Plant Environmental Adaptation || The Potential for Adaptive Evolution of Pollen Grain Size in Mimulus guttatus

The Potential for Adaptive Evolution of Pollen Grain Size in Mimulus guttatusAuthor(s): Ellen Lamborn, James E. Cresswell and Mark R. MacnairSource: New Phytologist, Vol. 167, No. 1, Plant Environmental Adaptation (Jul., 2005), pp.289-296Published by: Wiley on behalf of the New Phytologist TrustStable URL: http://www.jstor.org/stable/3694348 .

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P New SPhytologist Research

The potential for adaptive evolution of pollen grain size in Mimulus guttatus Ellen Lamborn*, James E. Cresswell and Mark R. Macnair

School of Biological and Chemical Sciences, Hatherly Laboratories, University of Exeter, Prince of Wales Road, Exeter, Devon EX4 4PS, UK; *Present address:

Laboratory of Biogeography and Cultural Ecology, Department of Geography, University of the Aegean, University Hill, Mytilene 81100, Greece

Summary

Author for correspondence: * We tested whether pollen grain size (PGS) shows heritable variation in three Mark R. Macnair independent populations of Mimulus guttatus by imposing artificial selection for this Tel: +44 (0)1392 263 791 character. In addition, we looked for correlated responses to selection in a range of Fax: +44 (0)1392 273 700 Email: [email protected] 15 other floral characters.

* Heritable variation in PGS was found in all three populations, with heritabilities Received: 6 Decembery2004 of between 19 and 40% (average 30%). After three generations, upward and

downward lines differed on average by 30% in pollen volume. * No consistent patterns of correlated response were found in other characters, indicating that PGS can respond to selective forces acting on PGS alone. * Possible selection mechanisms on PGS in this species could include intermale selection, if large pollen grains produce more competitive gametophytes; or optimization of patterns of resource allocation, if local mate competition varies.

Key words: correlated response, gametophytic selection, heritability, Mimulus guttatus, pollen grain size (PGS).

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? New Phytologist (2005) doi: 10.1111/j.1469-8137.2005.01403.x

Introduction Pollen grain size (PGS) varies enormously across angiosperm species, exhibiting a range of diameters from 5 to 250 ipm (Muller, 1979); in comparison, intraspecific variation is often limited (Cresswell, 1998; Torres, 2000; Sarkissian & Harder, 2001). Several adaptive hypotheses have been proposed to explain this variation, which refer to variation in resource allocation (Bertin, 1988); pollen transport mechanisms

(Burley & Willson, 1983; Harder, 1998); and postpollination mate competition (Burley & Willson, 1983; Torres, 2000). Selection could therefore act on PGS through either direct effects on the gametophyte (Harder, 1998), or competition with other gametophytes during the pollination process (Lau & Stephenson, 1993, 1994). Alternatively, if pollen size is correlated with sporophytic characteristics, then selection

acting on these could also affect PGS (Walsh & Charlesworth, 1992). To develop and test evolutionary theories about pollen size, it is necessary to identify (a) whether this character

displays heritable variation; and (b) whether this variation is

independent of other characters.

Many authors have argued that selection on PGS will occur

during the pollination process, in particular during postpollination events (Cruzan, 1990; Harder, 1998; Torres, 2000; Sarkissian & Harder, 2001). Many species do not exhibit pollen limitation, therefore the incidence of intermale competition is probably commonplace in plant populations (Burley & Willson, 1983; Snow, 1986; Walsh & Charlesworth, 1992). Pollen- competition experiments have shown a positive association between seed-siring success and pollen size, suggesting that larger grains have an advantage in competitive situations (Lau & Stephenson, 1993, 1994). In addition to pollen competition, pollen grain size may be interpreted as a functional barrier to reproduction among or within species, or between morphs (in the case of heterostylus species), as pollen grains that do not contain enough resources may not reach the ovules (Williams & Rouse, 1990; Lau & Stephenson, 1993; Torres, 2000; but see Manicacci & Barrett, 1995). If PGS determines the maximum

pollen tube length, then variation in style length could impose selection against low values of PGS (Torres, 2000).

Several other factors have been postulated to affect PGS. For example, the easy release of pollen from the anthers has

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been used to explain the particularly small grains found in explosive or buzz-pollinated species (Muller, 1979). Harder (1998) proposed that bee grooming could select for small grains by removing large grains to the corbiculae and out of the plant reproductive cycle, although he was unable to find any evidence for this in a comparative study of a number of bee- and bird-pollinated species. Another hypothesis predict- ing small grains refers to inbreeding (Bertin, 1988). When inbreeding is the predominant form of sexual reproduction in a species, plants tend to produce only a few, small pollen grains in small flowers, in contrast to outbreeding species, which often have large flowers producing numerous large pollen grains (Schoen, 1982; Lord & Eckard, 1984; Ritland & Ritland, 1989; Diaz & Macnair, 1999). Reduced male allocation in inbreeding species may reflect cost-effective allocation to male function (Bertin, 1988) or local mate competition (Charnov, 1982).

The relatively few studies of the heritability of PGS have produced contrasting results. Significant genetic variance has been found in three species which are more-or-less domesticated: Brassica rapa (Sarkissian & Harder, 2001), Phaseolus vulgaris (Montes-R & White, 1996) and the arable weed wild radish, Raphanus sativus (Mazer & Schick, 1991; Mazer, 1992; but see Young et al., 1994). No heritability has been found in wild species (Spergularia marina; Mimulus guttatus and Campanula rapunculoides) despite considerable heritable variation for other floral characters (Delesalle & Mazer, 1995; Fenster & Carr, 1997; Vogler et al., 1999). Sarkissian & Harder (2001) suggested that domestication might allow increased heritability in PGS relative to wild species.

The response to selection on PGS will be dependent on both its heritability and its genetic correlation with other characters also under selection, the so-called G matrix (Lande, 1979; Falconer & Mackay, 1997). Even characters with high heritabilities may not respond easily to natural selection if they share strong genetic correlations with other characters (O'Neill & Schmitt, 1993). Genetic correlations have two causes: pleiotropy and linkage disequilibrium (Falconer & Mackay, 1997). Pleiotropy occurs when two or more characters are controlled by the expression of the same genes, and has been identified as a cause of correlations among some floral characters in wild radish (Conner, 2002). Conversely, linkage disequilibrium involves a statistical association between alleles at different loci, either because of close linkage, or by chance through the unrepresentative choice of breeding individuals. Linkage disequilibrium is a common source of genetic correlations found in small populations (Lande, 1976) or intense selection experiments (Falconer & Mackay, 1997). Several traits are believed to share genetic correlations with PGS, the most common of which is a resource allocation trade-off with the number of pollen grains produced, which genetically constrains pollen size evolution (Vonhof & Harder, 1995; Sarkissian & Harder, 2001).

Variation in resource acquisition, however, can lead to positive genetic correlations between the PGS and pollen number in some species, including M. guttatus (Young et al., 1994; Fenster & Carr, 1997). Crucially, it is not known whether these correlations are caused by pleiotropic effects which will constrain the ability of this character to evolve independently.

Selection experiments are extremely efficient ways of testing for heritability and genetic correlations (Hill & Caballero, 1992). The choice of replication strategy can be used to address different questions. Replicated lines within a population will allow determination of the G matrix within the base population, but interpretation of whether correlations are caused by linkage disequilibrium or pleiotropy is difficult (Harper et al., 1997; Hill & Caballero, 1992). Replication at the population level, however, can resolve the interpretation of any genetic correlation - a correlation caused by linkage disequilibrium will probably not be found in all populations because linkage disequilibrium is generated by chance and population history. In contrast, pleiotropy is more likely to be manifested in all populations, particularly if it is caused by some absolute constraint that will inhibit independent evolution of characters.

The optimum experimental design might be to replicate both between and within populations, but the time, effort and space required for such a design precludes this. Replica- tion at the population level is to be preferred if the purpose is to investigate whether pleiotropy poses fundamental constraints to adaptive evolution of a trait. This study uses the

response to selection of three independent lines ofM. guttatus to test for the heritability of PGS and to identify genetic correlations between PGS and other floral traits.

Materials and Methods

Study species Mimulus guttatus Fischer ex DC is a self-compatible, annual or perennial hydrophilic herb native to North America. Plants

produce racemes of opposite pairs of zygomorphic yellow flowers, which are pollinated predominantly by bumble bees (Robertson et al., 1999).

Plants used in the selection lines were grown from seed, collected in bulk from many plants in three wild populations of M. guttatus in California between 1985 and 1995 (SB, Stinson Beach, Marin Co.; CL, Hunt Road, Calaveras Co.; C, Copperopolis, Calaveras Co.). These populations have been described previously (Macnair & Cumbes, 1990; Macnair et al., 1993). All populations comprise dense stands of plants with high rates of pollinator visitation. Plants were grown individually in pots on glasshouse benches as previously described (Harper et al., 1997). All seed was stored at constant temperature at 150C and the viability, even of seed collected in 1985, remains high.

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Pollen collection and preservation Pollen was collected from the third and fifth flowers produced on each plant, because resources for reproduction are limited

by investment in seed that competes directly with pollen and attractive structures in late flowers ofM. guttatus (Macnair & Cumbes, 1990; E.L., unpublished data). Anthers were dissected and transferred into 1.5 ml vials, with the anthers from the two flowers stored separately. The vials were agitated mechanically until pollen was clearly visible, 100 pt GAA (1: 3 glacial acetic acid: absolute ethanol) was added as a fixative, then each vial was sonicated for 15 min to ensure maximum release of pollen from the anther.

Quantification of pollen diameter

Pollen grain size was quantified using two methods: micro-

scope and automated particle sizer. While the selection lines were being established, mean pollen diameter in all the

parental populations and first generation of population CL was quantified by sizing 30 pollen grains per flower with a calibrated eyepiece on a compound microscope. Pollen diameters in the other offspring generations were quantified using a Coulter Counter Multisizer II (Beckman Coulter (UK) Ltd, High Wycombe, UK). The pollen and GAA mixture was first agitated to ensure an even mix, then half (50 pl) of this mixture was added to 20 ml particle-free saline solution (Isoton II, Coulter Electronics Ltd). The Coulter Counter produces a

frequency distribution of pollen grain sizes from two samples of 2 ml, which therefore relates to 10% of the total pollen from each flower. Concentrations of GAA and saline solution were always consistent, reducing the chance of differences in

conductivity which could produce errors in size measurements.

Regression analysis on pollen diameters of samples measured

by both methods was used to convert diameters measured by microscope to those measured by the Coulter Counter for

subsequent analysis. The r2 for this conversion (48%) was not large, but note that this conversion was required to calculate the response to selection only in one generation. In the common garden experiment (see below), PGS from all plants was determined using the Coulter Counter. The PGS of two flowers from each plant were measured separately and the value for a plant was calculated as the mean of the two.

Establishment of the selection lines

Pollen diameters (pm) were quantified in 150 plants grown from randomly sampled seed from each of the three parental populations and subsequent generations. The largest and smallest 10 plants per line were crossed together in a full diallel design (without selfs) to produce the upward (u) and downward (d) selection lines (90 crosses per line). Within each line, the seed from all capsules was pooled at each generation. The selection protocol was applied until three

generations were obtained for populations SB and CL, and two generations for population C.

Common garden experiment As no control line was grown to factor out environmental effects, 35 plants from each of the following populations were

grown in a subsequent common garden experiment: the three

parental populations (SB, CL, C); the three generations of the upward and downward lines of SB and CL; and the two generations of the upward and downward lines of C (19 populations in total). The plants were grown in a single randomized block, and flowered over a 4 month period (June-September 2001). In order to examine the patterns of

response and heritability of PGS, the results of the common

garden experiment were analysed at the population level (SB, CL and C) and at the species level (All) by pooling the

replicate populations to represent M. guttatus in general.

Calculating the response to selection

The average response to selection (R) was estimated from the regression coefficient of generation mean on generation number. The response was calculated separately for upward (u) and downward (d) lines and from the divergence between the lines, by subtracting the downward generation mean PGS from the upward (u-d).

The sampling variance of the response ((2) was estimated as:

Y = V,[(th2/N ) + (1/M)]

where Vp is the phenotypic variance of the M individuals measured; tis the number of generations; h2 is the heritability of the character (see below); and NA is the effective number of

parents of the lines (number of plants used for breeding +0.5; Falconer & Mackay, 1997).

The sum of the variances of the two lines was used when the response was calculated from the divergence between lines (Falconer & Mackay, 1997). A significant response was indicated when the value of the response was > 2 SE from zero.

Realized heritabilities of PGS

The selection differential (S) is a measure of the strength of selection applied to a character, and was calculated from the difference between the mean of the selected individuals and the generation mean before selection. Realized heritabilities (h2) of PGS were calculated for each line and the divergence between lines from the response (R) and cumulative selection differential (C S) according to the formula R = h2S(Falconer & Mackay, 1997).

Standard errors of realized heritabilities (oj) were approx- imated from the sampling variance of the response (0) divided

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by the cumulative selection differential (I S) (Falconer & Mackay, 1997). Heritabilities were considered significant, as above.

Correlated response to selection To determine whether other floral attributes responded to artificial selection on pollen diameter, 15 other characters were scored on each flower sampled from the common garden experiment. The mean number of pollen grains per flower was estimated using the Coulter Counter Multisizer II, from the same two samples used to measure pollen diameter.

Pollen tube growth rate (PTGR) was determined in vitro on pollen grains from a subsample of 10 plants per family. Previous experiments have shown that the rate of tube growth in media is comparable to that found in styles of M. guttatus (Diaz, 1994). Pollen tubes were grown on glass slides covered in Brewbaker-Kwack medium with no additional nutrients (Kearns & Inouye, 1993). Pollen for the pollen tubes was taken from flowers produced = 2 wk after flowering was initiated (PTGR is independent of date of flower production; E.L., unpublished data). The anthers from each flower were tapped over the slides to release an even layer of pollen, then slides were placed in Petri dishes and transferred for 18 h to a cooled incubator set at 220C. After 18 h the slides were removed and fixed with GAA before storing at 5'C until measurement. Pollen tubes were measured using a camera lucida (x 100), which projected images of the pollen tubes onto paper. The first 20 tubes encountered on systematic transects of each slide were drawn carefully onto paper then measured using a map measurer. In order to reduce the chances of density effects on either germination rates (Thomson, 1989) or tube growth rates (Cruzan, 1986), measurements were restricted to spatially isolated grains whose pollen tubes were longer than the diameter of the pollen grain. Mean pollen tube growth rate per hour (pm h-1) was calculated for use in subsequent analyses.

Other characters were measured on the fourth flower of each plant using electronic callipers: pedicel length; style length; pistil length; corolla height, width and length; lengths of upper and lower pairs of stamens; calyx length. Further details of the exact characters measured are given by Macnair & Cumbes (1989). Ovary length was calculated from the difference between pistil and style lengths. The spatial separation of male and female organs (herkogamy) was computed from pistil length minus upper stamen length. Stamen separation was calculated from the length difference between upper and lower stamens. Flowering time was quantified as the number of days from germination to first flower.

Identification of correlated characters

To identify genetically correlated characters, the average response of each character was examined following the same

methodology used to determine the response in PGS: by the regression of mean character value on generation to determine whether the lines increased or decreased on average. An ANCOVA

was conducted on each of the selection lines (excluding the parental populations) with generation as a covariate to determine the significance of any character changes. A significant line term would indicate a difference between the lines, while a significant generation x line term would indicate a divergence between the lines, and thus a genetic correlation (as the two PGS lines diverged in every case). The conservative sequential Bonferroni correction was applied to reduce the probability of a type I error in multiple tests (Rice, 1989).

Results

Variation in PGS within and among populations Variation in PGS was evident among individuals within each of the parental populations (ANOVA: SB, F136,4110 = 14.8, P< 0.001; CL, F124,3625 = 11.4, P< 0.001; C, F67,1972 = 14.4, P< 0.001), allowing selection intensities usually > 1.5 to be imposed per generation in each line (data not shown). In the common garden, the PGS varied among parental populations (SB, 29.9 ? 0.1 pm; CL, 27.9 ? 0.2 pm; C, 28.1 ? 0.2 pm; ANOVA F2,102 = 40.1, P< 0.001).

Response and realized heritability of PGS

Pollen grain size responded to selection in all three populations (Fig. 1). The average response ranged from 0.25 to 0.39 pm per generation (0.29-0.39 SD) under upward selection, and 0.45-0.66 pm per generation (0.33-0.93 SD) under downward selection in the three populations. Overall, PGS diverged in upward and downward lines by an average of

- 0.92 pm per generation (0.96 SD on average, Fig. 1), leading to a final difference in pollen grain volumes of 30% on average. Pollen grain size showed significant realized heritabilities in both up and down lines (Table 1), with an overall heritability of about 30%.

Correlated characters

As PGS diverged in all populations, if selection on PGS has produced a correlated response in one or more of these other characters, then the up and down lines should also be diver- gent for these characters (significant line x generation term in ANCOVA, Table 2). Pollen grain size shows no consistent genetic correlation with any floral character across all the populations.

There is some evidence for associations between characters and PGS within individual populations. For pollen grain number, both SB and C have significant line x generation interactions, but the divergence goes in opposite directions in the two populations. Thus C shows the predicted size/number trade-off, but in SB the upward selection line has a greater





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Generation Generation Fig. 1 Mean (? SE) pollen grain size (diameter in pm) in three independent selection lines of Mimulus guttatus, selected for increased (closed symbols) or decreased (open symbols) pollen grain size. (a) SB (Stinson Beach, Marin Co., CA); (b) CL (Hunt Road, Calaveras Co., CA); (c) C (Copperopolis, Calaveras Co., CA, USA); (d) populations combined.

Table 1 Realized heritabilities (h2 ? 2 SE) for pollen grain size in three separate selection lines of Mimulus guttatus (SB, CL and C) and combined data (All)

Population

Heritability SB CL C All

h2 u 0.19 ? 0.09 0.26 ? 0.11 0.21 ? 0.17 0.26 ? 0.11 h2 d 0.40 ? 0.10 0.31 ? 0.11 0.26 ? 0.19 0.35 ? 0.11 h2 u-d 0.30 + 0.10 0.28 + 0.11 0.23 ? 0.19 0.30 + 0.11

Heritabilities were calculated as given in the text, and are calculated for the upward (u) and downward (d) lines and for the divergence (u-d) between them. All values are significant (mean ? 2 SE does not include zero). SB, Stinson Beach, Marin Co., CA; CL, Hunt Road, Calaveras Co., CA; C, Copperopolis, Calaveras Co., CA, USA.

number of pollen grains. None of the lines differed in pollen tube growth rate.

Correlations with floral size characters (characters 4-12, Table 2) showed the greatest consistency, but they were still

not significant in all populations. In SB, the up and down lines differed in mean for a number of characters, and for some (pollen grain number, style and pistil length, stamen

length) the slopes of the regressions on generation were

significantly different. The flowers of the down line decreased

substantially in size, while the up line changed very little. In CL, the lines again differed in mean for most of these characters, with the up line again having larger flowers, but in none of them was the slope of the regression significantly different after Bonferroni correction. In C, no significant effects were found.

For the final four characters there was no consistent

pattern between populations. Pedicel length showed diver-

gent selection in two populations (SB and CL), but the directions were different (in SB the up line was longer, in CL, shorter). Two derived characters, herkogamy and the

separation between the stamens, decreased in the down line in SB in line with the other floral characters, but were not

significant in the other two populations. There were no

significant effects on flowering time, and the trends were in different directions.

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Table 2 Comparison of the response in 16 characters in six lines of Mimulus guttatus selected for increased (u) or decreased (d) pollen grain size

Population

SB CL C

Character u d u d u d

Pollen grain size +0.21 > -0.62 +0.47 > -0.66 +0.39 > -0.45 Number of pollen grains per flower -0.92 > -17.9 -6.56 -2.50 -13.3 < +2.43 Pollen tube growth rate +0.53 +0.25 +1.97 +0.32 +4.61 +0.27 Style length -0.03 > -1.12 +0.53 +0.50 -0.56 -0.74 Pistil length -0.15 > -1.33 +0.80 +0.53 -0.17 -0.66 Corolla height +0.01 > -0.15 +0.94 +0.76 -0.79 -0.97 Corolla width -0.18 > -0.99 +1.19 > -0.97 -0.50 -1.56 Corolla length -0.44 > -2.02 +2.01 > +0.32 -0.41 -1.30 Length of first pair stamens -0.17 > -1.20 +0.77 > +0.17 -0.34 -0.39 Length of second pair stamens -0.26 > -0.96 +0.66 > +0.16 -0.16 -0.44 Calyx length -0.38 > -0.81 +0.98 > +0.37 -0.41 -0.75 Ovary length -0.12 -0.21 +0.26 +0.02 +0.39 +0.09 Pedicel length -0.11 < +2.93 +1.93 > -0.80 -4.74 -2.52 Herkogamy +0.02 > -0.12 +0.03 +0.36 +0.17 -0.27 Stamen separation +0.08 > -0.24 +0.11 +0.07 -0.18 +0.04 Flowering time -0.98 > -5.06 -3.06 < -0.05 -0.70 -0.14

Response given as the regression coefficient of mean character value on generation. Where ANCOVA shows that there is a significant (after Bonferroni correction) line term, or line-generation interaction, the symbols > or < indicate whether or not the upward line has a higher value than the downward line. No symbol indicates that the ANCOVA was nonsignificant. Where there is a significant line-generation interaction, values are given in bold. SB, Stinson Beach, Marin Co., CA; CL, Hunt Road, Calaveras Co., CA; C, Copperopolis, Calaveras Co., CA, USA.

Discussion Artificial selection for increased and decreased pollen grain size was successful in each of three independent populations of M. guttatus, demonstrating a substantial realized heritability for this trait. No consistent correlated response was found with any other floral character, indicating that PGS is generally an independent character that could evolve without constraints imposed by selection acting on other floral attributes.

The level of heritable variation for PGS within populations found in our present study is comparable to that found in rapid cycling Brassica rapa (Sarkissian & Harder, 2001), and the broad sense heritability found by Fenster & Carr (1997) in M. guttatus. The latter authors were unable to detect any narrow sense heritability by regression on sires, however. Our heritability estimates indicate the maximum responsiveness of PGS to selection as laboratory conditions minimize environmental variation, so these results are generalized with caution to field environments where heritability is potentially reduced (Conner et al., 2003). It is possible, however, to estimate heritability in natural conditions assuming that environmental variability affects phenotypic variation, but not the mean or additive genetic variance, VA (Sarkissian & Harder, 2001). The experiments reported here enable us to estimate VA and we have also measured the phenotypic variance (Vp) in field- collected material (data not shown). Thus field heritabilities

can be estimated as VA /V and lie between 0.13 and 0.22, which indicates that there should be enough genetic variability for a response to selection under field conditions.

Although pollen size responded to selection, no consistent response was observed in any other floral character. Several characters showed divergence in keeping with the selection on pollen size, but none exhibited this in all three populations. This indicates that the likely cause of the associations found in some populations is linkage disequilibrium, as opposed to the effect of pleiotropic genes, which would have been observed in all the populations. This illustrates the necessity of replicat- ing at the population level to establish pleiotropy or linkage disequilibrium as the cause of genetic correlations (Harper et al., 1997). Our results suggest that pollen size in M. guttatus, when not in linkage disequilibrium, can evolve independently of other floral traits, which contrasts with studies of floral evolution in other species (O'Neill & Schmitt, 1993; Sarkissian & Harder, 2001). The reason for these different findings remains unclear. One possibility is that the power of our analyses is too low to detect an effect. However, our experiment has been able to show a consistent effect of selection on

pollen size; if this character was consistently correlated with another character studied, we would expect the other character(s) also to show a consistent trend, even if not statistically significant. The fact than none does so indicates that, if there are undetected genetic correlations, then the value of these is substantially < 1.





In M. guttatus the sizes of all the floral parts tend to be

positively correlated both phenotypically and genetically with smaller flowers having smaller reproductive organs (Macnair & Cumbes, 1989; Carr & Fenster, 1994; Mossop et al., 1994). Conner (2002) has shown that similar correlations in wild radish are caused by pleiotropy. Here, similar phenotypic correlations between the sizes of floral parts were also discernible, but they were not consistently associated with PGS. Various authors have suggested that the association between style length and pollen size, which are positively correlated across many species, may represent a functional relationship (Williams & Rouse, 1990; Kirk, 1993; Torres, 2000). The lack of consistent correlation in this study implies that the association is not inevitable and thus correlated selection may be a more likely cause.

The experimental design used involved pooling all the seeds produced by crossing all the parents of the next generation in each line. This means that if any parents produce, on

average, more seeds than others, then they have a higher probability of being represented in the seeds chosen to produce the next generation. This regime was chosen to allow any correlation between PGS and seed production to be manifested

properly. However, it also means that if there is genetic variation for flower size (and thus ovule number, as all floral

components are highly correlated, Macnair & Cumbes, 1989; Carr & Fenster, 1994), then natural selection could operate in this experiment to increase flower size. This could confound our results somewhat, but is unlikely to make a substantial difference over the short term of this experiment.

Trade-offs between pollen allocation and female function

(Mossop et al., 1994; Robertson et al., 1994) or within male function, between pollen size and number (Fenster & Carr, 1997), have never been found in M. guttatus. Likewise, our

study also found that plants producing smaller pollen grains do not reliably produce larger flowers, more pollen grains or

greater female investment. Selection on pollen grain size has appeared to change the total resource allocated to pollen production, rather than just the allocation between individual

grains. The consistent results of studies of Mimulus suggest that trade-offs in resource allocation are not inevitable

(Mossop et al., 1994; Robertson et al., 1994). We found no evidence for an association between PGS

and pollen tube growth rate in vitro. This is in contrast to a number of other studies (Lau & Stephenson, 1993; Diaz & Macnair, 1999) that have looked at the phenotypic correlation between the characters. Stephenson et al. (2003) demon- strate that, in a large number of studies, pollen performance in vitro and during the initial growth phase in vivo is strongly influenced by the ability of paternal sporophytes to provision their pollen grains during development. However, in the studies they review, the differences in PGS and in vitro or in vivo PGTR were induced by environmental manipulation of the sporophyte or by inbreeding. There have been few studies like ours, in which in vitro PGTR has been compared between

pollen grains which differ in PGS through genetics. Thus whether large pollen grains inevitably produce more com-

petitive gametophytes remains an open question. It is also

interesting to consider whether either PGS or PGTR are true

gametophytic characters, under the control of the gametophyte's genotype, or whether they are manifestations of the

sporophyte's genotype. Our selection regime has treated PGS as though a sporophytic character, and achieved divergence in PGS; note that Montes-R & White (1996) imposed selection on the gametophyte and also achieved divergence. It is possible that PGS and PGTR differ in the degree to which the pheno- type of an individual pollen grain is determined by the sporophyte's or gametophyte's genotype, and this might reduce

ability to detect a correlation between the two characters. The populations of M. guttatus studied in this paper

differed substantially in pollen size. The high heritability for PGS, and the independence of this character from other floral characters, mean that it is reasonable to search for an adaptive explanation of this difference. One possibility is that the

populations differ in the activity of pollinators, and thus in the degree of pollen competition for access to ovules. This

hypothesis is amenable to experimental testing. In addition, in M. guttatus and related species there is a consistent association between pollen size, flower size and breeding system (Ritland & Ritland, 1989; Diaz & Macnair, 1999). Our results

suggest that selection acting on pollen size itself has achieved a change in size subsequent to the change in breeding system. In this case there are two possible, nonexclusive hypotheses. First, an increase in local mate competition could result in a reduction of resource allocation to male function (Charnov, 1982). Second, the change in breeding system is often associated with a reduction in flower size, and thus style length (Macnair & Cumbes, 1990). IfPGS is associated with maximum tube length (Torres, 2000), this could also result in a relaxation of selection for large grains needed to penetrate the style. Future work should test these hypotheses.

Acknowledgements During the course of this work, E.L. held a NERC studentship, receipt of which is gratefully acknowledged.

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Plant Environmental Adaptation || The Potential for Adaptive Evolution of Pollen Grain Size in Mimulus guttatus