American Journal of Botany 95(9): 10721078. 2008.

POLLINATOR AND NONPOLLINATOR SELECTION ON RAY MORPHOLOGY IN LEUCANTHEMUM VULGARE (OXEYE DAISY, ASTERACEAE)1
Stefan Andersson2
Plant Ecology and Systematics, Department of Ecology, Slvegatan 37, S-223 62 Lund, Sweden Despite evidence that both pollinators and nonpollinator agents of selection can shape the evolution of oral characters, there have been few attempts to compare the strengths and directions of selection from pollinators and other agents in the same study system. In this investigation of Leucanthemum vulgare, a self-incompatible composite known for its conspicuous white rays, I obtained data from a ray removal experiment in the eld and from a segregating F2 population in an experimental garden to assess the role of pollinator and nonpollinator selection as stabilizing factors on oral evolution in this species. Removal of all rays reduced the pollination success of heads by 3135%, but did not signicantly affect the level of infestation by larvae of the y Tephritis neesii. Data from F2 plants indicated a potential for indirect selection on ray morphology, mediated through links between ray morphology and measures of vegetative size and plant vigor. The results of this study show that individuals of the normal, rayed phenotype have a clear selective advantage, in terms of both pollinator attraction and general plant vigor. Thus, there were no conicting selection pressures between the pollinators and the other selective agents considered in this study. Key words: Asteraceae; oral evolution; Leucanthemum vulgare; pollination; selection.

Understanding the selective mechanisms underlying the great diversity of oral sizes, shapes and rewards in the angiosperms, remains a primary challenge for plant evolutionary biologists. There is ample evidence that both pollinators and nonpollinator agents of selection can shape the evolution and diversication of oral characters (Grant, 1949; Stebbins, 1974; Faegri and van der Pijl, 1979; Galen, 1999b; Fenster et al., 2004; Strauss and Whittall, 2006). In the case of oral size variables, the patterns of selection are determined by (1) the extent to which large oral organs enhance the plants visual display to oral mutualists and antagonists (Bell, 1985; Brody, 1992; Galen, 1999a), (2) the importance of perianths as protective organs during the bud stage (Delph et al., 1996), (3) the existence of tradeoffs between the allocation of resources to oral advertising vs. fruit or seed production (Charlesworth and Charlesworth, 1987; Andersson, 2005), and (4) the pattern of genetic association between oral and nonoral size characters (Primack, 1987; Andersson, 1993, 1997). Thus, it is necessary to consider a broad variety of selective pressures and tness consequences to understand how pollinators and other ecological forces interact to determine the optimum oral phenotype of a species or population. Despite growing evidence for the importance of both pollinator and nonpollinator selection in oral evolution (Galen, 1999a; Strauss and Whittall, 2006), there have been relatively few attempts to compare the strengths and directions of selection from pollinators and other agents in the same study system (Strauss and Whittall, 2006). In this regard, it seems
1

Manuscript received 6 March 2008; revision accepted 15 July 2008.

The author thanks M. Wirn for collection of plant material, H. Persson and B. Jacobsson for technical assistance in the experimental garden, R. Bygebjerg for identication of the oral herbivore, H. Sheppard for linguistic advice, and C. Fenster and two anonymous reviewers for valuable comments on the manuscript. Financial support was provided by the Swedish Research Council. 2 E-mail: stefan.andersson@ekol.lu.se doi:10.3732/ajb.0800087

particularly relevant to study plants with generalist pollinators, given the inferred role of conicting selection pressures in preventing the evolution of specialization in generalized pollination systems (Fenster et al., 2004). Characters reecting ower morphology often show high phenotypic stability within and among plants of the same species or population (Stebbins, 1974), as evidenced by the frequent use of oral features as key characters of individual species, especially in taxonomic groups where pollination by animals predominates (Grant, 1949). While the relative constancy of owers may be a consequence of unifying selection imposed by local pollinators (Berg, 1960; Stebbins, 1974; Wolfe and Krstolic, 1999), there are other possible causes, for example, intrinsic developmental factors that limit the range of phenotypes that can be expressed in a oral character and negative (purifying) selection arising from the physiologically adverse effects that may be associated with major changes in ower development (Stebbins, 1974; Fenster and Galloway, 1997; Cresswell, 1998). Identifying agents of unifying selection is difcult when all or almost all individuals express the same phenotype. One way to circumvent this problem is to experimentally enhance the variation, either by articial manipulation or the use of segregating progenies derived from crosses between phenotypically distinct genotypes. Data from manipulative experiments afford great condence when inferring the functional (causal) relationship between phenotype and tness, provided that the experimental units can be randomized across treatment categories (MitchellOlds and Shaw, 1987). Segregating populations allow detailed analyses of selection when the expanded variation is more continuous, i.e., when hybrid intermediates bridge the gap between the extreme phenotypes. Although measures of pollination success generally decrease for plants with articially manipulated corollas (e.g., Nilsson, 1988; Sandvik and Totland, 2003; for exceptions, see Wilson, 1995; Herrera, 2001), there is still too little experimental data to draw general conclusions about the relative importance of pollinator and nonpollinator selection in preventing the invasion of aberrant oral morphologies.

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Specialized ray orets (hereafter referred to as rays), a striking feature of many species in the Asteraceae family, play a major role as pollinator attractants (Leppik, 1977; Bertin and Kerwin, 1998), as evidenced by the positive association between pollination success and the possession of rays found in ray removal experiments (e.g., Stuessy et al., 1986) and in populations polymorphic for rayed and discoid (rayless) individuals (Marshall and Abbott, 1982; Abbott and Irwin, 1988). Nevertheless, there have been several parallel reversals to discoid heads in this family (Bremer and Humphries, 1993), and genetic data indicate a considerable potential for selective forces other than pollinators to inuence the evolution of ray morphology, especially in self-fertilizing species (Abbott, 1986; Comes, 1998; Oxford et al., 1996; Abbott et al., 1998). In oxeye daisy (Leucanthemum vulgare Lam.), a normally rayed species with a generalized pollination system, the discoid type is rare throughout the geographic range of the species (Tutin et al., 1976; Bogle, 1983), suggesting a history of unifying selection in favor of the ancestral, rayed phenotype. In this study, I obtained data from a ray removal experiment and a segregating F2 population, established in a near-natural garden environment, to evaluate the role of pollinators, oral antagonists and intrinsic, developmental links with overall size and performance variables in preventing the spread of the derived, discoid phenotype. Specically, I asked: Are fully rayed plants of oxeye daisy more successful in terms of pollinator attraction or more likely to escape oral enemies than those with no or short rays? And, are measures of ray morphology correlated with stem height or measures of plant vigor?

The patchy distribution of stems, together with the close similarity in head size and phenology within patches (S. Andersson, personal observation), indicated that heads in the same pair could represent the same clone. About 4 wk later, I collected all heads and placed each head in a sealed paper bag. Many heads were infested by Tephritis larvae, as evidenced by the presence of large mines and a number of orets that failed to develop beyond the early bud stage (easily distinguished by their small size and dark color), and the emergence of adult ies from the heads stored in the sealed paper bags. To assess the level of infestation, I recorded the number of ies in each paper bag. A sample of 30 nonarrested orets from each head was used to determine pollination efciency, quantied as the proportion of orets that developed into achenes (hereafter called fruit set). Achenes from ray orets were excluded from the calculations of fruit set to allow comparison with heads on which all rays had been removed. A crossing experiment was carried out to determine whether fruit set is dependent on outcrossing and can thus serve as a measure of the extent of crosspollination achieved. In the autumn of 2006, I sowed a bulked sample of achenes from heads used in the ray removal experiment, planted 30 of the resulting seedlings in separate pots, and placed the pots in an unheated, pollinator-proof greenhouse. In 2007, when the majority of the plants reached anthesis, I marked two terminal heads on each individual and assigned the two heads to different pollination treatments: (1) hand-outcrossing with pollen from one or two other plants, and (2) self-pollination with pollen from the same head or another head on the same plant. Pollen was transferred with separate cotton swabs. Slow oral development made it necessary to repeat the pollination procedures every second or third day for 2 weeks. Fruit set was determined as in the eld study. Segregating populationThe segregating F2 population was established to investigate, rst, whether ray morphology covaries with vegetative size and performance variables, and second, how pollinators respond to differences in ray morphology when the oral variation is continuous rather than discrete, as in the ray removal experiment. This plant material originated from crosses between 10 greenhouse-grown plants, derived from a bulked sample of achenes from a few, scattered individuals of the discoid phenotype in a natural population of the rayed variant (about 75 km ESE of Lund). The progeny from the eld-collected achenes had rayed ower heads, but with varying degrees of reduction in ray length and number (S. Andersson, personal observation), indicating that their discoid parents in the eld had been outcrossed with pollen from plants of the normal, rayed variant. I assumed that the rayed, greenhouse-grown plants were heterozygous at loci with large effects on ray morphology and considered the outcrossed progeny of these (F1) parents as F2 segregants. In 2004, I planted about 730 F2 seedlings from a bulked sample of achenes, obtained by mixing achenes from all the successful F1 crosses, in separate pots and placed the plants in a sunny part of an experimental garden (University of Lund), where they were exposed to natural levels of pollination and ower herbivory. The potted plants were arranged in ve adjacent rows along a fence facing south on a bed of ne sand (~15 cm between rows, ~10 cm between pots within rows). Water was supplied as needed, but no fertilizer was applied. The garden is situated ~200 m S of the meadow site in a region where only the rayed morph has been recorded (S. Andersson, personal observation). In 2005, each F2 plant was classied into one of four performance categories: (1) dead before owering, (2) alive but remaining vegetative, (3) bolted but all heads wilted immediately before or during anthesis, and (4) bolted and at least one head developed to the fruit stage. Plants in category 4 were classied as rayed or discoid, and rayed plants were scored for ray length, based on the longest ray in the head terminating the tallest stem. This head was almost always the rst to ower. Early initiation of rays during the bud stage enabled the identication of the oral morph (rayed vs. discoid) for plants in category 3. In 2006, I scored each individual for performance (using the categories used in 2005) and oral morph and recorded the total number of heads initiated and the length and number of rays for the head terminating the tallest stem. If more than one owering stem was available, I also obtained data on ray length, ray number, and stem height for the shortest owering stem on each individual. The paired data for these variables made it possible to relate ray morphology and stem height both between and within individuals. To investigate the relationship between ray morphology and pollination success, I scored one head on each of 80 F2 plants for fruit set, as in the ray removal experiment. For 20 of these plants, I also assessed the fruit set of a head that had been outcrossed by hand to determine whether female fertility was limited by insufcient pollination in the garden environment. Infestation by Tephritis was too infrequent to allow assessment of oral herbivory in the F2 population.

MATERIALS AND METHODS

The plantOxeye daisy is an insect-pollinated, perennial plant found in open, grassy areas, both in its native geographic range (Eurasia) and in regions where the species occurs as a naturalized weed, e.g., North America. It owers from June to August when the branched rhizome develops a variable number of stalks, each having one or a few heads on long terminal peduncles. Heads of the normal, rayed type are up to 7 cm in diameter and consist of hundreds of 4 mm long hermaphroditic disc orets surrounded by a variable number of pistillate ray orets, each having a 1035 mm long, white ligule surmounting a short corolla tube. Fertilized orets develop into one-seeded, indehiscent fruits (achenes) with no special mechanism for dispersal (Howarth and Williams, 1968). Plants of oxeye daisy are more or less self-incompatible and require insect visitation to set fruit (see Results, The effects of ray removal). Flower visitors include a variety of generalist pollinators, mainly members of Coleoptera, Diptera, Hymenoptera and Lepidoptera. Several herbivores and orivores are associated with oxeye daisy (Howarth and Williams, 1968), including larvae of the y Tephritis neesii Meigen (Diptera: Tephritidae). Larvae of Tephritis make large mines in the receptacles causing the early arrest of owers in parts of the inorescences. Ray removal experimentThe ray removal experiment was done in the summer of 2006 at two sites in the northern part of the city of Lund (southern Sweden). The rst site (the meadow site) is an articially established hay meadow outside a university building (Department of Ecology, Lund University) about 1.6 km NE of the central railway station. The second site (the roadside site) is a grassy roadside about 1 km NE of the meadow site. Oxeye daisy is common at both sites, forming patches of varying size within communities dominated by grasses (e.g., Festuca rubra, Poa pratense) and other herbaceous perennials (e.g., Hypericum performatum). At the start of the experiment (early June), I marked 60 pairs of adjacent heads at each site, removed all the rays from one of the heads in each pair (before any of the orets had exposed their stigmas) and assigned the other head as a control. The two heads in each pair were separated by 1030 cm depending on plant density, and different pairs of heads were separated by a minimum of 1 m.

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[Vol. 95

Statistical analysisFollowing arcsine square-root transformation of fruit set, I performed a two-way ANOVA without replication for each eld site using treatment (rayed vs. rayless) as a xed factor and the identity of the head pair as a random block factor. A similar approach was used to assess the effects of open vs. hand pollination (xed factor) and plant identity (random block factor) in the F2 population. The F2 data were also analyzed with one-way ANOVA to compare the mean fruit set of rayed and discoid individuals and with multiple regression to determine whether ray morphology and other characters were under direct selection in the F2 population. A standardized regression coefcient (b) quantied the direct relationship between each phenotypic character and fruit set, holding all the other characters constant. The role of pollinators as selective agents in the F2 population was also evaluated by regressing the degree of pollen limitation (quantied as the difference in fruit set between handand open-pollinated heads) on ray length and ray number. The fruit set of self- and cross-pollinated heads in the greenhouse was compared using Wilcoxons matched-pairs signed-ranks test because transformation failed to improve normality. The data on oral herbivory (the level of infestation by Tephritis) from the ray removal experiment were too skewed to be used in ANOVA. For these data, rayed and rayless heads were compared using MannWhitneys U test (data pooled over head pairs). Associations between oral and nonoral variables in the F2 population were studied in three ways. First, ANOVAs or goodness-of-t tests were performed to compare rayed and discoid plants with respect to head number, stem height, survival rate and owering percentage. Second, the data from the rayed individuals were subjected to product-moment correlation analysis to search for subtle relationships between the quantitative variables. This analysis also provided tests for correlated reductions in stem height and ray morphology, quantied as the difference between the tallest and shortest stem within those plants that initiated more than one stem in the 2006 season. Third, I performed paired t tests (using plants as blocks) to compare oral measurements across years (ray length) and across tall and short stems on the same individual (ray length, ray number). Relevant analyses included row number (15) as a covariate to provide statistical control for spatial variation within the garden plot. The assumption of normality in ANOVAs and correlation analyses was fullled by log-transformation of head number. Descriptive statistics were based on data measured in their original scale. All analyses were carried out with SuperANOVA (1989) on a Macintosh computer.

Number and percentage of plants in different (A) performance and (B) oral morph categories in the F2 population of Leucanthemum vulgare in 2005 and 2006.
2005 plants 2006 plants % No. % No.

Category of plant

A) Performance status Dead before owering Alive but vegetative Heads wilted before fruit stage Heads developed to fruit stage B) Floral morph Rayed Discoid Unknown

0 91 85 556 529 111 92

0 12 12 76 72 15 13

4 100 42 586 570 58 104

1 13 6 80 78 8 14

Note: N = 732

The number of Tephritis larvae per head was higher and more variable at the meadow site (mean 1.95, range 08) and the roadside site (mean 0.46, range 05) than in the F2 population (mean 0.19, range 02), but was not signicantly affected by ray removal in the eld experiment (meadow: U = 1174.50, P = 0.593; roadside: U = 1197.50, P = 0.628). Patterns of variation in the F2 population As can be seen in Table 1, relatively few F2 individuals died before owering (01%), remained vegetative (1213%) or produced heads that wilted before the fruit stage (612%). Most of the plants (86 88%) produced heads that could be scored for the presence or absence of morphologically distinct ray orets (rayed vs. discoid). The percentage of rayed plants increased from 83% in 2005 (529 of 640 plants) to 91% in 2006 (570 of 628 plants). This increase was almost entirely due to individuals that shifted from being discoid in the rst year to rayed in the second year. Extensive variation was seen in all quantitative variables (Table 2), both in the F2 population as a whole and among individuals in the rayed category, with coefcients of variation ranging from about 30% (stem height) to 160% (head number). Rayed F2 plants varied greatly in both ray length (range 134 mm) and ray number per head (range 138), resulting in an almost continuous distribution between the discoid and the fully rayed phenotypes. The heads of rayed plants were more likely to develop to the fruit stage, both in 2005 (98% vs. 38% for discoid plants, 21 = 296.28, P < 0.001) and 2006 (100% vs. 32% for discoid plants, 21 = 398.42, P < 0.001). In 2006, the rayed plants had the tallest stems (mean 48.4 cm vs. 18.9 cm for discoid plants; F1,626 = 226.20, P < 0.001, ANOVA) and the largest number of heads (mean 8.77 vs. 3.22 for discoid plants; F1,626 = 27.92, P < 0.001).

RESULTS The effects of ray removal Greenhouse data show that oxeye daisy is self-incompatible: cross-pollinated heads had a much higher fruit set (mean 57%, range 093%, N = 20) than heads subjected to self-pollination (mean 2.5%, range 020%, N = 20; Z = 3.83, P < 0.001, Wilcoxons matched-pairs signedranks test). Two-way ANOVAs on data from the ray removal experiment revealed signicant differences in fruit set between head pairs (meadow: F49,49 = 4.57; roadside: F49,49 = 4.31, P < 0.001 in both cases) and a highly signicant decrease in fruit set following ray removal, both at the meadow site (mean 42% vs. 65% for control heads; F1,49 = 69.18, P < 0.001) and the roadside site (mean 41% vs. 59% for control heads; F1,49 = 37.06, P < 0.001).
Table 2.
Character

Descriptive data for quantitative characters measured in (A) 2005 and (B) 2006 in the F2 population of Leucanthemum vulgare.
Meana Rangea SDa CV (%)a

A) 2005 Ray length (mm) B) 2006 Ray length (mm) Ray number Stem height (cm) Head number

13.9 (17.1) 18.3 (20.2) 18.8 (20.6) 45.7 (48.4) 8.3 (8.8)

033 (233) 034 (134) 038 (138) 791 (991) 1164 (1164)

8.6 (6.2) 8.9 (7.0) 8.9 (7.0) 16.6 (14.7) 13.3 (13.9)

62 (36) 49 (35) 47 (34) 36 (30) 160 (158)

Note: N = 500628 plants, SD = standard deviation, CV = coefcient of variation aData in parentheses refer to rayed plants only.

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The differences in stem height and head number remained signicant after the inclusion of row number as a covariate (P < 0.001 in both cases). The possession of rays in 2005 was associated with a higher reproductive performance in 2006, especially when quantied as total head number (9.35 vs. 4.78 for discoid plants; F1,579 = 12.44, P < 0.001), but to some extent also when measured as owering percentage (95% vs. 81% for discoid plants, 21 = 29.10, P < 0.001) and the percentage of plants that developed their heads to the fruit stage (100% vs. 68% for discoid plants; 21 = 159.55, P < 0.001). The fraction of plants that remained alive in 2006 was not signicantly affected by the presence or absence of rays in the 2005 season (>99% in both groups). Correlation analysis of quantitative data from rayed F2 individuals revealed positive associations between variables (Table 3). Ray length and ray number per head were positively correlated with stem height and, to a lesser extent, total head number. All associations became weaker (especially those involving head number) but generally remained signicant after accounting for the effect of row number. Ray length and ray number also remained signicantly positively correlated with stem height after adjusting for variation in head number (r > 0.20, P < 0.0010.01). The ray lengths for individual plants were strongly positively correlated between years (r = 0.72, P < 0.001), and there was a strong positive correlation between the ray lengths on the tallest and shortest stem on each individual (r = 0.76, P < 0.001). A signicant between-stem correlation was also observed for ray number (r = 0.52, P < 0.001). Despite these consistencies, individual plants had a signicant increase in ray length from 2005 to 2006 (mean difference 4.95 mm; t530 = 21.40, P < 0.001) and a signicant decrease in both ray length (mean difference 7.31 mm; t451 = 32.76, P < 0.001) and ray number per head (mean difference 7.48; t451 = 23.92, P < 0.001) from the tallest to the shortest stem in the 2006 season. Rayed individuals that had a substantial decrease in stem height also had a considerable reduction in both ray length (r = 0.60, P < 0.001) and ray number (r = 0.50, P < 0.001). Judging from the results of two-way ANOVA, controlling for a signicant effect of plant identity (F24,24 = 7.01, P < 0.001), the F2 plants had a signicant increase in fruit set after hand pollination (mean 46%, range 087%, N = 25) relative to open pollination (mean 15%, range 057%, N = 79; F1,24 = 61.90, P < 0.001). Data from open-pollinated heads revealed a signicantly higher fruit set for rayed F2 plants (mean 17%, range
Table 3.

057%, N = 65) than for discoid F2 plants (mean 5%, range 030%, N = 14; F1,77 = 8.24, P < 0.01). Multiple regression analyses revealed a positive relationship between ray length and fruit set for open-pollinated heads (b = 0.67, P < 0.01), whereas the relationship for hand-pollinated heads failed to reach signicance (b = 0.70, P = 0.10). Ray number, stem height and total head number did not signicantly affect fruit set, regardless of pollination treatment (|b| < 0.20, P > 0.34). Neither ray length nor ray number were signicantly correlated with the degree of pollen limitation (|b| < 0.15, P > 0.49). DISCUSSION Despite growing evidence that both pollinator and nonpollinator selection can drive the evolution of oral characters (Grant, 1949; Faegri and van der Pijl, 1979; Galen, 1999b; Fenster et al., 2004; Strauss and Whittall, 2006), only a few investigations have explored how pollinators and other ecological forces operate simultaneously in exerting selection on oral size characters (Strauss and Whittall, 2006). In the study presented here, I have obtained data from a ray removal experiment in the eld and a segregating F2 population in a near-natural garden environment, to assess the roles of pollinators and nonpollinator agents of selection as stabilizing factors on oral evolution in oxeye daisy, a self-sterile composite known for its conspicuous white rays. My study not only provided insights into the direct effects of rays on pollination success and oral herbivory, but also enabled me to assess the potential for indirect selection through intrinsic, developmental links with vegetative size and performance variables. Plants of oxeye daisy almost always possess rayed ower heads, both in the native Eurasian range and in North America (Tutin et al., 1976; Bogle, 1983). The sparse and scattered occurrence of the discoid oxeye daisy morph suggests that the normal, rayed variant is optimal and that selection operates in a way that prevents the establishment and spread of aberrant oral phenotypes. The results of the present investigation demonstrate that the rayed phenotype has a clear selective advantage, in terms of both pollinator attraction and plant vigor and that it would be difcult for discoid mutants to establish and spread in the area studied. Interactions with oral herbivores (Tephritis) seem to play a minor role in exerting selection on ray morphology; thus, there were no conicting selection pressures between the generalist pollinators and the nonpollinator agents of selection considered in this study. Ray morphology vs. pollinator attraction and ower herbivory The results of this study agree with the inferred advantage of oral organs that enhance the plants visual display to pollinators (Leppik, 1977; Bell, 1985; Bertin and Kerwin, 1998). The percentage fruit set of open-pollinated heads decreased by 3135% after ray removal and was 71% lower for discoid plants in the segregating F2 population, which showed continuous rather than discontinuous variation between the discoid and the fully rayed phenotype. Thus, the reproductive advantage of possessing rayed ower heads was consistent not only across locations, but also between populations with widely different arrays of oral phenotypes. These consistencies point toward a predominant role for pollinator-mediated selection in maintaining the ancestral, rayed phenotype of oxeye daisy. The decrease in percentage fruit set after ray removal falls within the range observed for composites with few or solitary

Product-moment correlation coefcients for quantitative characters measured in the F2 population of Leucanthemum vulgare, based on data from rayed individuals that owered in the 2006 season.
Correlation coefcient 1 2 Partial Ordin. Partial Ordin. 3 Partial

Character

Ordin.

1. Ray length 2. Ray number 3. Stem height 4. Head numbera

0.57*** 0.53*** 0.66*** 0.60*** 0.71*** 0.64*** 0.32*** 0.23** 0.50*** 0.39*** 0.66*** 0.45***

Note: Ordin. = ordinary correlation coefcient, Partial = partial correlation coefcient when position is held constant (N = 491 plants). ** P < 0.01, *** P < 0.001. aLog-transformed before analysis

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heads (23% in Scalesia afnis, Rostgaard Nielsen et al., 2002; 64% in Helianthus grosseserratus, Stuessy et al., 1986), but exceeds values reported in species with a large number of heads massed together in dense, corymbiform inorescences (0% in Senecio jacobaea, Andersson, 1996; 12% in Achillea ptarmica, Andersson, 1991; 15% in S. integrifolius, Andersson and Widn, 1993). The current study therefore provides additional support for the idea that the possession of rays is most favorable when the basic attraction units are individual heads rather than clusters of small heads in large compound inorescences (Andersson, 1996). Consistent differences were found in percentage fruit set between pairs of adjacent stems in the eld (ray removal experiment) and between different plants in the F2 population, which agrees with results regarding other composites (Andersson 1991, 1996; Andersson and Widn, 1993). Thus, the use of head pairs or plants as blocks enhanced the statistical power to detect a functional relationship between ray morphology and pollination success by adjusting for variation in unmeasured parameters such as plant density, resource status, and degree of cross-fertility. In this context, it is necessary to stress that hand pollination signicantly increased fruit set in the garden plot; thus, female fertility was at least partly determined by the amount of cross-pollination achieved, a necessary assumption when using fruit set as a proxy for pollinator visitation. Moreover, the positive relationship between ray production and fruit set among F2 plants remained signicant when measures of plant vigor (e.g., total head number) were held constant in a multiple regression analysis. Because the amount of pollen donation (male success) is expected to be an increasing function of the visitation rate, it is also reasonable to assume that male and female fertility respond similarly to differences in ray morphology and that the results concerning pollination efciency therefore apply to both sexual functions (Andersson, 1991). Plants in the F2 population had no signicant relationship between ray length and the degree of pollen limitation, as would be expected if long-rayed heads had the highest visitation rates. Although this result argues against direct, pollinator-mediated selection on ray morphology, the overall response to ray removal or hand-pollination demonstrates clearly that the possession of rays is associated with increased pollinator visitation. The individual-level analyses probably lacked the power to detect a relationship between ray morphology and pollen limitation, given the small number of F2 plants subjected to both open- and hand-pollination. Floral antagonists, for example, ower herbivores, predispersal seed predators, and nectar thieves, sometimes act as strong selective agents on oral features that make plants more attractive to pollinators (Brody, 1992; Galen, 1999a; Strauss and Whittall, 2006). For instance, ovipositing females of the sunower moth Homoeosoma electellum have been found to discriminate against plants with small ower heads (Pilson, 2000). In this study of oxeye daisy, no detectable effect of ray removal was found on the level of infestation by Tephritis, despite the drastic reduction in oral display arising from the manipulation. As found in a previous study of this and other composites, the incidence of larval infestation seems to be strongly determined by the diameter of the disc (Fenner et al., 2002), raising the possibility that the oral antagonists use attributes of the disc, for example, the number, color, or scent of disc orets, rather than the possession of rays, when searching for their host plants.

The potential for indirect selection The data from the F2 population conrm previous observations from other plants that there is a potential for selective pressures other than pollinators and oral antagonists to impose selection on oral morphology through developmental associations between oral and nonoral characters (e.g., Primack, 1987; Andersson, 1997; Strauss and Whittall, 2006). First, plants with discoid or short-rayed heads initiated fewer heads, their heads were less likely to develop to the fruit stage, and they had a signicantly lower owering percentage and inorescence production in the subsequent year than those with well-developed rays. Second, I detected positive associations between stem height, ray length, and ray number per head among F2 plants in the rayed category, regardless of whether position (row number) or head number was held constant. The latter associations were also manifested as a correlated decrease in stem height and ray morphology within those plants that initiated more than one stem in the 2006 season. These ndings indicate the existence of two partly independent axes of covariation, one involving ray morphology and general plant vigor and another involving ray morphology and plant stature. Given that general performance variables such as head number are under positive selection and that the relationship between low plant vigor and raylessness can be generalized to other populations, there is considerable potential for indirect selection pressures to prevent the spread of the derived, discoid phenotype. The high interyear consistencies observed for ray morphology suggest that the oral variation had a strong genetic basis, conrming results from a previous study of oxeye daisy (Bogle, 1983). On the basis of the observed frequency of the discoid morph (about 10%), it is also reasonable to conclude that rayed heads are dominant over discoid heads, and that the suppression of rays is controlled by a relatively small number of genes, as observed in other species (Ford and Gottlieb, 1990; Andersson, 2001b; Gillies et al., 2002). However, it is not known whether the absence of rays represents a pleiotropic side effect of genes with deleterious effects on plant vigor, whether the reduction in vigor represents a side effect of genes suppressing ray development, or whether the observed associations stem from linkage equilibrium between loci controlling ower development and loci affecting vegetative growth parameters. In view of the strong linear relationship between stem height and quantitative measures of ray morphology, which remained signicant after adjusting for variation in plant vigor, it seems reasonable to attribute some of the covariation to loci expressed during stem and ower development, not solely to loci with general effects on plant vigor. Loci affecting levels of endogenous gibberellin activity are one possible source of stem heightower size associations, given the strong inuence of gibberellin on the growth of both oral and vegetative structures (Jones, 1973; Koning, 1984). A small fraction of F2 plants shifted from being discoid in the rst season to being rayed in the second season, and there was a slight (though signicant) increase in ray length between the two owering seasons. These observations indicate that the overall plant vigorand the ability to produce raysincreased with increasing age in the F2 population. This age effect was too weak to obscure the high interyear consistencies documented for ray morphology, but raises some concern about the discoid plants used as parents for this F2 population. However, the detection of discoid F2 segregants shows that the original parents must have been homozygous or heterozygous for allele(s) with negative effects on ray development.

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In this regard, it must be emphasized that the F2 plants represent a small number of parents and that the observed character associations may be specic to the population from which the parents originated. Bogle (1983), whose intermorph crosses involved oxeye daisy plants from a population in North America, presented no quantitative data on nonoral variables, but noted that the discoid progeny plants generally owered earlier than those with rayed ower heads, a pattern not observed in the present investigation (S. Andersson, personal observation). It will therefore be necessary to perform additional crosses (involving several unrelated genotypes of the discoid variant) before any broad generalizations can be made regarding patterns of covariance between ray morphology and other characters in oxeye daisy. Realized patterns of selection on ray morphologyThis study of oxeye daisy revealed no conicting selection pressures between the pollinators, the orivores, and the indirect, pleiotropic associations inferred from the correlation analyses; consequently, one would expect a strong persistent relationship between tness and the extent to which naturally occurring plants approach the fully rayed phenotype in this study system. These patterns contrast with the more variable or complex relationships documented in other species of the Asteraceae family. Larger, more conspicuous heads of sunower (Helianthus) species have been found to enhance the attraction of both pollinators (Stuessy et al., 1986) and oral antagonists (Pilson, 2000), suggesting that the optimum ray size is smaller than that favored by pollinators alone. A similar interpretation may be invoked in cases where the possession of rays entails a cost in terms of ower or fruit production, as observed in Achillea ptarmica (Andersson, 1999), Crepis tectorum (Andersson, 2006), and Madia sativa (Celedn-Neghme et al., 2007). Data for C. tectorum also indicate the existence of positive genetic correlations between plant height, achene size, and ower size, a pattern that should reduce the selective optimum at sites selecting for short stature and (or) small propagule size (Andersson, 1993). In Senecio jacobaea, in which the frequency of rayed and discoid individuals varies ecogeographically (van der Meijden, 1976), the optimum oral phenotype may be a compromise between several selection pressures, involving not only pollinators but also physiologically adverse effects of raylessness and spatially varying selection on germination characteristics (Andersson, 2001a), mediated through the close developmental link between ray and fruit dimorphism in this species (McEvoy, 1984). One would also expect morph-specic differences in germination behavior, as well as other characters, to affect the frequency of rayed and discoid individuals of S. vulgaris, given the strong, persistent linkage relationships between oral and nonoral characters in certain populations of this largely selng species (Abbott, 1986; Oxford et al., 1996; Abbott et al., 1998; Comes, 1998). The existence of such associations, coupled with the reduced selection pressures on oral size characters in selng taxa, suggests that nonpollinator selection has played a signicant role in the establishment and spread of discoid genotypes in S. vulgaris. With the results from other wild plant species (Galen, 1999b; Strauss and Whittall, 2006), the available data indicate a considerable potential for selective forces other than pollinators to shape the evolution and diversication of oral characters. LITERATURE CITED
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