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Keywords:

  • Cucurbita;
  • female function;
  • inbreeding depression;
  • male function;
  • soil nitrogen

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Year to year variation
  6. Nitrogen stress
  7. Results
  8. Year to year differences
  9. High and low nitrogen fields
  10. Discussion
  11. Acknowledgments
  12. References

We grew inbred and outcrossed Cucurbita pepo ssp. texana plants and measured inbreeding depression for several male and female fitness traits 4 years in a row in adjacent fields at the same field station under the same cultivation conditions. We found that the magnitude of inbreeding depression varied from 0.16 to 0.53 from year to year and that those traits which were most affected tended to vary with year. We also grew inbred and outcrossed C. pepo ssp. texana plants in two adjacent fields differing only in the presence of nitrogen fertilizer to examine the effect of nutrient limitation as a form of environmental stress on the magnitude of inbreeding depression. We found that inbreeding depression was more severe in the unfertilized field. Overall, this study illustrates the notion that any estimate of inbreeding depression represents a single point in a cluster of possible estimates that can vary (often dramatically) with growing conditions.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Year to year variation
  6. Nitrogen stress
  7. Results
  8. Year to year differences
  9. High and low nitrogen fields
  10. Discussion
  11. Acknowledgments
  12. References

Inbreeding is well known to reduce fitness in plants as a result of a decrease in the proportion of heterozygous loci. The magnitude of inbreeding depression is known to vary widely among species (Husband & Schemske, 1996; Byers & Waller, 1999; Crnokrak & Barrett, 2002), but recently attention has shifted away from estimating inbreeding depression at the species level and focused instead on investigating the range in the magnitude of inbreeding depression among populations (Eckert & Barrett, 1994; Hedrick et al., 1999; Carr & Eubanks, 2002), among maternal families within a population (Husband & Schemske, 1996; Dudash et al., 1997; Vogler et al., 1999; Willis, 1999), among individuals in a population (Holsinger, 1988; Schultz & Willis, 1995), and within a population over time (Dole & Ritland, 1993; Cheptou & Schoen, 2002). The magnitude of inbreeding depression may also vary among fitness traits, often stronger in some stages of the life cycle than others depending on prior history of inbreeding (Lande & Schemske, 1985; Husband & Schemske, 1996; Willis, 1999), and may differ with respect to male vs. female traits (Carr & Dudash, 1995, 1997; del Castillo, 1998; Chang & Rausher, 1999). Moreover, several studies have shown that environmental stress may influence the magnitude of inbreeding depression (e.g. Dudash, 1990; Schmitt & Ehrhardt, 1990; Wolfe, 1993; Carr & Dudash, 1995; Hauser & Loeschcke, 1996; Roff, 1997; Cheptou et al., 2000). Although theoretical work has begun to explore the consequences of inbreeding depression as a dynamic property that may vary over time and evolve in concert with the mating system (Schultz & Willis, 1995; Cheptou and Schoen, 2002), long-term studies are needed to quantify the variation in inbreeding depression among populations, families, and under a range of environmental conditions (Cheptou and Schoen, 2002).

In this study we examined temporal variation and the role of environmental stress on the strength of inbreeding depression in Cucurbita pepo ssp. texana (wild gourd). Unlike previous studies, here we examined the year to year variation in inbreeding depression using the same set of families grown under the same cultivation conditions at the same field station 4 years in a row. Our goal was to measure the variation in inbreeding depression for several male and female fitness traits while controlling for soil fertility, competition, and herbivory.

Although inbreeding depression is a consequence of increased homozygosity, the actual magnitude of inbreeding depression depends on the number and types of deleterious recessives that are exposed in the homozygous state, as well as the fitness costs of losing heterozygotes at overdominant loci (Charlesworth & Charlesworth, 1979). The magnitude of inbreeding depression further depends on how the resulting phenotype interacts with the environment, which may be benign or stressful, nutrient-rich or nutrient-limiting, crowded or sparse. Differences in fitness may be because of the joint fitness costs of a number of deleterious alleles of individually small effect placed under increased metabolic demand as a result of stress (Ramsey & Vaughton, 1998). Inbred plants may therefore be less buffered against stressful environmental conditions as a consequence of increased homozygosity (Mitton & Grant, 1984).

If inbred plants are more sensitive to environmental conditions than outcrossed plants, inbreeding depression should be more severe under stressful environmental conditions. To explore the role of stress on the magnitude of inbreeding depression, we experimentally varied the level of soil nitrogen. Nitrogen was chosen because it frequently limits growth and reproduction in plants; it is known to vary across microhabitats; and soil nitrogen affects aspects of both male and female function in plants (see Lau & Stephenson, 1993). Because a low nitrogen environment may be perceived as more stressful for inbred plants than for outcrossed plants, we expected there to be stronger inbreeding depression under the low nitrogen environment. The level of nitrogen is known to influence the number of staminate flowers, the number of pollen grains per flower, pollen diameter, and pollen performance (Stephenson et al., 1992; Lau & Stephenson, 1993), and when nitrogen is limiting there may be differences between inbred and outcrossed plants in their ability to accumulate, utilize, and allocate nitrogen for growth, maintenance, and reproduction.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Year to year variation
  6. Nitrogen stress
  7. Results
  8. Year to year differences
  9. High and low nitrogen fields
  10. Discussion
  11. Acknowledgments
  12. References

Cucurbita pepo L. ssp. texana (A Gray) Filov (Cucurbitaceae) (Lira et al., 1995) is an annual, monoecious vine with indeterminate growth and reproduction that is native to Texas and New Mexico and is thought to be the wild progenitor of cultivated squashes (Decker & Wilson, 1987; Decker-Walters, 1990). After a period of vegetative growth, one flower (either staminate or pistillate) is produced at most nodes. The large yellow flowers are bee-pollinated and are open and receptive for only one day.

Prior to the start of the experiment seeds were collected from plants growing in a natural population in Texas. In order to reduce the likelihood of sampling from related plants, seeds were collected from plants spaced no closer than 15 m along a linear transect. Seeds were grown in an experimental garden and plants were outcrossed to produce an initial population of f = 0 plants. We randomly selected five f = 0 progeny to found five maternal families, and reserved the remaining lines to serve as potential pollen donors. We performed single-sire outcross and self pollinations on each plant to produce f = 0 and f = 0.5 progeny, respectively, but because few studies of inbreeding use a coefficient of inbreeding >0.5, we performed an additional set of outcross and self pollinations in the following generation to produce f = 0 and f = 0.75 offspring. Pistillate flower buds were covered with cheesecloth bags prior to anthesis to exclude pollinators, and after fruit initiation the bags were removed.

Year to year variation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Year to year variation
  6. Nitrogen stress
  7. Results
  8. Year to year differences
  9. High and low nitrogen fields
  10. Discussion
  11. Acknowledgments
  12. References

To measure year to year variation in the strength of inbreeding depression, we grew plants representing five families and two levels of inbreeding (f = 0 and f = 0.75) at the same field station 4 years in a row (1998, 1999, 2000, and 2001). Each year in early May, 25 seeds from each family and each level of inbreeding were randomly selected and planted in potting soil at a depth of 1 cm in 10 cm ‘jiffy’ pots and arrayed in a randomized block design in a greenhouse. Most seeds germinated within one week, and after 2 weeks most seedlings had one or more expanded leaves. Depending on the year of the study, between 50 and 200 seedlings were transplanted to one or more adjacent 60 × 60 m plots in an experimental garden at the Pennsylvania State University Agricultural Experimental Station at Rock Springs, Pennsylvania. The fields were organized into 12 rows of 10 plants spaced 5–10 m apart, and plants were randomized into blocks to reduce the effect of variation within fields. All fields were fertilized with all essential micro and macro nutrients, including half the level of nitrogen, phosphorus, and potassium recommended for commercial squash production. Fields were sprayed with pesticides every 2 weeks or as needed to control Diabrotica beetle (cucumber beetle) and aphid populations. In each year we also grew f = 0 and f = 0.5 plants from the same families in an adjacent field. These plants were both selfed and outcrossed to produce the f = 0 and f = 0.75 plants that were used the following season.

Each day the number of new staminate and pistillate flowers was recorded to determine total flower production. Once per week staminate flower buds were lightly clamped with a twist-tie prior to opening to prevent pollen removal and then flowers were collected at anthesis. Anthers and loose pollen were removed and dried in scintillation vials in a drying oven at 45°C for 2 weeks before being rehydrated for 1 day in a 0.5% NaCl solution and then sonicated for 15 min to dislodge pollen from anthers. Pollen number and diameter were determined using an Elzone® EX180 particle counter (Particle Data, Inc. Elmhurst, IL, USA).

On one or more dates midway through each growing season, staminate flower buds were clamped prior to anthesis and then the next day dehisced pollen from each plant was collected and sprinkled onto Brewbaker and Kwack (Brewbaker & Kwack, 1963) pollen-germination media containing 10% sucrose and allowed to germinate and grow for 30 min, at which point 2 mL of 70% ethanol was applied to arrest growth. In vitro pollen tube growth rate was determined by calculating the mean length of thirty pollen tubes per plate using image analysis (Rich et al., 1989). When pollen was collected from the same plant on more than one date, the mean pollen tube growth rate was used for analysis.

After the first lethal frost in September in each year, the total number of mature fruits per plant was recorded and two fruits per plant were collected for seed extraction. Fruits were selected in a haphazard manner, although immature and damaged fruits were excluded. Seed number was measured either by counting (1998 and 1999) or calculated by dividing the total seed mass by the mass of a random sample of 25 seeds (2000 and 2001). The mean seed number of the two fruits was used in analysis.

Because fitness traits may be correlated, all traits were examined simultaneously using a multivariate analysis of variance with the following terms: coefficient of inbreeding (f), maternal family, year, and the two-way interactions. Individual mixed-effects analysis of variance models were then run using family as a random effect. Inbreeding depression (δ) was calculated for each trait for each year as 1 −ws/wo, where ws is the mean value of the trait among inbred plants and wo is the mean value of the trait among outcrossed plants.

Nitrogen stress

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Year to year variation
  6. Nitrogen stress
  7. Results
  8. Year to year differences
  9. High and low nitrogen fields
  10. Discussion
  11. Acknowledgments
  12. References

Inbred and outcrossed plants were grown under high and low nitrogen conditions to determine the effects of different stress levels on the severity of inbreeding depression. In May 1999 100 plants representing five families and two levels of inbreeding (f = 0 and 0.75) were grown in a greenhouse as above and transplanted into two adjacent fields. Both fields were prepared as above, except only one of the fields was fertilized with nitrogen. Both fields were sprayed with pesticides every 2 weeks to control beetle and aphid populations. We counted new staminate and pistillate flowers daily, and in late July we collected staminate flowers to measure in vitro pollen tube growth rate, pollen diameter, and pollen number per flower following the techniques described above. Fruits and seeds were counted following the first frost in September. All traits were examined simultaneously using a multivariate analysis of variance, and then separate mixed-model analysis of variance models were run for each field using coefficient of inbreeding, family, and the interaction as factors. Inbreeding depression was calculated for each trait as above.

Year to year differences

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Year to year variation
  6. Nitrogen stress
  7. Results
  8. Year to year differences
  9. High and low nitrogen fields
  10. Discussion
  11. Acknowledgments
  12. References

When all traits were considered simultaneously using manova, coefficient of inbreeding (f), family, and year were all significant, as were the interactions between f and year and between f and family (Table 1). In individual univariate tests (Table 1), means varied significantly among years for all traits. There was a significant effect of f for pollen number per flower and a marginally significant effect of f for seed number per fruit, whereas a significant f × year interaction was found for staminate flowers per plant and seeds per fruit. Pollen number per flower also showed a significant year × family interaction, but this may not be important, as the year × family interaction was not significant in the manova when all traits were analysed simultaneously. Significant f × family interactions were also found for staminate flowers per plant and seeds per fruit.

Table 1.  Analysis of variance for components of male and female fitness in a study to test for variation in the strength of inbreeding depression at the same field site over four consecutive field seasons (1998–2001). All response values were included in a multivariate analysis of variance to control for correlations among traits, and then individual univariate tests were performed using the coefficient of inbreeding (f), the year in which the plants were measured, the maternal line (family), and the two-way interactions. Staminate flowers per plant and fruits per plant were square-root transformed to meet statistical assumptions. Family was treated as a random effect.
Source of variationd.f.Wilks λFP
Multivariate anova
 f1800.769.74<0.001
 Year5090.1823.60<0.001
 Family6290.772.050.002
 f × year5090.782.57<0.001
 f × family6290.821.580.040
 Year × family9850.621.270.069
Source of variationd.f.MSFP
Staminate flowers per plant
 f162.532.450.181
 Year3111.98114.86<0.001
 Family429.2111.170.441
 f × year340.6864.520.004
 f × family430.73.410.009
 Year × family127.4430.830.622
 Error4968.996  
Pollen tube growth rate in vitro
 f1423673.10.101
 Year368886431.03<0.001
 Family4508922.810.095
 f × year3248121.480.221
 f × family4118310.710.589
 Year × family12226521.350.191
 Error23716778  
Pollen number per flower
 f12093708.820.018
 Year32091533.620.044
 Family4335310.630.654
 f × year39720.040.990
 f × family4233730.950.437
 Year × family12595362.410.005
 Error34124677  
Fruits per plant
 f142.6793.740.109
 Year387.34413.81<0.001
 Family47.0480.60.681
 f × year36.1460.980.401
 f × family412.842.050.086
 Year × family126.3291.010.438
 Error4746.265  
Seeds per fruit
 f1434774.620.089
 Year36133726.78<0.001
 Family4137751.50.356
 f × year380432.740.043
 f × family4112783.840.004
 Year × family1222550.770.684
 Error4262938  

Most traits showed relatively little inbreeding depression in 1998 and 1999 and greater inbreeding depression in 2000 and 2001, but not all traits followed this pattern (Fig. 1). The overall mean inbreeding depression was 0.14, whereas the mean inbreeding depression for male function was 0.12, and for female function the mean was 0.15. Although inbreeding depression was often greater across years for traits related to female function, inbreeding depression for staminate flower number jumped from 0.17 in 2000 to 0.5 in 2001, and inbreeding depression for in vitro pollen tube growth rate was stronger in 1999 than it was during any other year of the study and was also the trait exhibiting the strongest inbreeding depression in that year. When a multiplicative estimate of inbreeding was used, overall inbreeding depression varied by year from 0.16–0.20 to 0.34–0.53 between 1998 and 2001.

image

Figure 1. Variation in inbreeding depression for three male fitness traits (solid lines) and two female fitness traits (dashed lines) at the same field site over four consecutive field seasons. Inbreeding depression was calculated for each trait (e.g. staminate flowers per plant) for each year as 1−ws/wo, where ws is the mean value of the trait among inbred (f = 0.75) plants and wo is the mean value of the trait among outcrossed plants.

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High and low nitrogen fields

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Year to year variation
  6. Nitrogen stress
  7. Results
  8. Year to year differences
  9. High and low nitrogen fields
  10. Discussion
  11. Acknowledgments
  12. References

In the nitrogen experiment, three of 20 outcross plants and four of 17 inbred plants in the high nitrogen field (field 1) died and were excluded from the analysis, and in the low nitrogen field (field 2) one of 30 outcrossed plants and two of 30 inbred plants died. When all traits were considered simultaneously using manova, field was the only significant factor, indicating that differences between the two fields affected overall means for at least some traits (Table 2). Because field is confounded with nitrogen treatment in this study, it is not possible to interpret the interaction between f and field directly, so the two fields were analysed separately. Plants grown in field 1 showed no significant effect of inbreeding for any trait, whereas plants grown in the field 2 showed significant or marginally significant effects of inbreeding for staminate flower number, fruit number, pollen number per flower, and in vitro pollen tube growth rate (Table 2). Mean inbreeding depression was higher for all traits for plants grown in field 2 (0.25) compared with plants field 1 (0.13) (Table 3, Fig. 2). In field 2 mean inbreeding depression was slightly higher for female function (0.26) than male (0.23) function, but in field 1 mean inbreeding depression for both male and female function was the same (0.13).

Table 2.  Analysis of variance for components of male and female fitness in a study to test for variation in the strength of inbreeding depression among plants grown in two adjacent fields varying in the level of nitrogen fertilizer. A multivariate analysis of variance was used to test the effect of field when all traits were considered simultaneously, and separate univariate tests were performed using the coefficient of inbreeding (f), the maternal line (family), and the interaction between f and family. Because the field treatment was not replicated, the fields were analysed separately. Staminate flowers per plant and fruits per plant were square-root transformed to meet statistical assumptions, and family was treated as a random effect. Pollen tube growth rate was not measured in plants grown in the field fertilized with nitrogen.
  Source of variationd.f.Wilks λFP
Multivariate analysis of variance f230.6761.8350.136
Family810.5080.7280.808
Field230.5333.3520.016
f × family810.3401.2320.242
f × field230.8250.8130.571
Family × field810.4061.0010.475
TraitFieldSource of variationd.f.MSFP
Staminate flowers per plantHigh nf110.410.730.451
Family336.492.610.226
f × family3140.550.657
Error1925.63  
Low nf148.387.790.048
Family424.593.990.105
f × family46.170.480.754
Error4712.97  
Pollen number per flowerHigh nf1559701.640.284
Family387650.260.854
f × family3342341.110.395
Error930839  
Low nf11939815.80.064
Family4393071.160.444
f × family4337921.130.379
Error1529840  
Pollen diameterHigh nf1000.997
Family311.220.210.882
f × family352.741.030.423
Error950.97  
Low nf11.730.080.789
Family412.160.60.685
f × family420.320.530.714
Error1538.15  
Pollen tube growth rate  in vitroLowf13257111.820.023
Family440931.520.347
f × family426910.410.800
Error296569  
Pistillate flowers per plantHigh nf14.131.720.258
Family314.917.10.071
f × family32.10.150.929
Error1914.12  
Low nf117.774.430.101
Family47.5921.910.274
f × family43.9810.470.755
Error478.402  
Fruits per plantHigh nf13.2690.80.430
Family38.1242.050.285
f × family33.9650.480.699
Error198.225  
Low nf122.24.140.111
Family45.8281.090.469
f × family45.360.950.442
Error475.619  
Seeds per fruitHigh nf18820.220.671
Family373341.820.318
f × family340350.820.497
Error194903  
Low nf147483.630.128
Family442043.230.141
f × family413020.550.699
Error442358  
Table 3.  Means for male and female fitness traits for inbred and outcrossed plants grown under high and low nitrogen treatments.
TraitNitrogenf = 0f = 0.75
Staminate flowers per plantHigh119.9 ± 24.892.2 ± 18.0
Low74.0 ± 13.643.0 ± 7.5
Pollen number per flower (×30)High716.4 ± 50.8599.1 ± 51.0
Low578.7 ± 49.7435.7 ± 49.9
Pollen diameter (μm)High132.5 ± 1.4132.6 ± 2.9
Low140.5 ± 1.2139.7 ± 1.8
Pollen tube growth rate in vitro (μm)Highnot measured
Low297.4 ± 16.8234.7 ± 17.2
Pistillate flowers per plantHigh63.5 ± 13.350.4 ± 10.2
Low35.1 ± 6.621.8 ± 4.3
Fruits per plantHigh61.4 ± 11.152.5 ± 8.6
Low36.3 ± 5.123.8 ± 4.4
Seeds per fruitHigh250.1 ± 12.6237.4 ± 24.2
Low237.1 ± 10.9220.8 ± 7.5
image

Figure 2. Inbreeding depression for three male fitness traits (solid lines) and three female fitness traits (dashed lines) for plants grown in two adjacent fields differing in the level of nitrogen fertilizer. Inbreeding depression was calculated as in Fig. 1.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Year to year variation
  6. Nitrogen stress
  7. Results
  8. Year to year differences
  9. High and low nitrogen fields
  10. Discussion
  11. Acknowledgments
  12. References

In a study of fitness traits across 4 years, means were found to vary significantly from year to year for most traits, suggesting that growing conditions vary widely among years even at the same field site. The significant interaction between coefficient of inbreeding and year in the manova indicates that the severity of inbreeding depression varies among years for at least some traits when all traits are considered simultaneously. Although the coefficient of inbreeding is related to the degree of homozygosity, the actual magnitude of inbreeding depression depends on which alleles at which loci are homozygous and which traits they affect and when. Perhaps this varies with the type, timing, and magnitude of stress (e.g. drought, temperature stress, herbivory, and disease), which may vary from year to year and within years.

Within each year of the study traits differed significantly in their magnitude of inbreeding depression, although inbreeding depression was relatively low (<0.5) for all traits. This relatively mild level of inbreeding depression should favour alleles that increase the selfing rate in this otherwise predominantly outcrossing species (Robinson et al., 1979; Charlesworth & Charlesworth, 1987), although Cheptou and Shoen (2002) showed that fluctuating inbreeding depression may act as an additional cost of selfing and may help to stabilize mixed mating systems. Stable mixed mating systems may also be maintained when inbreeding depression is greater through female function than through male function (Rausher & Chang, 1999). We found that female function tended to show slightly greater inbreeding depression overall, although we have not tested for this statistically.

As the less ‘expensive’ function (Lloyd & Webb, 1997), male function may be less subject to environmental variation or be limited by different conditions, so in a resource-limited environment, female function may be more strongly affected, and in general, environmental factors may affect male and female fitness traits differently (Bertin, 1982; Schlichting & Devlin, 1989; Devlin & Ellstrand, 1990). Most studies show that male and female reproductive success are positively correlated (Broyles & Wyatt, 1990; Devlin & Ellstrand, 1990; Conner et al., 1996a,b), although different suites of genes are expressed during pollen and ovule/seed development (Coen & Meyerowitz, 1991; Meagher, 1992; Yanofsky, 1995) and some studies suggest that under stress conditions male and female fitness may be weakly or even negatively correlated (e.g. Bertin, 1982; Marshall & Ellstrand, 1986; Schlichting, 1986; Schlichting & Devlin, 1989). In a separate study we found that overall inbreeding depression is less severe for male function than for female function in C. pepo ssp. texana, and the few studies that have examined the relationship between male and female fitness in response to inbreeding have found little correlation or consistency between the magnitude of inbreeding depression in male function relative to female function (e.g. Carr & Dudash, 1997; Chang & Rausher, 1999; Melser et al., 1999).

Environmental variation is likely to affect the quantity and quality of resources that a plant is able to allocate to reproduction, particularly among inbred plants. Inbred plants tend to produce fewer or smaller seeds than outcrossed plants (e.g. Darwin, 1876; Husband & Schemske, 1996), presumably because inbred plants are less vigorous and less able to acquire and allocate resources to offspring. Inbreeding depression has also been detected in several aspects of pollen fitness, including pollen quantity, viability, pollen tube growth rate, and siring success (e.g. Willis, 1993; Carr & Dudash, 1995, 1997; Jóhannsson et al., 1998; Chang & Rausher, 1999). Although pollen translates and transcribes a large portion of its genome during development, germination, and pollen tube growth (Tanksley et al., 1981; Willing & Mascarenhas, 1984), it is not directly subject to inbreeding depression, as partial dominance and overdominance do not apply in haploid organisms. If inbred plants differ in the quantity or quality of resources provided to pollen during development, however, and if this reduces pollen competitive ability, then inbreeding depression can be said to extend to the gametophyte generation (Stephenson et al., 2001). Consequently adverse growing conditions or inbreeding depression in the pollen parent may reduce the quality and siring success of pollen, particularly in competition with outcross pollen.

Although inbreeding depression is no longer considered a static property of a species or population (Lloyd, 1979), most models of mating system evolution assume a threshold level of inbreeding depression (0.5 in the simplest case), below which the transmission advantage of selfing favours alleles that increase the selfing rate and above which inbreeding depression favours alleles that promote outcrossing (Lloyd, 1979; Lande & Schemske, 1985; Charlesworth & Charlesworth, 1990; Uyenoyama et al., 1993). Therefore, accurate estimates of inbreeding depression for individuals in a population at a given point in time are necessary in order to predict the evolutionarily stable selfing rate.

Accurately estimating inbreeding depression is difficult, however, because the same plant might exhibit different levels of inbreeding depression when grown under different conditions, as the magnitude of inbreeding depression can vary with the level of stress experienced by a plant in a particular environment and may be more severe under harsh environmental conditions (Pedersen, 1968; McCall et al., 1989; Dudash, 1990; Schmitt & Ehrhardt, 1990; Schmitt & Gamble, 1990; Wolfe, 1993; Carr & Dudash, 1995; Hauser & Loeschcke, 1996; Roff, 1997; Cheptou et al., 2000). In this study we found that the magnitude of inbreeding depression differed between plants grown in two fields that differed in their level of stress. The higher-stress, low-nitrogen field exhibited stronger inbreeding depression, although we cannot conclude that nitrogen availability alone is responsible for the differences in inbreeding depression because the nitrogen treatment is confounded with field in this experiment. There may be other differences between the fields, such as soil composition, drainage, or availability of other macro and micro nutrients; however, our goal was to examine the relative performance of outcross and inbred plants in two environments, and we have shown that the magnitude of inbreeding depression may vary depending on growing conditions.

Studies conducted under benign environmental conditions, such as those experienced in a greenhouse, may underestimate the level of inbreeding depression that would be observed under natural conditions. Conversely, studies conducted under unreasonably high stress conditions may reveal inbreeding depression that is rarely expressed under natural conditions, so ideally inbreeding depression studies should be performed under conditions typical to those experienced in a natural population (Charlesworth & Charlesworth, 1987). Koelewijn (1998) and Dudash (1990) found much greater levels of inbreeding depression under field conditions than under greenhouse conditions, and Ramsey & Vaughton (1998) found more severe and more comprehensive inbreeding depression in field-grown plants than plants grown under laboratory conditions. The magnitude of inbreeding depression may increase under conditions of crowding (Wolfe, 1993), drought stress (Hauser & Loeschcke, 1996), and nutrient stress (Helenurm & Schaal, 1996). As many environments vary in one or more respects from year to year, studies which measure inbreeding depression under a variety of environments are likely to better capture the range of inbreeding responses.

We found a significant interaction between coefficient of inbreeding and family for several traits (staminate flower number and seed number per fruit), suggesting that the strength of inbreeding depression may vary among families (Husband & Schemske, 1996; Dudash et al., 1997; Vogleret al., 1999; Willis, 1999), although we hesitate to generalize these results as we used only five families. When families vary in the strength of inbreeding depression, among-family selection may occur, leading to the loss of maternal lines (Willis, 1999), although the low levels of inbreeding depression in this species reduce this risk and make it a good candidate for long-term inbreeding studies.

Inbreeding depression may be less an intrinsic property of an individual or family than a measure of sensitivity to a given environment. Because growing seasons vary with respect to the amount of rainfall, wind, sun, pollinator availability and effectiveness, herbivory, pathogen exposure, soil quality, nutrient availability, intra- and interspecific competition, etc., the magnitude of inbreeding depression is likely to vary from year to year within a population. Variation over time in the strength of inbreeding depression may influence the evolution of the mating system. In low stress years, deleterious alleles may become fixed, thereby increasing the severity of inbreeding depression in future generations, whereas in high stress years, strong selection may purge deleterious alleles, thereby decreasing the severity of inbreeding depression in future generations by altering allele frequencies in addition to genotype frequencies.

This study suggests that the severity of inbreeding depression is variable across environments and across the range of conditions experienced in one location over several years. The estimated level of inbreeding depression therefore depends on the conditions in which plants are grown and the year in which inbreeding depression is measured.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Year to year variation
  6. Nitrogen stress
  7. Results
  8. Year to year differences
  9. High and low nitrogen fields
  10. Discussion
  11. Acknowledgments
  12. References

We thank Robert Oberheim and the Department of Horticulture for use of The Pennsylvania State University Agricultural Experimental Station at Rock Springs, PA. We thank Tony Omeis, Steve Breault, Mike Westerman, Brian Clark, Sara Simmers, and Laura Leist for field and lab assistance. This work was supported by NSF grants DEB 93-18224 and DEB 98-06691 to A.G.S.

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  5. Year to year variation
  6. Nitrogen stress
  7. Results
  8. Year to year differences
  9. High and low nitrogen fields
  10. Discussion
  11. Acknowledgments
  12. References
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