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Reproductive Biology |
Department of Biological Sciences, Indiana University South Bend, South Bend, Indiana 46634 USA
Received for publication July 7, 2005. Accepted for publication December 19, 2005.
ABSTRACT
Identifying ecological factors that affect seed number and seed size is key to understanding the persistence of large seed mass variation in some plant species. Pathogens may increase seed mass variation by increasing resource demand over the growing season such that late fruits experience higher resource competition than early fruits. We tested whether Fusarium sp. and Rhizoctonia sp., soil fungi that cause wilt, contributed to seasonal decline in flower size, seed number, or seed mass in Hydrophyllum appendiculatum and H. canadense. A third species not infected by these soil fungi, H. virginianum, was studied to determine how seasonal decline in floral traits and seed mass variation varies within this genus. Flower size declined seasonally for all species, but was greatest for H. appendiculatum, a monocarpic biennial with indeterminate inflorescences. Seed number decreased between first and last inflorescences in H. appendiculatum, but not in H. canadense or H. virginianum, perennials with determinate inflorescences. Seed mass varied most in H. appendiculatum and H. canadense (4–20-fold in 50% of individuals) and least in H. virginianum (4–8-fold in >30% of individuals). Fungal infection increased seed mass variation among diseased plants in H. canadense and H. appendiculatum. However, within plants fungal infection only increased seasonal decline in flower size, seed number, and seed mass in H. appendiculatum when flowers received supplemental pollination.
Key Words: fungal pathogens • Fusarium • Hydrophyllum • life history • offspring size • Rhizoctonia • seed mass • seed number
Variation in seed size within species is commonly observed in wild populations (Mazer, 1987
; Winn, 1988
; Thompson and Pellmyr, 1989
; Wolfe, 1995
; Alonso-Blanco et al., 1999
; Kliber and Eckert, 2004
; Halpern, 2005
). The persistence of this variation is intriguing because both empirical and theoretical studies predict that within species, seed size should vary less than other reproductive traits due to finite resources and trade-offs in seed size and seed number. Models of optimum offspring size show that parental fitness is maximized by investing equally in all offspring such that an organism should respond to variable resources by varying offspring number not seed size (Smith and Fretwell, 1974
; McGinley et al., 1987
; Haig and Westoby, 1988
). Results from studies of some plant species follow this prediction to the extreme. For example, wheat grown over an 816-fold range of densities varied 833-fold in seed number, but only 1.04-fold in mean mass per grain (Harper et al., 1970
). Larger seeds typically germinate sooner, have a higher probability of germinating, and yield larger seedlings that have a higher probability of surviving, which selects against producing small seeds (Schaal, 1980
; Dolan, 1984
; Ouborg and Van Treuren, 1995
; Turnbull et al., 1999
; Susko and Lovett-Doust, 2000
; Moles and Westoby, 2004
). Yet in some plant species seed mass varied up to 10-fold, even though larger seeds often had higher fitness in these species as well (Mazer, 1987
; Wolfe, 1995
; Vaughton and Ramsey,
1998; Alonso-Blanco et al., 1999
; Halpern, 2005
).
Seed number and seed size production patterns can be more complex than these models suggest due to changes in resources over the season and physiological and developmental constraints that limit optimal floral resource allocation (Diggle, 1997
, 2002
; Sakai and Harada, 2001
). In addition, phylogenetic constraints and life history traits, such as number of times a plant reproduces and duration of flowering, can also affect seed number, seed size, and variance in seed mass. Theoretical and empirical studies show that annuals put more energy into reproduction than perennials and tend to produce larger seeds and a higher number of seeds per reproductive bout compared to perennials (Young, 1990
; Roff, 2002
). Seed mass variation may be higher in monocarpic species due to relatively stronger selection on maximizing seed number in a single reproductive bout compared to seed size. Within a season, species that produce flowers over a longer period of time may experience a greater range of environmental conditions, which can contribute to seed mass variation within individuals (Cavers and Steel, 1984
). Comparative studies have demonstrated significant effects of phylogeny and life history on seed mass and seed mass variance (Mazer, 1989
; Hodkinson et al., 1998
; Moles et al., 2005
). For example, a study of 507 genera of Indiana dune angiosperms showed that plant family, life history, and duration of flowering contributed 13%, 13%, and 1%, respectively, to seed mass variance (Mazer, 1989
). Each of these factors was significant in explaining seed mass variance, but together only explained a small part of the seed size variation observed in this study. Within species, most seed size variation occurs within plants, rather than among plants (Michaels et al., 1988
; Wolfe, 1995
; Vaughton and Ramsey,
1998; Halpern, 2005
). In addition, some species have low additive genetic variation for seed mass (Wolfe 1995
). Together, this indicates that environmental factors during flower or fruit development, rather than genetic differences between plants, are primarily responsible for phenotypic variation in seed size within plants, although we are still in the early stages of identifying which ecological factors contribute to seed mass variation (Wolfe, 1995
; Paz et al., 1999
; Halpern, 2005
).
One phenomenon that increases seed mass variation within individuals is decline in size of reproductive structures over the flowering season. Abiotic resources and developmental constraints have been investigated most often for their role in declining size of reproductive structures over time (Cavers and Steel, 1984
; Wolfe, 1992
; Diggle, 1997
; Ashman and Hitchens, 2000
; Kliber and Eckert, 2004
). Damage by herbivores and pathogens also changes over time, which could contribute to the temporal decline in flower size, seed size, and seed number (Thompson and Pellmyr, 1989
; Krannitz, 1997
; Paz et al., 1999
; Stephenson et al., 2004
). Therefore, extrinsic factors (resources and species interactions) can dampen or accentuate intrinsic factors (life history, genetic, and developmental constraints) that determine the final distribution of phenotypes among the offspring.
Hydrophyllum appendiculatum L. is a common understory herb in deciduous forests throughout the midwestern United States (Gleason and Cronquist, 1991
). Previous studies of H. appendiculatum have shown 10-fold variation in seed mass (Wolfe, 1993a
, 1995
). Although reports of seed mass variation in wild populations are not unusual, the extreme variation observed in H. appendiculatum is unusual; up to four-fold variation in seed size within populations is more common (Michaels et al., 1988
). Studies of this species also showed that larger seeds had higher fitness as they resulted in larger seedlings with higher survival (Wolfe, 1993a
, 1995
). Much of this seed mass variation was attributed to differences between self and outcross pollination, amount of light received by the maternal plant, and time of season in which seeds were produced (Wolfe, 1992
, 1993a
, 1995
). Of these factors, decline in flower size over the flowering season contributed the most to seed mass variation within individuals (Wolfe, 1995
). Hydrophyllum appendiculatum is infected by a soilborne fungal pathogen that causes stems to wilt; the proportion of infected individuals has been reported to be as high 90% in Illinois populations (Wolfe, 1990
) and between 20 and 50% of the population is commonly infected in northern Indiana populations (D. L. Marr, unpublished data). Despite the commonness of this disease, little is known about the pathogen or its effect on reproduction in species of Hydrophyllum. We were interested in whether other species of Hydrophyllum also showed high seed mass variation and whether wilt disease might contribute to this variability. Specifically, we compared seed mass variation in three species (H. appendiculatum, H. canadense L., H. virginianum L.). These understory herbs differ in life history and duration of flowering. Both H. virginianum and H. canadense are perennials, and individuals flower for 2–3 wk. Hydrophyllum appendiculatum is the only monocarpic species in the genus (usually a biennial), and individuals flower for 4–6 wk. We also tested whether fungi that cause wilt affected temporal decline in flower size, seed number, or seed size variation in H. appendiculatum and H. canadense.
MATERIALS AND METHODS
Life history and floral traits of study species
The genus Hydrophyllum L. (Hydrophyllaceae) includes eight species, four species in eastern and four species in western North America (Beckmann, 1979
; Ferguson, 1999
). Three eastern species, H. appendiculatum, H. canadense, and H. virginianum, co-occur in maple and beech forests in northern Indiana. Hydrophyllum appendiculatum and H. canadense are sister species, whereas the sister species of H. virginianum appears to be the western species Hydrophyllum tenuipes (Constance, 1942
; Beckmann, 1979
; Ferguson, 1999
). Flowering phenology for these species is sequential, with some overlap: H. virginianum from early May to June, H. appendiculatum from mid-May to early July, and H. canadense from June to July (Table 1).
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). However, H. canadense only blooms after the canopy is fully developed; thus its first and last inflorescences bloom under more uniform light conditions. This species also grows clonally via rhizomes. All three species are self-compatible, but selfing is most common in H. appendiculatum (Beckmann, 1979
; Wolfe, 1993a
).
The number of compact cymose inflorescences is determinate in H. virginianum and H. canadense. Hydrophyllum virginianum usually has two major inflorescences that extend above the foliage with 10–20 flowers per inflorescence. Flowers are white to pale purple and protandrous with stamens extending above the petals (Gleason and Cronquist, 1991
). Each flower contains up to four ovules and seeds mature in 4–5 wk (D. L. Marr, unpublished data). Plants die back in July. Inflorescences of H. canadense are also composed of compact cymes, but hang below the leaves; the maple-like leaves of H. canadense form a canopy similar to Podophyllum peltatum. Typically there are two major inflorescences with up to 20 flowers per inflorescence. Flowers are white, protandrous, and stamens extend above the petals. Each flower contains four ovules, that usually yield two seeds. Seeds mature in 3 mo, dehiscing in late September or early October. Aboveground foliage dies back after frost in late October and November.
In H. appendiculatum, the number of cymose inflorescences is indeterminate, ranging from 3 to 15 or more inflorescences. Cymes are less compact and extend above the foliage, and each inflorescence contains 10–20 lavender flowers. Flowers are protandrous, but unlike the other two species, stamens are not exserted. Individual flowers last 2–3 d (Wolfe, 1993b
), and inflorescences last approximately 7–10 d. Outermost inflorescences open first, and later inflorescences develop at basal nodes on central stems of the plants. Each flower contains one ovule, and seeds mature in 4–5 wk and then dehisce from capsular fruit. Flowering plants die in July.
Bumble bees and honey bees are the primary floral visitors, but flowers are also visited by Andrenidae and Halictidae bees (LaBerge, 1977
; Beckmann, 1979
; Wolfe, 1993b
; D. L. Marr, personal observation). Weevils lay eggs in flowers of all three species, but the number of seeds eaten by weevils was relatively small (<10% of all seeds) in the years of this study (D. L. Marr, personal observation).
Study sites
This study was done in two county parks: St. Patrick's County Park and Bendix Woods, located in St. Joseph County, Indiana, USA. The two parks were located 30 km from each other. St. Patrick's County Park (SPCP) is a secondary, maple-dominated woods, whereas Bendix Woods (BW) is a mix of secondary and old-growth maple-beech dominated woods. All three species of Hydrophyllum were present at BW, but only H. virginianum and H. appendiculatum were present at SPCP.
Observations of wilt and descriptions of fungi
Each species was monitored for signs of wilt for 4 yr (2002–2005). Hydrophyllum virginianum was monitored for signs of wilt at the two sites, SPCP and BW; 30–50 individuals were randomly chosen each year within approximately a 100-m2 area at each site. For H. appendiculatum, 20 individuals were marked in each of four plots at SPCP and two plots at BW for a total of 120 plants; each plot was approximately 20 m2. Fifty individuals of Hydrophyllum canadense were monitored at BW in two areas that were approximately 20 m2 each. The number of wilted stems, leaves, and inflorescences was recorded twice per week (approximately every 3–4 d) throughout the flowering period for H. appendiculatum and H. canadense. Differences in plot size reflect differences in plant distribution.
Wolfe (1990)
reported wilting symptoms in H. appendiculatum, but the causal agent has not been previously described. Fusarium sp., Rhizoctonia sp., and Pythium sp. were most commonly isolated from root, stem, and leaf tissue in both H. appendiculatum and H. canadense (D. L. Marr and A. Truex, unpublished data). Comparison of DNA sequences in GenBank of the internal transcribed spacer rDNA region (ITS1-5.8s-ITS2) with known pathogenic and nonpathogenic fungi suggested that Fusarium (possibly F. oxysporum) and a binucleate, undescribed species of Rhizoctonia were the most likely species causing wilt in H. appendiculatum and H. canadense (G. Abad, North Carolina State University, personal communication). The species of Pythium appears to be a secondary infection (G. Abad, personal communication). Both Fusarium and Rhizoctonia are soil pathogens that commonly infect roots (Agrios, 1997
). Conidial spores are transported in vascular tissue and can block vascular tissue when spores reach high numbers. The frequency with which both Fusarium and Rhizoctonia are present in diseased plants is unknown. It is also unknown whether disease symptoms can be caused by a single fungal species.
Does the pattern of temporal decline in flower size, seed number, or seed mass differ between healthy and diseased H. appendiculatum?
We tested whether the pattern of decline in floral size, seed number, or seed mass differed between healthy and diseased plants. In 2003, 20 plants were marked in each of four plots located at the two sites; plots were 10 x 20 m. Within plots, plants were haphazardly chosen while they were in the bud stage so that all areas of the plot were sampled. A total of 80 plants from the four plots were followed from onset of flowering through seed maturation. Disease status (number of wilted stems, leaves, and inflorescences) was recorded once per week, and each inflorescence was marked with a uniquely colored wire when it began to flower. One pistillate flower with dehisced anthers was collected from each inflorescence on each plant every 5–7 d and placed in an Eppendorf tube with FAA fixative (10 ml 40% formaldehyde, 5 ml acetic acid, 50 ml 95% ethyl alcohol and 35 ml dH2O) (Kearns and Inouye, 1993
). Petal length, filament length, and ovary width were measured with digital calipers to the nearest 0.01 mm. One petal was removed per flower, flattened, and measured from base to tip. Filament length was measured from base of filament to the base of the anther. Dehisced anthers fall off the filament when placed in liquid; thus we found it more reliable to measure filament length rather than stamen length. Ovary width was measured at the midpoint of their length. Ovaries were cylindrical and tapered at either end; thus measurement error was reduced by measuring the midpoint.
The effect of wilt on seed number and seed mass was also measured in these 80 plants. To ensure that differences in pollinator activity did not affect differences in seed set across sites, flowers were hand-pollinated every 3–4 d in 2003. Individual flowers last 2–3 d, so most flowers were pollinated at least once. Pollen was collected from two different plants located at least 10 m away, although the distance of pollen donor did not affect seed size in another population of H. appendiculatum (Wolfe, 1995
). Pollinating all flowers maximized demand on resources for fruiting, which has been shown to increase the decline in seed mass in H. appendiculatum (Wolfe, 1995
). Developing seeds were bagged with bridal veil netting to prevent loss of seeds due to dehiscence. All seeds were collected when ripe in late July. The number of seeds was counted for each inflorescence, and the colored wires were used to note whether the inflorescence had flowered relatively early or late. Each seed was weighed to the nearest 0.1 mg using a Mettler (Greifensee, Switzerland) Toledo AG104 balance. Seeds consumed by weevils or aborted in development were not weighed.
Temporal decline in flower size and differences between wilted and healthy plants were analyzed using a repeated-measures ANOVA. Time (early and late) was entered as a within-subjects factor and disease status as a between-subjects factor. Early was defined as inflorescences that began flowering in the first week and late was defined as inflorescences that began flowering in the last week. Grouping plants into only two categories of healthy and wilted obscured differences among plants with low and high levels of wilt. Therefore we grouped plants into one of three categories: healthy (0% wilted inflorescences), low wilt (5–49% wilted inflorescences), and moderate to high wilt (50–100% wilted inflorescences). More finely described categories (0%, 5–25%, 25–50%, 50–75%, and 75–100% wilt) produced similar results as the three categories, but sample sizes were smaller and unequal. No difference was found between plots or sites, so results from all 80 plants were combined in the analyses presented. The assumption of normality and homogeneity of variances was met for each variable (petal length, filament length, and ovary width). Box's test indicated that data met the assumption of equal covariance matrices (Zar, 1996
).
A repeated-measures ANOVA was used to compare the number and mass of seeds produced by early- and late-blooming inflorescences in healthy and diseased plants. Seed mass met assumptions of homogeneity of variances. Seed number had unequal variances for the early inflorescences, equal variances for late inflorescences, and equal covariance matrices based on Box's test. A ln transformation caused seed number variances to be equal for early inflorescences and unequal for late inflorescences. Results of both nontransformed and transformed data were similar, so the nontransformed results are presented here. A univariate ANOVA was used to test differences in total seed number and mean seed size in healthy and wilted plants. The more finely described categories of wilt were used in this analysis to get a better idea of how wilt affected seed production at the plant level. Seed number and seed mass were square-root transformed to meet assumptions of equal variances. Scheffé post-hoc comparisons were used to test differences among categories. Significance levels were set at 0.01 to account for differences in sample size among groups.
Patterns of flower size decline and seed mass variation in three species of Hydrophyllum
To determine whether temporal decline in floral size and high seed mass variation in H. appendiculatum are unique to this species, flower size and seed mass variation were measured in H. virginianum (HV), H. canadense (HC), and H. appendiculatum (HA) in 2004. Due to the number of flowering plants and our avoidance of working in very public areas of the park, we were unable to do the entire study at Bendix Woods. Twenty-five plants of HV were marked in BW and SPCP, 40 plants of HC were marked in Bendix Woods, and 40 plants of HA were marked in SPCP. A previous 2-yr study showed no differences between these sites in flower size, seed size, or seed number when plant size was controlled (D. L. Marr, unpublished data and the 2003 results for HA). Deer herbivory reduced final sample sizes to 49 HV, 40 HC, and 30 HA. Disease status and beginning of flowering for each inflorescence were recorded, and one flower was collected from each inflorescence on each plant every 5–7 d. Petal length, filament length, and ovary width were measured with digital calipers to the nearest 0.01 mm as described.
Flowers were open-pollinated in all three species to determine the relative magnitude of temporal decline in reproductive structures under natural conditions. Developing seeds were bagged with bridal veil netting to prevent loss of seeds due to dehiscence. Seeds were collected when ripe. Seed number was counted for each inflorescence, and each seed was weighed to the nearest 0.1 mg. Floral size, seed mass, and seed number data for H. virginianum were analyzed using a paired t test to compare early and late inflorescences. Floral size, seed mass, and seed number data for H. canadense were analyzed using a repeated-measures ANOVA as described or H. appendiculatum. A Welch ANOVA, assuming unequal variances, was used to compare mean seed mass among the three species. The coefficient of variation (CV) was used to compare seed mass variation among species. Interpretation of the coefficient of variation can be problematic as the mean approaches zero (Lande, 1977
; Sokal and Rohlf, 1995
). Means in our analysis varied from 11 to 25 mg and did not approach zero relative to the standard deviation, suggesting that this problem should have minimal effects on CV in this data set. All analyses were done in SPSS, version 11.0 for Macintosh (SPSS, Chicago, Illinois, USA).
RESULTS
Description of wilting symptoms
Wilting symptoms were only observed in H. appendiculatum and H. canadense (Table 1). In H. appendiculatum, stems, leaves, and inflorescences bent or collapsed and eventually turned brown with dark necrotic spots on the stem and leaves. Symptoms appeared 1–2 wk after flowering began in a population. Flowers on wilted stems continued to open and produce seed. Wilted plants were present each year from 2001 to 2005, but wilt was rare in 2002 when few H. appendiculatum flowered, most likely due to cold temperatures in May. In H. appendiculatum, wilt occurred primarily in flowering plants and was rare in vegetative plants.
In H. canadense, leaves and buds were healthy in April and May, but wilting symptoms appeared with the beginning of flowering in early June in each year of the study (2002–2005). Both vegetative and flowering genets showed signs of wilt; vegetative plants produce one stem with one leaf, whereas flowering stems arise from the junction of two principal leaves. In flowering genets, typically one leaf was wilted and one appeared healthy. The wilted leaf bent sharply at the junction between the blade and petiole and the leaf eventually turned brown to black. Unlike H. appendiculatum, stems remained upright in wilted plants, but often had dark black spots along the stem. Inflorescences adjacent to wilted leaves typically shriveled and turned black, although some flowers continued to produce seed. Throughout the remainder of this paper, wilted or diseased plants refer to individuals that showed wilt symptoms as described.
Temporal decline in flower size, seed number, and seed mass in healthy and diseased H. appendiculatum
There was a significant decline in petal length, filament length, and ovary width between inflorescences produced in the first and the last week of flowering in both 2003 and 2004 (Table 2, Fig. 1). The effect of wilt on declining flower size was greater in 2003 than in 2004. In 2003, plants received supplemental pollination, and wilted plants had a greater decline than healthy plants in petal length and filament length (Fig. 1), as indicated by the significant interaction between time (within subjects) and wilt status of the maternal parent (between subjects) (Table 2). However, differences between subjects due to wilt were only significant for filament length, which declined 30% in wilted plants and 15% in healthy plants. In 2004, the direction and magnitude of the decline in size of filaments and ovaries was similar between flowers from healthy and wilted plants, and there was no significant difference between subjects for effect of wilt on flower size. Only petal length had a significant interaction between flowering time and wilt status (Table 2). Similar to 2003, the decline in petal length was greater in wilted plants compared to healthy plants. In both years, early flowers were very similar in size regardless of disease status, but larger differences between health and diseased plants were found in size of late-blooming flowers (Fig. 1).
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In H. canadense, timing of flowering did not have a significant effect on either seed mass or seed number. However, wilted plants produced smaller seeds both early and late in the season and nearly five times fewer seeds compared to healthy plants (Table 3, Fig. 2). In H. virginianum, seeds were on average 1.5 times larger in early inflorescences compared to late inflorescences, but there was no difference in number of seeds produced early vs. late (Table 3, Fig. 2).
Mean seed mass differed among species (Table 4; Welch ANOVA: F2,54.8 = 14.2, P < 0.0001); H. virginianum seeds were significantly smaller than H. appendiculatum and H. canadense seeds (Scheffé post-hoc comparison: HV < HA, P = 0.0001; HV < HC, P = 0.03; HC = HA, P = 0.08). Seed mass variation was highest in H. appendiculatum with 24% of the individuals producing seeds that varied 7–20 times in mass, and the proportion of individuals producing highly variable seeds was more consistent across years (Table 5, Fig. 4). Seed mass variation was also high in H. canadense in which individuals produced seeds that varied 7–16 times in mass. Hydrophyllum virginianum produced seeds that varied the least in mass. Wilted plants produced seeds that varied more in seed mass in both H. appendiculatum and H. canadense (Table 4). The coefficient of variation was ~15% higher in wilted H. appendiculatum in both years and 30% higher in H. canadense.
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There are three major results of this study. First, there were significant temporal declines in petal length and ovary width in all three species of Hydrophyllum. Temporal decline of filament length was not consistent across species. A temporal decline in seed number per inflorescence was only found for H. appendiculatum, and seed mass declined from first to last inflorescence in two of the three species, H. appendiculatum and H. virginianum. Of the three species, H. appendiculatum had the largest temporal decline in all reproductive structures that were measured. Temporal decline in flower size and seed production is not unusual and has been well documented in several plant species (Diggle, 1997
; Ashman and Hitchens, 2000
; Mazer and Dawson, 2001
; Williams and Conner, 2001
; Kliber and Eckert, 2004
; Halpern, 2005
). Second, variation in seed mass was greater in plants with wilt disease in H. appendiculatum and H. canadense. Third, the effect of wilt on temporal decline in flower size, seed number, and seed size differed between H. appendiculatum and H. canadense. No wilt was observed in H. virginianum, and this species showed the least plasticity in flower size and seed production over the flowering season. The latter two results are novel because few studies have identified biotic ecological factors that contribute to seed mass variation or seasonal decline either within or between species (Thompson and Pellmyr, 1989
; Krannitz, 1997
; Halpern, 2005
). Below, we discuss how fungal pathogens, pollination, and life history contribute to the patterns of flower and seed production in Hydrophyllum.
Pathogens can greatly affect resource levels and plant development, altering the parent's ability to provision offspring and ultimately offspring phenotype (Burdon, 1987
; Levri and Real, 1998
; Stephenson et al., 2004
). We found that two of three species of Hydrophyllum were commonly infected by Fusarium sp. and Rhizoctonia sp. that cause wilt. Resource levels in plants with wilt can be lowered due to decreased photosynthetic ability because leaves lose chlorophyll shortly after initial symptoms appear. In addition, the collapsed leaves and stems probably limit transport of water and possibly nutrients. Therefore, we were interested in whether wilt disease affected offspring number or seed mass beyond existing developmental constraints on offspring size.
In H. appendiculatum, flower size tended to have a greater decline in wilted plants, and seed production continued even under remarkable levels of resource stress with up to 70% of the inflorescences affected by wilt. Only plants with more than 70% wilted inflorescences showed significant decreases in seed number and seed mass (Fig. 3). A consequence of continuing seed production in infected plants was increased variation in seed mass; seed mass variation was highest in plants with moderate levels of wilt and high levels of pollination. Pollination treatments differed between years, thus it is possible that a factor other than pollination contributed to the difference in the rate of decline in healthy and wilted plants between years. However, our results are similar to Wolfe's (1992), who reported greater changes in flower size and seed production in healthy plants when supplemental pollen was added, compared to natural pollination levels. In our study, supplemental pollination accentuated differences in seed production between healthy and wilted plants. Plants with low to moderate amounts of wilt responded like healthy plants by increasing seed production when pollinated, but plants with high amounts of wilt produced significantly fewer seeds when pollinated. Under natural pollination conditions, the number of seeds was similar between healthy and wilted plants, but healthy plants produced heavier seeds.
Modifying seed mass rather than seed number in H. appendiculatum is contrary to other studies in which the environment of the maternal plant primarily affected number of offspring, but had little effect on quality of offspring (Sultan, 1996
; Weiner et al., 1997
). However, several studies have found that some maternal environments increased variation in offspring size. For example, seed size variation in Purshia tridentata increased with proportion of twigs browsed (Krannitz, 1997
). In Lupinus perennis, within-plant seed size variation increased under conditions of high plant density and clipping that simulated effects of herbivory (Halpern, 2005
). In Cucurbita pepo subsp. texana, foliar herbivory and viral infections had a greater effect on seed number than seed mass, but seed quality was lower in maternal plants exposed to both herbivory and pathogens (Stephenson et al., 2004
). Seeds produced by these plants had a lower chance of germinating, that was attributed to poor provisioning by the maternal plant, not differences in seed size (Stephenson et al., 2004
).
In contrast to the effects of wilt on reproduction in H. appendiculatum, in H. canadense temporal decline in flower size did not differ significantly between healthy and diseased plants, and seed number was greatly reduced in all diseased plants. The ability of wilted plants to increase seed production in response to supplemental pollination was unique to H. appendiculatum. Hand-pollination of diseased plants in H. canadense did not increase seed production; in fact, seed set was near zero because most flowers aborted (D. L. Marr, unpublished data). This suggests that low seed production in diseased H. canadense was not due to decreased visitation by pollinators between wilted and healthy inflorescences. Seed mass variation was greater in wilted plants, but the primary effect was to decrease seed number. The difference in response could reflect variation in the fungal strains that infected the two species, resulting in greater virulence of the fungi in H. canadense compared to H. appendiculatum. Alternatively, H. canadense may be more sensitive to resource stress or able to more finely regulate seed production through selective abscission than H. appendiculatum. We cannot test these hypotheses with the current set of data. The three plant species are sympatric; thus fungi present in the soil could potentially infect all three species. Both Fusarium and Rhizoctonia have been isolated from H. appendiculatum and H. canadense, but these species may differ in their susceptibility to each fungus. More work is needed to understand how the physiology of the plants is specifically altered when infected by each fungus. Interestingly, H. virginianum appears to be resistant to infection by these fungi.
We found differences in the timing of the appearance of wilt symptoms relative to the start of flowering, which could explain some of the differences in how wilt affected each species. Wilt symptoms occurred 1–2 wk after flowering began in H. appendiculatum, whereas wilt symptoms coincided with the beginning of flowering in H. canadense. This pattern was consistent over 4 yr of observation. Flowering in H. appendiculatum begins several weeks before H. canadense, thus an environmental cue, such as warm soil temperature, may be necessary before fungal spores can germinate and infect plants. Regardless of the cause, the delayed onset of wilt in H. appendiculatum increased resource differences between early and late inflorescences. Some plant species have a narrow window in which they can regulate seed number through selective abscission of young fruits (Stephenson, 1981
; Marr and Pellmyr, 2003
). Therefore, if seed development has already begun in early inflorescences when wilt symptoms appear, the plant may have limited ability to conserve resources through selective abscission.
Some of the differences in seed production among the three species are probably attributable to life history traits. Differences in duration of flowering and positional effects among species may contribute to differences in reproductive plasticity. Hydrophyllum appendiculatum had the greatest plasticity in flower size, seed number, and seed mass. The flowering period of H. appendiculatum was twice as long compared to the other species; thus early and late inflorescences have a higher probability of experiencing different environments than plants that flower within a short period (Cavers and Steel, 1984
; Diggle, 1997
). There is also a greater chance for positional effects (or architectural effects) in H. appendiculatum due to its indeterminate flowering habit where outermost inflorescences develop first and basal inflorescences develop later (Diggle, 1997
). Both H. canadense and H. virginianum have a determinate number of inflorescences and little variation in the position of inflorescence nodes. Furthermore, unlike the perennial species, the monocarpic H. appendiculatum dies once seeds are released, so there is no trade-off between placing resources into current reproductive effort vs. reserving some resources to maintain growth for future reproduction (Young, 1990
).
If life history were the main factor contributing to seed mass variation, then one would expect seed production patterns to be similar between the perennials H. canadense and H. virginianum. In addition, the two perennial species have similar fruit structure with four ovules per flower, whereas H. appendiculatum only has one ovule per flower. Ovule number per flower could affect seed size variation if resource allocation is unequal among flowers, but equivalent among ovules. However, we found that H. appendiculatum and H. canadense had greater similarity in mean seed size and seed mass variation (Tables 4 and 5). Seed mass variation was greatest in H. appendiculatum (up to 20-fold), followed by H. canadense (up to 16-fold), and H. virginianum (only up to 8-fold). Furthermore, the number of individuals that produced seeds varying at least four-fold in size was greater in H. appendiculatum and H. canadense (50%) compared to H. virginianum (>30%). In addition to wilt susceptibility in H. appendiculatum and H. canadense, it is possible that common ancestry contributed to the similarity in seed production between these sister species (Beckmann, 1979
; Ferguson, 1999
). Other seed traits such as size, shape, and seed coat texture are relatively conserved and even have been used to determine species relationships within the Hydrophyllaceae (Chuang and Constance, 1992
).
In summary, seed mass variation within species is common, but the relative contribution of ecological, architectural, and genetic factors to seed mass variation is still being sorted out (Wolfe, 1995
; Diggle, 1997
; Alonso-Blanco et al., 1999
; Kliber and Eckert, 2004
; Halpern, 2005
). Our results show that each of these factors contributes to seed mass variation in Hydrophyllum. Architectural constraints and life history are contributing to the patterns of temporal decline in seed number and seed mass in all three species. Differences in seed production, particularly between H. appendiculatum and H. canadense, illustrate that responses to resource stress (supplemental pollination in this study) can be highly variable within closely related species. Furthermore, infection by fungal pathogens contributes to the unusually large seed mass variation observed in two species and increases the seasonal decline in flower size and seed production in one species of Hydrophyllum.
FOOTNOTES
1 The authors thank G. Abad at North Carolina State University for her expertise in identifying the fungi infecting plants; E. Kirkwood, Superintendent of St. Joseph County Parks, for permission to use the field sites; M. Holland and G. Politowicz for help with initial surveys of wilt in H. appendiculatum; T. Greenslee and J. Martz for help with studies on pollination and seed production in H. virginianum and H. canadense; and K. Kubalanza and S. Orr for help with flower measurements and seed weighing for the 2004 data; and two anonymous reviewers for helpful comments on an earlier version of this manuscript. This study was supported by a Faculty Research Grant to D.L.M. and an Indiana University–South Bend Summer Research Fellowship to M.L.M. 
2 Author for correspondence (e-mail: dmarr{at}iusb.edu
)
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