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Reproductive Biology |
2 Department of Biology, The Pennsylvania State University, University Park, Pennsylvania 16802 USA 3 Department of Biology, The Hainan Normal University, Haikou, Hainan 571158 China 4 Department of Biology, The Pennsylvania State University, Altoona, Pennsylvania 16601 USA 5 Department of Biology, The Center for Chemical Ecology and The Center for Infectious Disease Dynamics, The Pennsylvania State University, University Park, Pennsylvania 16802 USA
Received for publication 8 June 2007. Accepted for publication 11 November 2007.
ABSTRACT
Herbivory is a ubiquitous component of terrestrial communities that reduces plant growth and reproduction. Consequently, a goal of evolutionary ecology is to identify the causes and consequences of variation in herbivory within plant populations. This three-year study examined the effects of inbreeding on the resistance of wild gourd plants (Cucurbita pepo subsp. texana) to herbivory by cucumber beetles and the impact of the timing of herbivory on reproduction. We grew families of inbred and outbred gourds and recorded beetle damage at three developmental stages, incidence of beetle-vectored wilt disease, survival, and reproduction. While total beetle damage significantly depressed flower and fruit production, damage until mid-July did not depress any measure of reproduction, indicating that these gourds are tolerant of moderate levels of herbivory for most of the growing season. However, beetle damage accumulating after mid-July significantly depressed reproduction, indicating that plants have reduced tolerance during peak reproduction. Early damage, however, did increase the probability of contracting a deadly wilt disease that is vectored by the beetles, suggesting that tolerance and resistance are not alternative defense strategies. Inbreeding significantly reduced resistance to herbivory and, independently of beetle damage, reproductive output. Finally, we found additive genetic variation for both resistance and tolerance that varies with ontogeny.
Key Words: bacterial wilt disease • cucumber beetles • Cucurbita • Cucurbitaceae • herbivory • inbreeding • inbreeding depression • ontogeny • resistance • tolerance
Leaf herbivory is a ubiquitous component of terrestrial communities (McNaughton et al., 1989
; Louda et al., 1990; Marquis, 1992) and has been shown in a great variety of species to adversely affect vegetative growth, reproduction through both the female (fruit and seed) and male (pollen production and pollen performance) functions, and survival (e.g., Marquis, 1992; Delph et al., 1997; Juenger and Bergelson, 1997; Lehtilä and Strauss, 1999). Moreover, both the amount of herbivory and the impact of herbivory on fitness often vary among individuals within populations. One of the primary goals of evolutionary ecology is to identify the causes and consequences of this variability. At least some of this variation has been shown to have a genetic basis. Several studies have demonstrated genetic variation among plants for resistance (the ability to reduce the amount of herbivory) and the traits responsible for resistance (e.g., leaf hairs, unpalatable secondary chemicals in leaves), and for tolerance (the ability to reduce the fitness consequences of herbivory) and the traits responsible for tolerance (e.g., shifts in patterns of resource allocation and meristem production) (e.g., Kennedy and Barbour, 1992; Strauss and Agrawal, 1999; Tiffin and Rauscher, 1999; Strauss et al., 2002; Hare et al., 2003; Weinig et al., 2003). Because herbivores often vector pathogens and leave wounds where pathogens can enter plants, variation in resistance to herbivores also affects exposure to diseases and the establishment and spread of diseases within populations.
The expression of traits associated with resistance is known to change during the growing season and across developmental stages. Leaf shape, defensive structures such as thorns and hairs, and the type and quantity of unpalatable secondary chemicals can change from juvenile to adult stages and with the age of the leaf (e.g., Feeny, 1970; Poethig, 1990; Karban and Baldwin,1997; Jarzomski et al., 2000). Such changes have been shown to alter the behavior of foliar herbivores, the types of herbivores that feed, the rates of predation on the herbivores, and the growth rates of foliar herbivores (e.g., Schultz, 1988; Turlings et al., 1990; Coley and Barone, 1996; De Moraes et al., 1998; Fenner et al., 1999). Similarly, seasonal and ontogenetic changes in patterns of resource allocation, stored reserves, plant architecture, and nutrient availability are expected to alter the tolerance of plants to foliar herbivores (see Boege and Marquis, 2005).
Inbreeding is also common in flowering plants (it is estimated that 50% of all species self-pollinate 20% or more of the time [Barrett and Eckert, 1990; Vogler et al., 1999]), and inbreeding has been shown in a great variety of species to reduce growth, reproduction, and survival (Husband and Schemske, 1996). Because inbreeding increases homozygosity, thereby exposing deleterious recessives to selection (Charlesworth and Charlesworth 1987), inbreeding could both directly affect the biochemical pathways underlying resistance and tolerance and indirectly affect resistance and tolerance by reducing the rate of resource acquisition for use in both defense pathways and compensatory responses to herbivory (Stephenson et al., 2004). A few recent studies have shown that both the amount of herbivory (resistance) and the impact of herbivory on fitness (tolerance) are affected by inbreeding in species with mixed mating systems (e.g., Carr and Eubanks, 2002; Hayes et al., 2004; Stephenson et al., 2004; Hull-Sanders and Eubanks, 2005). It is also possible that the impact of inbreeding on resistance and tolerance can vary across developmental stages.
Here, we report the results of a three-year experimental field study of the intensity and timing of herbivory by cucumber beetles (Acalymma vittatum F. and Diabrotica undecimpunctata howardi Barber) on survival, incidence of a deadly disease vectored by the herbivores, and reproductive output of inbred and outbred wild gourd plants (Cucurbita pepo L. subsp. texana Andres). Specifically, we address four questions: (1) Does the intensity of cucumber beetle damage change with developmental stage? (2) What is the impact of cucumber beetle damage during each developmental stage on fitness and incidence of disease? (3) What are the combined effects on fitness of beetle damage across developmental stages? (4) Is there genetic variation for resistance and tolerance across developmental stages? Moreover, we predict that inbreeding reduces resistance to herbivory and that the adverse effects of inbreeding on tolerance to herbivory will be greatest during the peak reproductive period of the life cycle (i.e., when resource demand from the developing reproductive organs is greatest).
MATERIALS AND METHODS
Study species
The wild gourd, Cucurbita pepo subsp. texana (also known as C. pepo subsp. ovifera var. texana and var. ozarkana; Cucurbitaceae), is an annual monoecious vine with indeterminate growth and reproduction. It is native to northern Mexico, Texas, and the lower Mississippi River drainage area and is thought to be either the wild progenitor of the cultivated squashes (C. pepo subsp. pepo, also known as C. pepo subsp. ovifera var. ovifera) or an early escape from cultivation (Decker and Wilson, 1987; Decker-Walters, 1990; Lira et al., 1995; Decker-Walters et al., 2002). After germination and seedling emergence, there is a period of vegetative growth (5–7 nodes). Thereafter, most nodes produce one large yellow flower (either staminate or pistillate) in the axils of each leaf. The flowers last for only one morning and are pollinated by bees, especially squash bees of the genera Peponapis and Xenoglossa. The fruits are round to oval with a volume of 175–450 mL and typically contain 150–300 seeds that weigh 20–40 mg/seed (Winsor et al., 2000; Avila-Sakar et al., 2001).
The leaves and other organs of this wild gourd produce extremely bitter compounds called cucurbitacins (oxygenated tetracylic triterpenes) that deter most herbivores (Tallamy, 1985; Metcalf and Rhodes, 1990). However, cucumber beetles are adapted to feed on cucurbitacins in the leaves of Cucurbita species and are found throughout the native ranges of Cucurbita species (Robinson and Decker-Walters, 1997). Cucumber beetles feed on leaves and other organs and cause a characteristic pattern of holes (typically 1.0–1.5 cm in diameter) in the portions of the leaves serviced by the smallest veins. Leaf damage over the entire growing season by cucumber beetles has been shown to substantially reduce yield in cultivated squash (e.g., Tallamy and Krischik, 1989) and reduce reproductive output in the wild (free-living squash) gourd (Quesada et al., 1995; Stephenson et al., 2004). Cucumber beetles are also the only known vector of the bacterial pathogen Erwinia tracheiphila Smith (Yao et al., 1996), the causative agent of bacterial wilt disease. The bacterium is transmitted via the feeding of striped and spotted cucumber beetles. Erwinia from the fecal pellets of the cucumber beetles enter the beetle-damaged leaves and proliferate in the xylem where they secrete an exopolysaccharide (mucilaginous) matrix that blocks the water supply, resulting in wilting and eventual death of the plant. Wilt symptoms typically develop 10–15 d after infection, and the disease is nearly always fatal once symptoms appear (Sherf and MacNab, 1986). Bacterial wilt disease is found throughout the native ranges of species in the C. pepo complex, and it is the most economically important disease of cultivated cucurbits (cucumbers, melons, watermelons, and squash) in the eastern USA (Fleischer et al., 1999).
Experimental field design
Over the past five years, we have investigated the interrelationships among inbreeding, herbivory, and disease in C. pepo subsp. texana. In 2002, 2004, and 2005, we planted 2–4 fields (0.4 ha each) at The Pennsylvania State University Agriculture Experiment Station at Rock Springs, Pennsylvania (PA) with inbred (F = 0.5) and outbred (F = 0) offspring. In each year, we germinated 30 selfed (S) and 30 outcrossed (X) seeds from each of five maternal families in 4-L pots in a greenhouse and transplanted the resulting seedlings (at the 1–2 true leaf stage) into a 1-ha field between 18 and 22 May. In 2002 there were two randomized blocks per 0.4-ha field (two 0.4-ha fields each with two blocks each with 15 plants per family per S/X and one 0.2-ha field with one block). In 2004 and 2005, there were three randomized blocks per 0.4-ha field (10 plants per family per S/X). In 2002, half of the inbred and half of the outbred plants from each family in each field were hand-sprayed with a contact insecticide, Asana XL (DuPont, Wilmington, Delaware, USA). The sprayed plants were eliminated from all of the analyses in this paper. In 2004 and 2005, no plants in the experimental fields were sprayed. In all years, the fields were fertilized prior to transplanting with 50% of the recommended levels of N-P-K for the commercial production of squash. The seeds used in this study originally came from seeds sampled randomly from a natural population in Texas, USA. In the summer of 2001, five unrelated plants were grown from seed at the Rock Springs, PA site and were both outcrossed to one (of five) unrelated pollen donors and self-pollinated. Each pollination was repeated 10 times on each plant to generate sufficient self and outcross seeds. Seeds from these five families were used in the 2002 fields, and self and outcross seeds for subsequent years were generated in the same manner. Consequently, all seeds used during a given year were produced during the previous growing season, and all self seeds were F = 0.5. In each year, we used different 0.4-ha fields, and none of the fields that we used were planted with a cucurbit crop during the previous growing season.
In each field during each year, we recorded the number of staminate and pistillate flowers on each plant twice weekly until 25–31 August. Because flowers last for only one morning, this provided an unbiased estimate of total flower production per plant. When we counted flowers, we also recorded any plants that had died and any plants that had symptoms of bacterial wilt disease. After the last flower count (31 August), we continued to record death and wilt symptoms once per week until 15 September. Field diagnosis of bacterial wilt disease is unambiguous because the symptoms are distinctive and because the diagnosis can be confirmed visually by the presence of the exopolysaccharide spanning the cut ends of the stem of a wilted branch. In addition, we confirmed the presence of Erwinia by isolating the bacterium from a sample of 6–8 infected plants per year. The isolates were cultured and then used to produce cell suspensions, which produced wilt symptoms in inoculated seedlings grown in a greenhouse (see Ferrari et al., 2007). At the end of each growing season (typically early October after the first hard frost), we counted the number of mature fruits produced per plant. In late July or early August of each year, we identified one staminate flower bud on each plant in at least one of the experimental fields that we thought would open the next day, and we enclosed it in bag made of fine mesh thule (bridal veil) to prevent insect visitation. The following morning, we collected the staminate flowers and brushed a small sample of the pollen onto petri plates containing a modified Brewbaker and Kwack 1963 medium (see Stephenson et al., 2001). We recorded the lengths of the first 30 pollen tubes encountered on a left to right transect through the middle of the plate using an image analysis system (Rich et al., 1989).
Three times during each year (mid-June = BD1; mid-July = BD2; mid-August = BD3), we nondestructively recorded the amount of beetle damage on the new growth along the main stem of each plant using a 0–5 scale (0 = most leaves with no beetle damage and no leaf with >5% of the leaf area removed, and 5 = all leaves damaged and at least one leaf with >50% of the leaf area removed; see Stephenson et al., 2004). The mid-June assessment measured beetle damage during the prereproductive seedling stage of development, just prior to the anthesis of the first flower; because growing conditions varied from year to year, this assessment occurred between 10 and 20 June. The mid-July assessment (30 d after the mid-June assessment) measured beetle damage when plants were growing rapidly, producing buds and flowers, and making their initial investments in fruit maturation. The mid-August assessment (31 d after the mid-July assessment) measured beetle damage during peak flower production and when large numbers of fruits were in various stages of maturation on the vines.
Statistical analyses
To determine whether resistance (amount of beetle damage) differed across the growing season, we used the type III sums of squares from a repeated measures analysis of variance (PROC GLM REPEATED; SAS program, version 9.1; SAS Institute, 2002) to measure the effects of year, time of beetle damage measurement (BD1, BD2, and BD3), field nested within year, breeding (F = 0 = cross pollination; F = 0.5 = self pollination), maternal family nested within year, and their interactions on the amount of beetle damage to new growth. For those plants that survived the entire season, we also determined the correlations among BD1, BD2, and BD3 using Pearson correlation coefficients. Separate mixed effects model ANOVAs (PROC MIXED; SAS Institute, 2002) were used to determine the effects of maternal family nested within year (random), year, field nested within year, breeding, and the interaction of maternal family and breeding nested within year (random) on the amount of beetle damage in mid-June (BD1), mid-July (BD2), and mid-August (BD3) and total beetle damage (BDT = BD1 + BD2 + BD3). For each analysis, we used all of the plants that survived until the census date for that measure of beetle damage. Type III sums of squares were used to test the significance of the fixed effects, and restricted maximum likelihood estimates were used to test the significance of the random effect and relevant interactions in the model. These analyses allowed us to identify which plant factors influence beetle damage (resistance) and to partition the variance resulting from uncontrolled environmental factors (field and year).
Separate fixed effect model analyses of covariance were conducted to determine the effects of maternal family nested within year, year, field nested within year, breeding, BD1 (covariate), BD2 (covariate), BD3 (covariate), and all possible two-way interactions (excluding those involving year and field nested within year) on the four components of fitness (total staminate flowers produced, pollen tube growth rate in vitro, total pistillate flowers produced, and total fruit production (PROC MIXED; SAS, version 9.1). These analyses allowed us to determine the independent effects of beetle damage at three times during the growing season (and total beetle damage) on reproductive performance (tolerance) over the entire growing season. It should be noted that these measures of tolerance reflect both differences in resource availability from herbivory and plant compensatory responses to herbivory. For these analyses, we used only those plants that survived until all three measures of beetle damage were obtained. In separate analyses, BD1 + BD2 (the two measurements added together) (a covariate) or BDT (a covariate) replaced BD1, BD2, and BD3 in the model. These analyses allowed us to determine the effect of all beetle damage prior to mid-July and total beetle damage throughout the growing season on tolerance.
We next performed a series of analyses designed to determine whether beetle damage had a transitory effect on reproduction (that is, over the following month). Separate fixed effect model analyses of covariance were also conducted to determine the effects of maternal family nested within year, year, field nested within year, breeding, BD1 (covariate), and all possible two- and three-way interactions (excluding those involving year and field nested within year) on the number of male and female flowers produced during the one month between the measurement of BD1 and the measurement of BD2 (PROC MIXED; SAS Institute, 2002). Similar analyses assessed the impact of BD2 on the number of staminate and pistillate flowers produced during the one month between the measurement of BD2 and BD3. In addition, similar analyses assessed the impact of BD1 + BD2 on the number of staminate and pistillate flowers produced during the one month between the measurements of BD1 and BD2 and, separately, the month between the measurements of BD2 and BD3. These analyses included only those plants that survived until the subsequent beetle damage measure. Finally, t tests were used to compare (1) the mean BD1 estimate of those plants that died before BD2 to those plants that survived until BD2; (2) the mean BD2 estimate of those plants that died before BD3 to those plants that survived until BD3; (3) the mean BD1 + BD2 estimate of those plants that died before BD3 to those plants that survived until BD3; (4) the mean BD3 estimate of those plants that died before 15 September to those plants that survived until 15 September; and (5) the mean BDT estimate of those plants that died before 15 September to those plants that survived until 15 September. All the one-month-interval analyses included only the 2004–2005 data because the twice weekly flower counts and survival data were no longer retrievable from 2002.
RESULTS
During each year, a few plants were eliminated from the analyses because they died following transplantation to the field, were damaged by hand-cultivation, or were growing in a field location that had standing water for three or more days. A repeated measures analysis of variance revealed that the amount of beetle damage varied significantly with year, breeding (S or X), time of the growing season (BD1, BD2, or BD3) and the interaction of time with maternal family (Table 1).Tukey pairwise comparisons with the overall probability adjusted for multiple comparisons revealed that the amount of damage at BD1 exceeded that at BD2 and BD3 (Fig. 1). However, the largest mean difference between any two assessments (BD1–BD3) was only 0.26. For those plants that survived the entire year, BD1 was not significantly correlated with BD2 (r = 0.073; P = 0.07); BD2 was positively correlated with BD3 (r = 0.09; P = 0.019); and BD1 was positively correlated with BD3 (r = 0.117; P = 0.003). Although positive, all three correlations among our estimates of beetle damage were relatively weak in terms of the proportion of variation they explained.
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Although BD1 and BD2 independently had no significant effects on either total reproductive performance or reproductive performance over the next month, they did have a strong effect on the probability of survival over the next month (Fig. 3). Analysis with a t test revealed that the plants that died during the month following a beetle damage assessment had significantly more beetle damage than those that survived for the next month. A few plants that survived transplantation were so heavily damaged by striped (mostly) cucumber beetles that they died prior to the first BD measurement, and only four plants after this time were also so heavily damaged that they died. The remaining plants died of bacterial wilt disease caused by Erwinia tracheiphila.
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This study quantified leaf herbivory by the primary herbivores (Acalymma vittatum and Diabrotica undecipunctata howardii) of C. pepo subsp. texana over the lifetime of inbred (F = 0) and outbred (F = 0.5) plants and determined the impact of inbreeding and the time of damage (from seedling to late reproductive stages of development) by these beetles on overall fitness. The leaves and other organs of Cucurbita species are chemically protected with cucurbitacins that deter most herbivores but stimulate feeding by cucumber beetles (e.g., Metcalf et al., 1980). The cucumber beetles sequester and chemically modify the cucurbitacins and use them for their own protection, while the males also transfer some of the modified cucurbitacins to the females in their seminal fluid, which is used to chemically protect the eggs (Ferguson et al., 1985; Nishida and Fukami, 1990; Tallamy et al., 1998). The only other leaf herbivores that we have observed on wild gourds (over nearly two decades of research) are aphids (several species) and squash bugs (Anasa nistis). Although squash bugs are not uncommon on some bush varieties of cultivated squash growing in other fields at Pennsylvania State University Agriculture Experiment Station, we rarely observe them on the wild gourd. Consequently, this study examined natural herbivory, which is known to have larger impacts on fitness than simulated herbivory in most species (e.g., Baldwin, 1988), and this study was relatively free of the complications caused by multiple herbivores that inflict different types and patterns of damage and thereby obscure the effects of the timing of herbivory on fitness. Although this study occurred outside the native range of this wild gourd (C. pepo subsp. texana), the entire plant–herbivore–disease pathosystem is present because of the long-time cultivation of squash (C. pepo subsp. pepo) and other cucurbits (cucumbers, melons, watermelons) in the region of our study.
Our findings showed that the amount of total beetle damage significantly depressed total pistillate flower and fruit production for those plants that survived at least until the mid-August measurement of beetle damage. This finding is not surprising given that we also found that our first (mid-June) and second (mid-July) measurements of beetle damage were positively correlated with our third (mid-August) measurement. Consequently, the differences among plants in the total amount of damage increased over time. Moreover, our second and third assessments of beetle damage underestimated beetle damage because beetle damage continued to accumulate on the older leaves and we only measured damage on the new growth that occurred since the previous measurement. Because the cucurbitacin content of Cucurbita leaves has both a constitutive and an herbivore-inducible component (Tallamy and Krischik, 1989) and because cucurbitacins stimulate feeding by cucumber beetles, we suspect that the positive correlations in beetle damage over the growing season were due to initial phenotypic differences in cucurbitacin content among plants that were reinforced by the induction of additional cucurbitacins. It should be noted, however, that any genetically determined resistance trait would result in these correlations.
Our data also showed that there were biologically small but statistically significant differences in the amount of beetle damage to new growth among our three measurements across developmental stages: damage at BD1 exceeded that of BD2 and of BD3 (Fig. 1). Even when we removed those 26 plants that died before the BD2 measurement (Fig. 3), BD1 was still statistically larger than BD2 or BD3 (data not shown). Neither the amount of beetle damage in mid-June nor the amount in mid-July (or even BD1 + BD2) had a significant effect on any of the measures of total reproductive performance (Appendices S1–S3: see Supplemental Data accompanying online version of this article) or reproductive performance over the next month (Appendices S2–S4: see Supplemental Data accompanying online version of this article). These findings indicate that wild gourds are tolerant (unaffected reproductively by the resource loss or able to compensate for the loss) of low to moderate levels of herbivory for most of the growing season. These findings are also consistent with previous studies showing that the pattern of simulated herbivory (dispersed vs. concentrated) has little effect on the growth or reproduction of the wild gourd when 5–15% of the leaf area is removed (Avila-Sakar et al., 2003; Avila-Sakar and Stephenson, 2006). In contrast, we found that the amount of beetle damage that accumulated between mid-July and mid-August (BD3) significantly affected reproduction through both the male and female functions (Table 4). It is during this time period (mid-July until late August) that most of the total reproduction occurred on these plants and that resource expenditures on pollen and developing fruits/seeds were the greatest (Avila-Sakar et al. 2001). Given that BD1 + BD2 (damage prior to mid-July) did not affect these measures of reproductive output, it appears that the timing and intensity of the additional damage with respect to the ongoing developmental processes (pollen and fruit/seed maturation) is very important to the overall fitness of the plant. Consequently, estimates of tolerance of these plants to damage by cucumber beetles in wild gourds depended strongly on when the damage was assessed.
Curiously, total beetle damage (BDT) affected only the female function (pistillate flower and fruit production) of plants (Table 5), while both the male and the female functions were affected by BD3. Because most studies of the impact of total herbivory on fitness measure only fruit/seed production (while hermaphroditic plants achieve half of their fitness, on average, through the male function), differences in the impact of herbivory on the male and female functions can result in misleading estimates of the impact of herbivory on fitness (tolerance).
The few published studies of ontogenetic changes in tolerance all demonstrate that the impact of herbivory on fitness changes across developmental stages (see Boege and Marquis, 2005). Consequently, selective pressures for plant defense traits (i.e., traits that promote resistance and tolerance) are also expected to vary across developmental stages (Tiffin, 2002; Boege and Marquis, 2005). While ontogenetic changes in tolerance have been demonstrated, the pattern of these changes in tolerance is less clear. For example, del-Val and Crawley (2005) examined several grassland species and found that the intermediate stages of development are least tolerant to artificial clipping. On the other hand, studies of grassland populations of Primula veris found that plants were least tolerant to grazing in the spring but that later grazing increased the probability of flowering and seed set (García and Ehrlén, 2002; Brys et al., 2004). In Ipomoea purpurea, herbivore damage early or late in the growing season had a greater impact on fitness than damage in midseason. Interestingly, Thomson et al. (2004a, b) found that a cultivated cucumber (Cucumis sativus cv. Lebanese) can compensate for early season damage by a snail, but damage during heavy reproduction reduces reproductive output.
Although we found that the intensity of beetle damage until mid-July had little effect on reproductive output, our data show that the amount of beetle damage did affect the probability of survival. The plants that died between mid-June and mid-July had significantly higher beetle damage to their leaves in mid-June than those that survived until mid-July. From mid-July until mid-August, 14% of the plants died over the three field seasons, and these plants had significantly greater beetle damage in mid-July than the plants that survived until mid-August (Fig. 3). Nearly all of the plants that died over our three field seasons had contracted bacterial wilt disease. Because cucumber beetles vector the causative agent of wilt disease, Erwinia tracheiphila, and the bacteria (present in the fecal pellets of the beetles) enter the plants via wounds caused by beetle feeding (Yao et al., 1996; Fleischer et al., 1999), it is reasonable to assume that exposure to this deadly pathogen increased with beetle damage to the leaves. Although beetle damage during the seedling, prereproductive, and early reproductive stages of wild gourd development had no significant direct effects on fitness, these findings indicate that beetle damage had profound indirect effects on fitness via the transmission of bacterial wilt disease. In contrast, the amount of beetle damage in mid-August had both a large impact on flower and fruit production, and those plants that died from mid-August to mid-September also had significantly greater beetle damage in mid-August than those that survived. These findings suggest that although the plants were tolerant of the levels of beetle herbivory that we observed over three years during the early and middle stages of development, there could still be strong selection for resistance to reduce exposure to Erwinia tracheiphila. Because herbivores vector pathogens and/or leave wounds where pathogens may enter a plant, resistance and tolerance to herbivory should not be considered alternative defensive systems (Fornoni et al., 2004).
Our findings also showed that inbred plants experienced higher levels of herbivory than outbred plants and that the effects of inbreeding on beetle damage varied by maternal family. These findings indicate that there was genetic variation for inbreeding depression among the families used in our study (Table 2). Moreover, inbreeding significantly reduced reproductive output throughout the life of the plant (Table 3). These results are consistent with previous studies of the effects of inbreeding on resistance to herbivores and pathogens in a variety of species; these studies found that resistance to herbivores and pathogens changes with inbreeding and that the changes occur in a family/population-specific manner (e.g., Strauss and Karban, 1994; Ouborg et al., 2000; Carr and Eubanks, 2002; Carr et al., 2003; Hayes et al. 2004; Stephenson et al., 2004; Hull-Sanders and Eubanks, 2005). Inbreeding increases homozygosity and thereby exposes deleterious recessives to selection (e.g., Charlesworth and Charlesworth, 1987; Husband and Schemske, 1996). Consequently, inbreeding could directly affect any of the myriad genes involved in plant defense pathways and indirectly affect resistance by reducing the resources available for defense pathways and by slowing the rate of growth during vulnerable stages of the life cycle.
Because both inbreeding and herbivory reduce plant vigor and the resources potentially available for reproduction (e.g., Marquis, 1992; Husband and Schemske, 1996), we expected strong interactions between inbreeding and beetle damage on reproductive performance, especially late in the growing season when the resource demand for flower, fruit, and seed production is the greatest (mid-July to mid-August). However, we found no significant interactions between BD3 (or BDT) and breeding for any of our measures of reproductive performance (Tables 4 and 5). These findings suggest that the direct effects of inbreeding and beetle damage were independent. There were, however, indirect effects of inbreeding on the amount of beetle damage (Table 1), which, in turn, affected reproductive performance (Tables 4 and 5).
Finally, our study (which used only five families per year) found that the amount of beetle damage among families differed significantly over the growing season (time by maternal family interaction; Table 1). This finding indicates that there was additive genetic variation for resistance that varied across developmental stages (i.e., there were genetically based differences in resistance among families that varied by developmental stage). Curiously, however, there were no significant main effects of maternal family on the total amount of beetle damage, indicating that there was no broad sense heritability for overall resistance to cucumber beetles among families (Table 1). These findings suggest that studies focusing on herbivory during a single developmental stage or on total herbivory across developmental stages can miss potentially important genetic variation for resistance.
Our study also revealed significant interactions among families and beetle damage during peak reproduction (the BD3 estimate) (Table 4) for pollen tube growth rate, female flower production, and fruit production. These findings indicate that there was broad sense heritability for tolerance to beetle damage across developmental stages, even with our limited sample of families (i.e., there were genetically based differences in tolerance among families that varied by developmental stage). Because there is additive genetic variation for resistance and tolerance to cucumber beetles across developmental stages in this wild gourd, natural selection can potentially operate on this variability in plant defenses. Future studies should examine the chemical ecology underlying these differences (e.g., genetic differences in constitutive and inducible cucurbitacin content of leaves) and the evolutionary consequences of these differences in resistance and tolerance across environments.
FOOTNOTES
1 The authors thank T. Kinney, J. Thaller, N. Myers, M. Sasuclark, G. Stephenson, and B. Miller for field and greenhouse assistance; R. Oberheim and his staff for use of the Pennsylvania State University Agriculture Experiment Station at Rock Springs, PA (Horticulture Farms); and A. Omeis for use of the Biology/Buckhout Greenhouse. This research was supported by NSF grant DEB02-35217 to A.G.S. and J.A.W. and the research of M.S. and A.D. was supported by REU supplements to NSF grant DEB02-35217. All experiments conducted in this study comply with U.S. Federal Law and the Guidelines of the Pennsylvania State University. 
6 Author for correspondence (e-mail: as4{at}psu.edu), phone 1-814-863-1553
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of P < 0.05 adjusted for multiple comparisons.
