possible that all of these species possess heritable variation for pollen size, but it was not detected for three of them because the studies considered limited samples. Clearly, resolution of whether significant additive genetic variation for pollen size persists in most plant species requires studies based on large samples of a greater variety of species, preferably under natural conditions.
Correlated responses to selection
Despite significant heritable variation for pollen diameter, which should facilitate pollen-size evolution, the correlated responses by other floral characters reveal that pollen size cannot evolve independently. Such associations between traits could arise from gametic-phase disequilibrium and/or pleiotropy (Falconer & Mackay, 1996). Regardless of the genetic mechanism, genetic correlations between floral traits may be maintained by selection as adaptive trait combinations if they produce effective reproductive phenotypes (Stanton & Young, 1994).
The positive genetic correlation between pollen size and style length observed in B. rapa (Fig. 6c) may represent a gametic-phase disequilibrium that arises commonly in angiosperms because of nonrandom mating. In particular, a positive genetic correlation should evolve when large pollen has a higher probability of siring seeds in pistils with long styles because of faster germination and/or faster or more prolonged pollen-tube growth (see, Cruzan, 1990; Lau & Stephenson, 1993, 1994; T.S. Sarkissian, L.D. Harder & N.M. Williams, unpublished data). This mating pattern will cause alleles coding for large pollen grains to become associated with alleles that promote long styles, creating gametic-phase disequilibrium. In contrast, for heterostylous species in which plants with contrasting style lengths have a higher probability of exchanging pollen, disassortative mating should create a negative genetic correlation between pollen size and style length. Indeed, negative phenotypic and genetic associations between pollen size and style length typify heterostylous species (Darwin, 1884; Ganders, 1979; Dulberger, 1992). Although the disequilibrium responsible for such associations could arise solely from male–male competition, it also provides the genetic architecture needed for the evolution of female mate choice (see Fisher, 1958; O’Donald, 1980; Lande, 1981; Kirkpatrick, 1982). Both of these aspects of sexual selection should maintain associations between pollen size and style length. Such sexual selection has probably produced the widespread positive association between species with respect to pollen size and style length (reviewed by Torres, 2000) as an incidental outcome of the evolution of mating patterns within species.
The other genetic correlations that we detected, involving pollen number, flower size and ovule number, could all reflect the genetic control of resource allocation within and between flowers. The only negative genetic correlation detected during this study involved pollen size and number (see Figs 4 & 5), which has also been found in other studies (Mazer & Hultgård, 1993; Stanton & Young, 1994; but see Fenster & Carr, 1997). This inverse relation probably arose simply from division of a fixed expenditure of resources among pollen grains within individual flowers, because total allocation to pollen did not change relative to the control lines in seven of the eight selection lines (Fig. 4b). Such a fixed expenditure of resources among pollen grains within individual flowers enables two opposing allocation options –‘more, smaller’ or ‘fewer, larger’ pollen grains (Fig. 5). This negative genetic association is probably responsible for the parallel phenotypic tradeoff between size and number observed within species for pollen production (see Vonhof & Harder, 1995). Furthermore, such a tradeoff probably governs the optimal size-number combinations that benefit reproductive success in specific pollination and fertilization environments (Harder, 1998), resulting in the negative phenotypic association between pollen size and number observed between species (see Vonhof & Harder, 1995).
A genetic size-number tradeoff may help maintain heritable variation in pollen size if selection on mating success varies spatially and/or temporally (see Haldane & Jayakar, 1963; Via & Lande, 1987; Gillespie & Turelli, 1989). Changes in pollinator type and abundance (see Galen, 1989; Schemske & Horvitz, 1989; Kelly, 1992; O’Neil & Schmitt, 1993; Robertson et al., 1994) and/or changes in population structure may mediate changes in the intensity and/or direction of selection on pollen size (but see Harder, 1998). For example, suppose plants in a sparse population attract few pollinators and suffer insufficient pollination, whereas plants in a dense population receive large pollen loads from multiple donors. The first environment places a premium on more (smaller) pollen grains, whereas the second environment may favour larger (fewer) grains, if large pollen has an advantage during postpollination processes. If the optimal pollen size varies with these different pollination conditions, then the direction and magnitude of selection will also differ. Extensive pollen and/or seed dispersal between such populations would maintain genetic variation in pollen size.
In contrast to pollen number, flower size and ovule number exhibited positive genetic associations with pollen size. Such associations are consistent with current models of the genetic control of flower development, with individual genes influencing the phenotypic expression of many floral organs (reviewed in Coen & Meyerowitz, 1991; Weigel & Meyerowitz, 1994; see Xie et al., 1999 for a pollen-specific example). However, such control must obey the physical conservation of matter, so that these positive correlations must reflect one of the two classes of differences between plants with large vs. small pollen. In one class, plants with large pollen are more proficient in their acquisition or conversion of nutrients, so that they have more resources per flower to invest in petals and ovules than plants with small pollen. In the alternate class, all plants invest equally in reproduction, but plants with large pollen (and larger flowers with relatively more ovules) produce fewer flowers and/or smaller ovules. Unfortunately, we did not measure ovule size, flower number, or total reproductive effort, so that we cannot distinguish among these contrasting allocation responses. Nevertheless, the correlated responses of flower size and ovule number despite no contrasting response of total pollen volume remind us that the hierarchical nature of allocation in plants complicates reproductive tradeoffs greatly (also see Mazer, 1992; de Jong, 1993; Venable, 1996; Koelewijn & Hunscheid, 2000; Worley & Barrett, 2000).
The observed genetic characteristics of pollen size indicate that the evolution of pollen size in a particular reproductive environment will involve many floral traits. Suppose that large pollen grains compete more successfully for fertilizations (e.g. T.S. Sarkissian, L.D. Harder & N.M. Williams, unpublished data), particularly in long-styled pistils. Because pollen size is heritable, the resulting sexual selection will tend to increase average pollen size within the population and create a positive correlation between pollen size and style length. However, the increase in pollen size cannot continue unchecked, because it reduces the number of pollen grains produced, which limits a plant’s pollen export opportunities. This resource tradeoff creates an optimal pollen size that balances the advantages of large pollen size for gametophytic competition against the fecundity disadvantages of fewer pollen grains. Whether selection eventually brings the population to this optimum depends on the mating consequences of correlated changes in flower size and ovule number (and perhaps reproductive effort, flower number and ovule size). Hence, a plant’s pollen size is but one component of the integrated floral design that determines mating success in a particular pollination and fertilization environment.