Sex Plant Reprod (2001) 14:77–83

© Springer-Verlag 2001

O R I G I N A L A RT I C L E

A.G. Stephenson · C.N. Hayes · M.H. Jóhannsson J.A. Winsor

The performance of microgametophytes is affected by inbreeding depression and hybrid vigor in the sporophytic generation

Received: 29 October 2000 / Accepted: 3 May 2001

Abstract Inbreeding reduces the level of heterozygosity, thereby exposing deleterious recessives to selection and simultaneously reducing the number of loci expressing heterosis (overdominance). In contrast, hybridization increases the level of heterozygosity, thereby masking deleterious recessives and simultaneously increasing the number of loci expressing heterosis. Most studies of inbreeding depression/hybrid vigor have focused on sporophytic performance such as survivorship, vegetative growth rates, fruit and seed production and (rarely) pollen production. Because the genetic mechanisms that underlie inbreeding depression/hybrid vigor are relevant only to the diploid stage of the life cycle, most studies have tacitly assumed that they have no effects on pollen performance (pollen germination, pollen tube growth rate, ability to achieve fertilization under conditions of pollen competition). However, we reasoned that because pollen is dependent upon the sporophyte for the resources necessary to develop, germinate and initiate tube growth, the level of heterozygosity (vigor) in the pollenproducing parent can affect pollen performance by affecting the ability of the sporophyte to provision its pollen. In a series of studies conducted under field conditions over 7 years, we experimentally varied the level of heterozygosity in wild gourd (Cucurbita pepo) plants (four levels of inbreeding, f = 0.75, 0.50, 0.25, 0 and a zucchini × wild gourd F1). We found that sporophytic vigor (e.g., flower and fruit production) increased with the level of heterozygosity and that the level of heterozyA.G. Stephenson (✉) · C.N. Hayes Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA e-mail: [email protected] Tel.: +1-814-863-1553, Fax: +1-814-865-9131 M.H. Jóhannsson The Soil Conservation Service of Iceland, Gunnarsholt, 851 Hella, Iceland J.A. Winsor The Pennsylvania State University, Altoona, PA 16601-3760, USA

gosity of the sporophyte affects the in vitro and in vivo performance of the microgametophytes it produces. These findings are analogous to the “maternal environmental effects” frequently observed in seeds. Keywords Breeding systems · Cucurbita · Inbreeding depression · Hybrid vigor · Pollen tube growth · Pollen performance

Introduction Inbreeding reduces the level of heterozygosity, thereby exposing deleterious recessives and partial recessives to selection while simultaneously reducing the number of overdominant (heterotic) loci. Over the last 125 years, inbreeding depression (the reduction in fitness of inbred progeny relative to outcrossed progeny) has been documented for scores of species possessing a wide range of mating systems (e.g., Darwin 1876; Husband and Schemske 1996; Vogler et al. 1999; Willis 1999 and references therein). Typically, these studies have measured survivorship, vegetative vigor, and/or reproductive output through the female function (fruit and seed production) (see Husband and Schemske 1996) although a few studies have also examined the effects of inbreeding on pollen production and/or pollen viability (e.g., Ritland and Ganders 1987; Krebs and Hancock 1990; Willis 1993, 1999; Carr and Dudash 1995, 1997; Johannsson et al. 1998; Vogler et al. 1999). In contrast, hybridization between two populations with fixed deleterious alleles at some loci (usually inbred families) increases the level of heterozygosity, thereby masking the effects of deleterious recessives while simultaneously increasing the number of loci exhibiting heterosis. Hybrid vigor (an increase in the vigor of the F1 progeny compared to the parental populations) is well documented for a variety of sporophytic traits such as vegetative growth, fruit and seed production, and (rarely) pollen production (see references in Charlesworth and Charlesworth 1987) and, coupled with inbreeding to

78

purge deleterious recessives and selection for desirable individual traits, forms the basis of modern plant breeding (Falconer 1989). Over the past 15 years, studies using both protein electrophoresis and mRNA-cDNA hybridization techniques have shown that a large portion of the genome of the microgametophyte (20,000–23,000 genes) is transcribed and translated, and that most of these genes are expressed during both the sporophytic and gametophytic stages of the life cycle (e.g., Tanksley et al. 1981; Willing and Mascarenhas 1984; Willing et al. 1988; Hamilton and Mascarenhas 1997). Haploid gene expression occurs throughout microsporogenesis and continues until the pollen tube enters the ovule (Mascarenhas 1993). In short, microgametophytes require expression of their own genes in order to develop, germinate and grow. Moreover, a few recent studies have shown that pollen performance (speed of germination and pollen tube growth rate) is influenced by the genotype of the microgametophyte, that selection can act on microgametophytes and that selection on microgametophytes can alter the performance of the resulting sporophyte (see review by Sari-Gorla and Frova 1997). Although gene expression by microgametophytes is well-documented, most investigators tacitly assume that inbreeding depression/hybrid vigor does not extend to microgametophytic performance, because pollen grains are haploid (hence no dominance relationships) and the genetic causes of inbreeding depression/hybrid vigor are relevant only to the diploid stage of the life cycle. Recent studies, however, have revealed that pollen performance is not only influenced by the microgametophyte’s own genome but also by the ability of the pollenproducing sporophyte’s ability to provision its pollen grains during development. These studies have shown that soil fertility (Young and Stanton 1990; Lau and Stephenson 1993, 1994), leaf herbivory (Quesada et al. 1995; Strauss et al. 1996; Mutakainen and Delph 1996; Delph et al. 1997), temperature stress during development (Zamir et al. 1981, 1982; Zamir and Vallejos 1983; Jakobsen and Martens 1994; Jóhannsson and Stephenson 1998a), level of mycorrhizal infection (Lau et al. 1995; Stephenson et al. 1998) and prior fruit development on a plant (Stephenson et al. 1994) can affect the size, chemical composition and/or performance of the pollen produced by a plant. Because inbreeding and hybridization affect sporophyte vigor, we reasoned, they could also affect the quantity or quality of the resources the sporophyte can provide to its pollen grains during development. Consequently, we initiated a series of studies designed to determine if the level of heterozygosity in the sporophyte has an effect on the performance of the microgametophytes that it produces.

Materials and methods To determine if inbreeding depression and hybrid vigor extends to the microgametophytic stage of the life cycle, we used Cucurbita

pepo ssp. ovifera var. texana, a wild gourd, formerly C. texana (Decker 1988), and C. pepo ssp. ovifera var. ovifera, the Black Beauty bush cultivar – an inbred line of zucchini. Cucurbita pepo ssp. ovifera is an annual monoecious (separate staminate and pistillate flowers on the same plant) vine with indeterminate growth. The variety texana grows on dry roadsides, waste places, and along watercourses in Texas and New Mexico (Decker and Wilson 1987). It is thought to be either the wild progenitor of the cultivated squashes, gourds and pumpkins, or an early escape from cultivation (Decker and Wilson 1987; Decker-Walters 1990); it is completely cross-compatible with C. pepo ssp. ovifera var. ovifera, and the F1 hybrids are completely fertile (Quesada et al. 1993). The C. pepo ssp. ovifera var. ovifera cv. (zucchini) used in these experiments is an inbred cultivar developed in the northeastern United States and is not found in the wild. Decker and Wilson (1987) reported a mean expected heterozygosity (Hs) for the wild gourd of 0.086 which is higher than for zucchini but still rather low for an herbaceous outcrossing annual (Gottlieb 1981). In the first of two studies (Johannsson et al. 1998) designed to examine the effects of inbreeding on pollen performance, seeds from eight families developed from seeds collected from a natural population of the wild gourd were sown into an experimental garden at the Pennsylvania State University Agricultural Experiment Station at Rock Springs, Pa, USA (the field site) in the summer of 1993. Female flowers on each plant were cross- and self-pollinated. To measure the effects of selfing on progeny performance, seeds from the selfed and outcrossed fruits were germinated in a greenhouse and transplanted into an experimental garden at the field site in the summers of 1994 and 1995. During both years, we recorded flower and fruit production on the plants and we measured in vitro pollen tube growth. On 5 separate days, when most plants were in bloom, a single male flower was collected from each plant that had an open male flower and brought into a laboratory. Pollen was sprinkled onto 50-mm petri dishes containing Brewbaker and Kwack (1963) growth medium with 3% BactoAgar (Difco Laboratories, Detroit, Mich.) and 12% sucrose. To examine differences in pollen performance in vivo, we performed a pollen mixture experiment in 1995 in which equal amounts of pollen from the selfed progeny and a tester line (zucchini) were deposited together onto stigmas of female flowers on the tester line (zucchini). On other flowers, we deposited pollen from the outcrossed progeny and the tester line. During the summer of 1996, we scored the resulting seeds for paternity (the progeny sired by the wild gourd differed from the progeny sired by the tester line in seven easily identified single gene morphological traits). In the second study, initiated during the summer of 1998, selfed and outcrossed seeds from seven families were germinated in peat pots in the greenhouse and transplanted after 10 days into an experimental garden at the field site. Female flowers on each outcrossed plant were cross-pollinated, self-pollinated or mated to a full sibling. Female flowers from each selfed plant were cross- or self-pollinated. This created progeny with coefficients of inbreeding (f) of 0, 0.25, 0.50, and 0.75. To measure the effects of these four levels of inbreeding on progeny performance, 100 seeds (in both 1999 and 2000) of each level of inbreeding from each family were germinated in a greenhouse and a random sample of ten seedlings was transplanted to the field site. During both years, we recorded flower and fruit production on each plant and we measured in vitro pollen tube growth on four dates. To examine differences in pollen performance in vivo, we performed a pollen mixture experiment in both years in which pollen from either outcrossed (f = 0) or highly inbred (f = 0.75) plants were deposited onto zucchini stigmas along with an equal amount of pollen from the tester line as described above (see Johannsson et al.1998 for details). In order to examine the effects of hybrid vigor on pollen performance (Johannsson and Stephenson 1998b), zucchini and wild gourd plants were grown at the field site in the summer of 1993. Pollen was collected from male flowers on the wild gourd and deposited onto stigmas of zucchini to produce F1 plants. The F1 seeds were collected from mature fruits, dried and stored until the

79 Fig. 1 Mean in vitro pollen tube growth rates for pollen produced by plants with coefficients of inbreeding 0, 0.25, 0.50 and 0.75. Pollen was collected from each plant on four dates over the course of the 1999 growing season, germinated on Brewbaker and Kwack (1963) medium and permitted to grow for 20 min. Fifty pollen tubes were measured per plant per date in 1999

Table 1 Effects of four levels of inbreeding on flower, fruit, and seed production in the wild gourd during the summer of 1999. Mean ± SE

Trait

f=0

f = 0.25

f = 0.50

f = 0.75

Regression

Males Females Fruits Seeds Seed mass (mg)

91.0±12.8 46.0±6.7 45.6±5.5 245.6±10 45.4±1.6

76.7±10.5 36.4±4.5 45.4±4.4 240.0±7.7 38.8±1.0

69.0±8.1 37.6±4.6 43.9±3.6 230.8±7.1 40.7±0.7

58.6±8.4 30.8±4.9 32.9±4.5 245.7±13.6 39.0±0.8

P=0.030 P=0.085 P=0.065 P=0.720 P=0.001

following summer. To monitor sporophytic traits, we grew 50 zucchini plants, 144 wild gourd plants (part of a larger screening project), and 50 F1 plants at the field site in the summer of 1994. Male and female flower production on each plant was monitored daily until 31 August 1994, the number of mature fruits and seeds per fruit were counted on each plant at the end of the growing season, and seed mass was determined. To estimate gametophytic vigor, we germinated pollen in vitro on two separate days in 1994.

Results In the first study of the effects of selfing on sporophytic and gametophytic performance (see Johannsson et al. 1998), a mixed-model analysis of variance revealed significant differences in the number of male flowers and the number of fruits produced by the selfed and outcrossed plants growing in the field. Compared to the outcrossed progeny, the selfed progeny produced 25% (1994) and 48% (1995) fewer male flowers and 47% (1994) and 46% (1995) fewer fruits, indicating that inbreeding adversely affects reproductive output (including one measure of the male function) of the progeny. We also found that pollen from the outcrossed progeny grew significantly faster in vitro than that of the selfed progeny. Finally, we found that pollen from the selfed plants fertilized about 49% of the seeds when in competition with pollen from the tester line, while the pollen from the outcrossed plants fertilized over 60% of the seeds. A chi-square test revealed that the probability that wild gourd pollen will sire a seed is not independent of the type of pollination that produced the plant (df = 1;

P = 0.021; n = 553; G2-test for heterogeneity among fruits, G2 = 9.94, NS, df = 10 for self pollen vs. tester line fruits; G2 = 21.06, NS, df = 14 for outcross pollen vs. tester line fruits). The second study examined the effects of three levels of inbreeding on sporophytic and gametophytic performance. Regression analysis revealed that in 1999 most measures of reproductive output (except seed number per fruit) decreased significantly or nearly significantly with the level of inbreeding (Table 1). Compared to the outcrossed plants, the plants with a coefficient of inbreeding of 0.75 produced 55% fewer male flowers, 49% fewer female flowers, and 39% fewer fruits that contained seeds that weighed 16% less. At this time, the data from the summer/autumn of 2000 were not complete but preliminary analyses of the flower production data through August revealed the same trends as 1999. In both 1999 and 2000, we also found that the in vitro growth of pollen tubes (pollen tube length after 20 min of in vitro growth) decreased significantly with the level of inbreeding (Figs. 1 and 2). The pollen tubes from plants with an f = 0.75 were 27% (1999) and 12% (2000) shorter than pollen tubes from outcrossed (f = 0) plants. Paternity analyses of the progeny resulting from the in vivo pollen performance experiment have not yet been completed. In the study of the effects of hybridization on sporophytic and gametophytic performance (Johannsson and Stephenson 1998b), a non-parametric Kruskal-Wallis test revealed that the zucchini, wild gourd and F1 plants differed significantly in the number of male flowers, female flowers and total seed mass per plant (Table 2). More-

80 Fig. 2 Mean in vitro pollen tube growth rates for pollen produced by plants with coefficients of inbreeding 0, 0.25, 0.50 and 0.75. Pollen was collected from each plant on four dates over the course of the 2000 growing season, germinated on Brewbaker and Kwack (1963) medium and permitted to grow for 20 min. Fifty pollen tubes were measured per plant per date in 2000

Table 2 Number of flowers and fruits produced by zucchini, wild gourd, F1 plants, (Mean ± SE). Values that share a lower case letter are not significantly different, others are significant at P<0.05

Zucchini Wild gourd F1 plants

Male flowers by August 31

Female flowers by August 31

Total seed mass per plant (g)

21.6±1.3 a 48.0±4.3 b 110.4±12.2 c

4.0±0.4 a 9.8±0.8 b 11.6±1.0 c

128.5±12.2 b 99.2±8.8 a 336.4±28.3 c

over, the F1 plants produced significantly more male and female flowers and had a significantly greater total mass of seeds than either the zucchini or the wild gourd plants (Table 2). Pollen tube growth in vitro was measured on two dates. An analysis of variance (proc GLM, SAS 1990) revealed that the F1 and two parental types of plants differed significantly in average pollen tube growth (see Johannsson and Stephenson 1998b). On average, the in vitro pollen tube lengths of the F1 hybrids were 15% greater than those of zucchini and 10% greater than those from the wild gourd.

Discussion In these studies we experimentally varied heterozygosity from a level that greatly exceeds that found in outcrossed plants in a natural population of wild gourds (the F1 plants), through the level typical of wild plants (Hs = 0.086), to levels corresponding to inbreeding coefficients of 0.25, 0.50, and 0.75. An f = 0.75 indicates a 75% decrease in average heterozygosity compared to that of outcrossed plants. As with other studies of hybrid vigor and inbreeding depression, we found that the vigor of the sporophytic generation (measured in terms of reproductive output) decreases with average heterozygosity. Unlike other studies of hybrid vigor/inbreeding depression however, we examined the effects of heterozygosity in the sporophytic generation on the performance of their microgametophytes. We found, in

a series of replicated experiments conducted under field conditions over a period of 7 years, that the in vitro and in one case the in vivo performance of the microgametophytes decreases with the heterozygosity of the pollen producing parent. It should be noted that in Cucurbita pepo, we have repeatedly observed that in vitro pollen performance parallels in vivo performance (e.g., Quesada et al. 1995; Johannsson and Stephenson 1997). We suspect that the differences in the performance of pollen from inbred, outcrossed, and F1 plants are due to differences in the ability of the plants to provide resources to their developing pollen grains. Because of the decrease in general vigor associated with inbreeding, the inbred plants are presumably less able to provision their pollen while the F1 plants exhibit an enhanced ability, even relative to the outcrossed plants. Conversely, it is difficult to imagine a genetic mechanism that could account for the differences in the performance of pollen from inbred, outcrossed, and F1 progeny, even though pollen does transcribe and translate a large portion of its haploid genome during development, germination, and tube growth (e.g. Tanksley et al. 1981; Willing and Mascarenhas 1984). While inbreeding would lead to an increase in homozygosity in the sporophytic generation, it would not alter allele frequencies and, of course, dominance relationships are not relevant in haploid systems. It should also be noted, however, that inbreeding depression/hybrid vigor could have direct genetic effects on species that produce diploid pollen (see Mok and Peloquin 1975). It is well known that environmental (e.g., herbivory and poor soil fertility), developmental (e.g., prior fruit production), and genetic (e.g., inbreeding) stresses can adversely affect the size and chemical composition of seeds by reducing the maternal plant’s ability to provide resources during seed development (see Roach and Wulff 1987; Stephenson 1992; Hauser and Loeschke 1995; Vogler et al. 1999 and references therein). Moreover, these differences in seed size lead to differences in seedling vigor that, in turn, can lead to differences in off-

81

spring fitness, especially under competitive conditions (Roach and Wulff 1987). As with seeds, mature pollen grains are packed (primed) with enzymes and mRNAs (see Stephenson et al. 1992) and they contain energy (e.g., starches and lipids) and nutrient (e.g., phytate) storage products (see Stephenson et al. 1992; Clément et al. 1996; Piffanelli et al. 1998). These storage products, or their precursors, are secreted by the tapetal “nurse” cells of the anther during pollen development (e.g., Wang et al. 1992 a,b; Clément et al. 1996) and, as with seeds, these products are known to be metabolized upon germination and pollen tube growth. These storage products are thought to play important roles during germination and the initial stages of pollen tube growth (see Vasil 1974; Mulcahy and Mulcahy 1982; Wetzel and Jensen 1992). In short, pollen grains, like seeds, require resources from the parent plant to develop, germinate, and initiate growth. Recently, a few studies have demonstrated that environmental stress during pollen development can also affect pollen performance. For example, in C. pepo, pollen from plants grown in high phosphorus (P) soils in the field was 5% larger and sired more seeds than pollen from plants grown in low P soils when equal amounts of the two types of pollen were applied together on a stigma (Lau and Stephenson 1994). Chemical analyses of the pollen in this study also revealed that the pollen from the high P soils contained a greater concentration of phosphorus than pollen from the low P soils, perhaps indicating that soil fertility affects the chemical composition of pollen that, in turn, affects pollen performance and the ability to achieve fertilization. Because pollen from several conspecifics is frequently deposited onto the same stigma in outcrossing plants (e.g., Ellstrand 1984; Dudash and Ritland 1991) and because the number of pollen grains deposited onto stigmas frequently exceeds the number of ovules in the ovary in natural populations of Cucurbita (Winsor et al. 2000) and other species (see review by Stephenson et al. 1995), differences in the performance of pollen from selfed, outcrossed and F1 plants can lead to differences in the number of seeds sired. Consequently, inbred wild gourd plants not only have fewer male flowers to donate pollen to their neighbors, but those pollen grains that are deposited onto the stigmas of their neighbors are less likely to fertilize an ovule. Similarly, the increased pollen production (male flower number) and faster pollen tube growth of F1 plants has the potential for rapidly spreading genes between wild and cultivated species of Cucurbita pepo (Wilson and Payne 1994) and, if they hold for other species as well, these findings have important ecological implications for biotechnology. Inbreeding depression has been invoked as a major force in the evolution of plant mating systems. Because a selfed seed carries twice as many of its parents genes than an outcrossed seed, there is an inherent genetic advantage to selfing (i.e., an outcrossing plant would have to produce twice as many seeds or have increased pollen

donation to conspecifics in order to get the same number of genes into the next generation). This genetic advantage is counterbalanced by the adverse effects of selfing (e.g., reduced survival and reproduction of the progeny and, perhaps, a decreased probability of siring seeds). Consequently, the magnitude of inbreeding depression is thought to be important in determining the spread of alleles affecting floral traits that promote selfing or those that enhance outcrossing (e.g., Darwin 1876; Charlesworth and Charlesworth 1979, 1987; Lloyd 1979; Holsinger et al. 1984). Because the magnitude of inbreeding depression is usually measured as the product of the relative fitness (e.g., typically, percent survivorship from fertilization to reproduction × total seed production) of selfed and outcrossed progeny at each life history stage (e.g. Husband and Schemske 1996), then differences in the relative fitness of plants through the male function (e.g., survivorship × seed production × number of seeds sired) can have profound effects on the estimate of inbreeding depression. If differences in pollen production and pollen performance due to inbreeding are widespread, then the magnitude of inbreeding depression is greater than all previous studies of inbreeding depression have indicated. This could substantially alter the rate of evolution of floral traits that influence the breeding system of plants in natural populations. Acknowledgements We thank Robert Oberheim and the Department of Horticulture for use of The Pennsylvania State University Agricultural Experiment Station at Rock Springs, Penn. We thank Brian Clark and Michael Westerman for field and lab assistance. This work was supported by NSF grant DEB98–06691 to A.G.S. and J.A.W. This paper is dedicated to Prof. Joseph Mascarenhas who has led, inspired, and mentored a generation of plant reproductive biologists.

References Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot 50:747–858 Carr DE, Dudash MR (1995) Inbreeding depression under a competitive regime in Mimulus guttatus: consequences for potential male and female function. Heredity 75:437–445 Carr DE, Dudash MR (1997) The effects of five generations of enforced selfing on potential male and female function in Mimulus guttattus. Evolution 51:1797–1807 Charlesworth D, Charlesworth B (1979) The evolutionary genetics of sexual systems in flowering plants. Proc R Soc London Ser B 205:513–530 Charlesworth D, Charlesworth B (1987) Inbreeding depression and its evolutionary consequences. Annu Rev Evol Syst 18: 237–268 Clément C, Burrus M, Audran J-C (1996) Floral organ growth and carbohydrate content during pollen development in Lilium. Am J Bot 83:459–469 Darwin C (1876) The effects of self and cross fertilisation in the vegetable kingdom. Appleton, New York Decker DS (1988) Origin(s), evolution and systematics of Cucurbita pepo (Cucurbitaceae) Econ Bot 42:4–15 Decker DS, Wilson HD (1987) Allozyme variation in the Cucurbita pepo complex: C. pepo var. ovifera vs. C. texana. Syst Bot 12:263–273

82 Decker-Walters DS (1990) Evidence for multiple domestication of Cucurbita pepo. In: Bates DM, Robinson RW, Jeffrey C (eds) Biology and utilization of the Cucurbitaceae. Cornell University Press, Ithaca, NY Delph LF, Johannsson MH, Stephenson AG (1997) How environmental factors affect pollen performance: ecological and evolutionary perspectives. Ecology 78:1632–1639 Dudash MR, Ritland K (1991) Multiple paternity and self-fertilization in relation to floral age in Mimulus guttatus (Scrophulariaceae). Am J Bot 78:1746–1753 Ellstrand NC (1984) Multiple paternity within the fruits of the wild radish, Raphanus sativus. Am Nat 123:819–828 Falconer DS (1989) Introduction to quantitative genetics, 3rd edn. Longman, UK Gottlieb LD (1981) Electrophoretic evidence and plant populations. Prog Phytochem 7:1–46 Hamilton DA, Mascarenhas JP (1997) Gene expression during pollen development. In: Shivanna KR, Sawhney VK (eds) Pollen biotechnology for crop production and improvement. Cambridge University Press, New York Hauser TP, Loeschke V (1995) Inbreeding depression in Lychnis flos-cuculi (Caryophyllaceae): effects of different levels of inbreeding. J Evol Biol 8:589–600 Holsinger KE, Feldman MW, Christiansen FB (1984) The evolution of self-fertilization in plants: a population genetic model. Am Nat 124:446–453 Husband BC, Schemske DW (1996) Evolution of the magnitude and timing of inbreeding depression in plants. Evolution 50:54–70 Jakobsen HB, Martens H (1994) Influence of temperature and ageing of ovules and pollen on reproductive success in Trifolium repens. Ann Bot 74:493–501 Jóhannsson MH, Stephenson AG (1997) Effects of pollination intensity on the vigor of the sporophytic and gametophytic generation of Cucurbita texana. Sex Plant Reprod 10:236–240 Jóhannsson MH, Stephenson AG (1998a) Effects of temperature stress during microsporogenesis on pollen performance in Cucurbita pepo. Int J Plant Sci 159:616–626 Jóhannsson MH, Stephenson AG (1998b) Variation in sporophytic and gametophytic vigor in wild and cultivated varieties of Cucurbita pepo and their F1 and F2 generations. Sex Plant Reprod 11:265–271 Jóhannsson MH, Gates MJ, Stephenson AG (1998) Inbreeding depression affects pollen performance in Cucurbita texana. J Evolution Biol 11:579–588 Krebs SL, Hancock JF (1990) Early-acting inbreeding depression and reproductive success in the highbush blueberry, Vaccinium corymbosum L. Theor Appl Genet 79:825–832 Lau T-C, Stephenson AG (1993) Effects of soil nitrogen on pollen production, pollen grain size, and pollen performance in Cucurbita pepo (Cucurbitaceae). Am J Bot 80:763–768 Lau T-C, Stephenson AG (1994) Effects of soil phosphorus on pollen production, pollen size, pollen phosphorus content, and the ability to sire seeds in Cucurbita pepo (Cucurbitaceae). Sex Plant Reprod 7:215–220 Lau T-C, Lu X, Koide RT, Stephenson AG (1995) Effects of soil fertility and mycorrhizal infection on pollen production and pollen grain size of Cucurbita pepo (Cucurbitaceae). Plant Cell Environ 18:169–177 Lloyd DG (1979) Some reproductive factors affecting the selection of self-fertilization in plants. Am Nat 113:67–79 Mascarenhas JP (1993) Molecular mechanisms of pollen tube growth and differentiation. Plant Cell 5:1303–1314 Mok DWS, Peloquin SJ (1975) Three mechanisms of 2 N pollen formation in diploid potatoes. Can J Genet Cytol 17:217–225 Mulcahy GB, Mulcahy DL (1982) The two phases of growth of Petunia hybrida pollen tubes through compatible styles. J Palynol 18:1–3 Mutakainen P, Delph LF (1996) Effects of herbivory on male reproductive succes in plants. Oikos 75:353–358 Piffanelli P, Ross JHE, Murphy DJ (1998) Biogenesis and function of the lipidic structures of pollen grains. Sex Plant Reprod 11:65–80

Quesada M, Winsor JA, Stephenson AG (1993) Effects of pollen competition on progeny performance in a heterozygous Cucurbit. Am Nat 142:694–706 Quesada MR, Bollman K, Stephenson AG (1995) Leaf damage decreases pollen production and hinders pollen performance in Cucurbita texana. Ecology 76:437–443 Roach DA, Wulff D (1987) Maternal effect in plants. Annu Rev Ecol Syst 18:209–236 Ritland K, Ganders FR (1987) Crossability in Mimulus guttatus in relation to components of gene fixation. Evolution 41:772– 786 Sari-Gorla M, Frova C (1997) Pollen tube growth and pollen selection. In: Shivanna KR, Sawhney VK (eds) Pollen biotechnology for crop production and improvement. Cambridge University Press, New York, USA. pp 333–351 SAS Institute (1990) SAS user’s guide. SAS Institute, Cary, NC Stephenson AG (1992) The regulation of maternal investment in plants. In: Marshall C, Grace J (eds) Fruit and seed production. Cambridge University Press, Cambridge UK Stephenson AG, Lau T-C, Quesada M, Winsor JA (1992) Factors that affect pollen performance. In: Wyatt R (ed) Ecology and evolution of plant reproduction. Chapman and Hall, New York Stephenson AG, Erickson C, Lau T-C, Quesada MR, Winsor JA (1994) Effects of growing conditions on the male gametophyte. In: Stephenson AG, Kao T-h (eds) Pollen pistil interactions and pollen tube growth. American Society of Plant Physiologists, Rockville, Md Stephenson AG, Poulton JL, Lau T-C, Koide RT (1998) Effects of soil phosphorus level and mycorrhizal infection on the male function of plants. In: Lynch JP, Deikman J. (eds) Phosphorus in plant biology: regulatory roles in molecular, cellular, organismic and ecosystem processes. American Society of Plant Physiologists, Rockville, Md Stephenson AG, Quesada MR, Schlichting CD, Winsor JA (1995) Consequences of variation in pollen load size. Monogr Syst Bot 53:233–244 Strauss SY, Conner J, Rush SL (1996) Foliar herbivory affects floral characters and plant attractiveness to pollinators: implications for male and female plant fitness. Am Nat 147:1098– 1107 Tanksley SD, Zamir D, Rick CM (1981) Evidence for extensive overlap of sporophytic and gametophytic gene expression in Lycopersicon esculentum. Science 213:453–455 Vasil JK (1974) The histology and physiology of pollen germination and pollen growth on the stigma and in the style. In: Linskens HF (ed) Fertilization in higher plants. North-Holland, Amsterdam, Netherlands Vogler DW, Filmore K, Stephenson AG (1999) Inbreeding depression in Campanula rapunculoides L. I. A comparison of inbreeding depression in plants derived from strong and weak self-incompatibility phenotypes. J Evol Biol 12:483–494 Wang C-S, Walling LL, Eckard KJ, Lord EM (1992a) Patterns of protein accumulation in developing anthers of Lilium longiflorum. Am J Bot 79:118–127 Wang C-S, Walling LL, Eckard KJ, Lord EM (1992b) Immunological characterization of a tapetal protein in developing anthers of Lilium longiflorum. Plant Physiol 99:822–829 Wetzel CLR, Jensen WA (1992) Studies of pollen maturation in cotton: The storage reserve accumulation phase. Sex Plant Reprod 5:117–127 Willing RP, Mascarenhas JP (1984) Analysis of the complexity and diversity of mRNAs from pollen and shoots of Tradescantia. Plant Physiol 75:865–868 Willing RP, Bashe B, Mascarenhas JP (1988) Analysis of quantity and diversity of messenger RNAs from pollen and shoots of Zea mays. Theor Appl Genet 75:751–753 Willis JH (1993) Effects of different levels of inbreeding on fitness components in Mimulus guttatus. Evolution 47:864– 876 Willis JH (1999) The role of genes of large effect on inbreeding depression in Mimulus guttatus. Evolution 53:1678–1691

83 Wilson HD, Payne JS (1994) Crop/weed microgametophyte competition in Cucurbita pepo (Cucurbitaceae). Am J Bot 81: 1531–1537 Winsor JA, Peretz S, Stephenson AG (2000) Pollen competition in a natural population of Cucurbita foetidissima (Cucurbitaceae). Am J Bot 87:527–532 Young HJ, Stanton ML (1990) Influence of environmental quality on pollen competitive ability in wild radish. Science 248: 1631–1633

Zamir D, Vallejos EC (1983) Temperature effects on haploid selection of tomato microspores and pollen grains. In: Mulcahy D, Ottaviano E (eds) Pollen: Biology and implications for plant breeding. Elsevier, New York Zamir D, Tanksley SD, Jones RA (1981) Low temperature effect on selective fertilization by pollen mixtures of wild and cultivated tomato species. Theor Appl Genet 59:235–238 Zamir D, Tanksley SD, Jones RA (1982) Haploid selection for low temperature tolerance of tomato pollen. Genetics 101:129–137