Annals of Botany 85 (Supplement A): 211-219, 2000 I A doi: 10.1006/anbo. 1999.1033, available online at http://www.idealibrary.com on I E

Interrelationships Among Inbreeding Depression, Plasticity in the Self-incompatibility System, and the Breeding System of rapunculoides L. ()

ANDREW G. STEPHENSON*, SARA V. GOOD and DONNA W. VOGLER Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA

Received: 21 July 1999 Returned for revision: 9 September 1999 Accepted: 20 October 1999 Downloaded from https://academic.oup.com/aob/article/85/suppl_1/211/102914 by guest on 29 September 2021

The evolution of breeding systems in is often viewed as a balance between the adverse consequences of selfing (inbreeding depression and the loss of opportunities to sire seeds on conspecifics) and the benefits of selfing (a genetic transmission advantage and reproductive assurance when cross pollen limits seed production). In this paper we examine the genetic and environmental causes of variation in the expression of self-incompatibility (SI) in Campanula rapunculoidesand explore the consequences of this variation on the breeding system. Campanularapunculoides has an S-RNase based S1 system similar to that described in the Solanaceae. However, our studies of plants from two natural populations have revealed that the flowers of most individuals are self-incompatible when they first open but become more self fertile as the flowers age. Moreover, when both cross and self pollen are deposited onto the stigmas of older flowers, the cross pollen tubes grow faster and sire a disproportionate number of the seeds. In short, self-fertilization occurs only after most opportunities for outcrossing have occurred. We also found that there is significant heritable genetic variation in the population for the strength of SI in young and old flowers and for the amount of breakdown in SI indicating that natural selection could operate on the strength of SI and its breakdown. In a multigenerational study, we used controlled crosses to create families of plants with a range of inbreeding coefficients (0, 025, 0.5 and 0.75). We found that fitness declined significantly over the range of inbreeding coefficients and that the decline in fitness was less for families derived from weak SI phenotypes. Consequently, it is only advantageous for C. rapunculoides to produce selfed seed when seed production is limited by the availability of cross pollen. Because of plasticity in the SI system, C. rapunculoides has a breeding system that combines the best of both worlds. (C 2000 Annals of Botany Company Key words: Campanula rapunculoides, self-incompatibility, pseudo self-compatibility, heritability, S-RNase, gameto- phytic self-incompatibility, breeding system, mating system, selfing, outcrossing, inbreeding depression.

INTRODUCTION resources, to produce selfed seed (e.g. fewer resources are expended to attract and reward pollinators) (Schemske, Since the publication of Darwin's (1876) classic book, 1978; Waller, 1979; Schoen and Lloyd, 1984). Recent The effects of cross- and self-fertilisation in the vegetable theoretical advances have demonstrated that the genetic kingdom, inbreeding depression (the reduction in fitness of and environmental advantages of selfing are not only inbred progeny relative to outcrossed progeny) has been counterbalanced by inbreeding depression (e.g. Maynard invoked as a major force in the evolution of mechanisms Smith, 1977; Charlesworth, 1980a) but also by the adverse that promote outcrossing. On the other hand, natural effects of selfing on pollen dissemination to conspecifics historians have long noted that selfing is advantageous if it (pollen discounting: e.g. Nagylaki, 1976; Holsinger, Feld- provides reproductive assurance when pollinators are scarce man and Christiansen, 1984) and by a decrease in outcross or unreliable (e.g. Baker, 1955; Stebbins, 1957; Schoen, seed production that may accompany an increase in selfing Morgan and Bataillon, 1996), and evolutionary biologists (seed discounting: Lloyd, 1992). have long noted that there is an inherent transmission Because self-incompatibility (SI) is regulated by S-genes, advantage to selfing because a donates two haploid plant scientists have tended to think of SI as a qualitative sets of chromosomes to each selfed seed and only one set to trait of the breeding system-if a species has SI, it is an each outcrossed seed (e.g. Fisher, 1941). Consequently, obligate outcrosser. In reality, however, SI is often a genetic modifiers that promote self-fertilization without quantitative and phenotypically plastic trait. That is, many reducing cross-fertilization through the male (pollen) species exhibit marked phenotypic variation in the expres- function should increase in the absence of inbreeding sion of SI that is often influenced by environmental depression (e.g. Nagylaki, 1976; Charlesworth, 1980b; Lande and Schemske, 1985; Campbell, 1986). Selfing may conditions. For example, the expression of SI is known to vary (a) with the action of specific S-alleles (e.g. also be advantageous if it maintains adaptive combinations weak and strong alleles); (b) with the expression of alleles at different loci (e.g. Stebbins, 1957; Solbrig, of modifier genes; 1976), and if it costs less, in terms of energy and other (c) with the composition of the pollen load (pure self-pollen vs. mixtures of self and cross pollen); (d) with external * Fax +1-814-865-9131, e-mail [email protected] or svglCpsu.edu or environmental conditions such as temperature; and (e) with [email protected], respectively. internal stylar conditions such as the age of the flower, time 0305-7364/00/0A0211 + 09 $35.00/00 © 2000 Annals of Botany Company 212 Stephenson et al.-Self-incompatibility in Campanula rapunculoides of the year, or presence of developing fruit (see Ascher and (from bottom to top) developing fruits, female phase Peloquin, 1966; de Nettancourt, 1977; Stephenson and flowers, male phase flowers, and flower buds. By recording Bertin, 1983; Mulcahy, 1984; Lloyd and Schoen, 1992; bumblebee movements within an inflorescence, we found Levin, 1996; Vogler, Das and Stephenson, 1998; and refer- that bumblebees usually begin foraging at or near the ences therein). These variations in the expression of SI have lowest open flower and that there is a strong tendency for many names in the literature (incomplete SI, weak SI, leaky the bees to forage upwards on an inflorescence (85% of the SI, partial SI, partial self-compatibility, pseudo self- movements by bumblebees within an inflorescence were to a compatibility, pollen prepotency etc.) and probably higher flower on the inflorescence). Consequently, most represent a constellation of related phenomena involving bees moved from female phase flowers to male phase subtle pollen pistil interactions that affect the relative flowers within an inflorescence. The combination of floral performance of self and cross pollen (i.e. speed of germina- protandry (temporal separation of the sexes within flowers)

tion and/or pollen tube growth rates). For the most part, and upward foraging by bumblebees on inflorescences with Downloaded from https://academic.oup.com/aob/article/85/suppl_1/211/102914 by guest on 29 September 2021 variations in the expression of SI have been documented by acropetal development would tend to promote outcrossing. geneticists and plant breeders who view them as nuisances On the other hand, the occasional downward movement by or curiosities that confound attempts to determine genetic a bumblebee could result in geitonogamy (between flower specificities in cultivated plants (Ascher, 1984). Evolution- self-pollination) and geitonogamy also occurs when a ary biologists have only recently begun to explore the bumblebee moves between inflorescences of the same potential roles of plasticity and plant-to-plant variation in genotype (rhizomatous cluster). Moreover, when the pollen the expression of SI in the origin and maintenance of the is not removed from the flowers during the male phase, the breeding system, especially mixed mating systems (e.g. fully reflexed stigmas of older female phase flowers come Stephenson and Bertin, 1983; Becarra and Lloyd, 1992; into close proximity of the pollen and autonomous self- Lloyd, 1992; Latta and Ritland, 1993; Levin, 1996). pollination sometimes occurs (pers. obs.). However, little is known about the genetic and molecular mechanisms that underlie variations in the strength of SI FLORAL AGE DEPENDENT VARIATION IN nor the consequences of such variation for the breeding EXPRESSION OF SI systems of the plants possessing them. For the past decade, our lab has examined variations In a preliminary study in a natural population of in the expression of SI in Campanula rapunculoides L. C. rapunculoides, a series of cross- and self-pollinations on (Campanulaceae). The goals of this paper are: (a) to young (first day of the female phase) and old (fourth day of document variation in the expression of SI; (b) to review the female phase) flowers revealed that: (a) self-pollination our recently published findings concerning the genetic and of young flowers results in very low fruit and seed set; environmental causes and population level consequences of (b) self-pollination of old flowers results in a significant variation in the expression of SI; (c) to present our recent increase in fruit and seed set; and (c) fruit and seed set advances in the molecular biology of the pistil proteins decreases slightly from young to old flowers following involved in SI in C. rapunculoides; (d) to demonstrate that cross-pollinations (Richardson et al., 1990; Stephenson there is heritable genetic variation for the expression of SI et al., 1992). In addition, we repeated the series of cross- in C. rapunculoides; and (e) to explore the interrelationships and self-pollinations on young and old flowers and among inbreeding depression, variation in the expression of harvested the styles 6, 12 or 24 h after pollination. These SI, and the evolution of mixed mating systems. styles were stained with decolourized analine blue and pollen tube growth was examined using fluorescent microscopy. We found that pollen tube length increased NATURAL HISTORY AND FLORAL significantly from 6 to 24 h. More importantly, we found BIOLOGY OF CAMPANULA that cross pollen tubes grew significantly more rapidly (and RAPUNCULOIDES there was less pollen tube bursting) in the styles of young Campanula rapunculoides is a naturalized, perennial herb flowers but the differences in the growth of cross and self that grows along roadsides and in open woods in the pollen tubes decreased significantly in the styles of old Northeastern United States and Canada (Rosatti, 1986). It flowers (Stephenson et al., 1992). In short, these preliminary overwinters as a rosette and, in midsummer, each rhizo- studies indicated that the flowers of most individuals are matous cluster produces one to eight flowering shoots self-incompatible when they first open but become more (racemes) of 10-60 flowers that mature acropetally (bottom self-fertile with floral age. upwards). The blue, bell-shaped flowers are bumblebee We have now performed controlled pollinations on pollinated and protandrous. Thus, at anthesis, the flowers young and old flowers on more than 120 plants that were are staminate (male phase) and the stigmatic lobes are collected from two natural populations (Centre Co., PA, tightly expressed. After 1-3 d, during which the pollen is and Duanesburg, NY, USA). With only a couple of removed by the bees, the stigmatic lobes reflex and pollen exceptions, the self compatibility index (SCI; self/cross deposition can occur (female phase). Richardson and seed set following controlled pollinations) increased from Stephenson (1989) demonstrated that pollen removal (either young flowers to old flowers. For both populations, we have manually by the investigators or by natural pollinators) found continuous variation in the SCI among plants hastens the onset of the female phase. At any given time ranging from 0 to 060 for young flowers and 002 to 095 during the flowering season, an inflorescence will consist of for old flowers (see Table 1). For many of our studies Stephenson et al.-Self-incompatibility in Campanula rapunculoides 213

TABLE 1. The self-incompatibility index (SCI) fr a sample of Campanula rapunculoides plants

Plant 1 3 4 5 6 8 13 14

Day I pollinations 0.21 048 0.12 0.14 0-05 0-09 0-03 0.55 Day 4 pollinations 0.26 0.95 0.17 0.45 0.28 0-22 0-07 0.84

The SCI is the number of selfed seeds produced per hand-pollination divided by the number of cross seeds produced per hand-pollination. Day I refers to pollinations on flowers during their first day in the female phase while day 4 refers to pollinations on flowers during their fourth day of the female phase. Data from the plants used by Vogler et al., 1998. Downloaded from https://academic.oup.com/aob/article/85/suppl_1/211/102914 by guest on 29 September 2021 described below, we arbitrarily defined three classes of were standardized by the method of Richardson et al. (1990) plants: weak SI plants (about 15% of the total plants), in to deliver approx. 500 pollen grains-an amount just which the young flowers were relatively self-compatible sufficient to produce a full complement of seeds when (SCI > 040) and became more so as the flowers aged; cross-pollen is used. Capsules were allowed to ripen over a strong SI plants (about 10% of the total plants), in which I month period; they were then collected and the seeds the old flowers remained highly SI (SCI < 015); and counted. typical breakdown plants (35% of total plants), in which We found that floral position within an inflorescence had the young flowers had strong SI (SCI < 015) and the old no effect on the strength of SI but we did find that clones in flowers had weak SI (SCI > 040). The remaining plants the NO FRUIT treatment had a stronger expression of SI had intermediate SI phenotypes. on young flowers and a weaker expression of SI on old flowers than clones in the FRUIT treatment [i.e. there was a significant treatment (FRUIT/NO FRUIT) x floral age PLASTICITY IN THE EXPRESSION OF SI interaction] (Vogler et al., 1998). Because the presence of Because developing fruits can draw resources from later few or no developing fruit on a plant would be indicative of flowers in an inflorescence, they have the potential to alter low pollinator activity in nature, the weaker expression of SI the expression of self-incompatibility in later developing on old flowers on the NO FRUIT clones is likely to flowers on that inflorescence (Becarra and Lloyd, 1992; promote selfed seed set under conditions of pollinator Reinhartz and Les, 1994). In this study, we examined the scarcity. Because seed set following cross-pollinations on effects of floral position within the inflorescence, floral age, old flowers was greater on the clones in the NO FRUIT genotype and prior fruit-set on the strength of the self- treatment than on clones in the FRUIT treatment, we incompatibility system in C. rapunculoides (Vogler et al., suspect that the increase in self seed on the NO FRUIT 1998). By treating these factors in a factorial design, we plants was due to a greater longevity of the ovules which tested for genetic variation and phenotypic plasticity in self- permitted the slower growing self-pollen to achieve fertil- incompatibility (as main effects in the analysis of variance) ization. Finally, data analyses revealed that plant genotype and as interactions among factors. explained a significant amount of the total variation in the During the summer of 1994, multiple clones of eight expression of SI and that there was a significant geno- genotypes were propagated by cuttings and type x treatment interaction. Although we used only eight brought to flower in a greenhouse. Eight to ten replicate genotypes in the study, these findings strongly indicate that clones of each genotype were matched for overall size and there is broadsense heritability for the magnitude of SI and vigour and split into two treatment groups: FRUIT, in that SI is a phenotypically plastic trait upon which natural which all flowers on the lower third of the raceme were hand- selection could operate. pollinated with cross-pollen, and NO FRUIT in which all flowers on the lower third of the raceme were prevented from GENETICS AND MOLECULAR BASIS OF SI being pollinated by removing the stigmatic lobes. In the IN C. RAPUNCULOIDES remaining two-thirds of the raceme, most of the flowers of each plant continued to receive the appropriate FRUIT/NO Our preliminary studies of SI in C. rapunculoides indicated FRUIT treatments, but four-12 flowers were selected and that SI was likely to be gametophytic and that rejection marked to receive one of four experimental pollinations: (1) (and/or retardation of growth) occurred in the styles. In self day 1, selfed on the first day of the female phase; (2) out order to determine if C. rapunculoides produced S-locus day , outcrossed on the first day of the female phase; (3) self glycoproteins such as those in the Solanaceae, we gathered day 4; and (4) out day 4. The order of these four treatments three types of preliminary evidence. We found that pistil was randomized for each plant, and each pollination was extracts run on SDS-PAGE had an abundant protein performed on flowers that were bracketed by other flowers (molecular weight approx. 32 kD) in l-d-old pistils that was receiving the appropriate FRUIT/NO FRUIT treatment for still present in 4-d-old pistils, but was not present in extracts that plant. Even with that spacing, at least one, and often from leaf and bud samples (Fig. 1). We found that the two replicates could be made for each of the four 32 kD pistil protein cross-reacted with antibodies to Petunia experimental pollinations per plant. We used fresh pollen S-locus glycoproteins (provided by T-h Kao) and that this from male-phase flowers for our pollen sources. Pollen loads protein exhibited RNase activity (see Blank, Sugiyama and 214 Stephenson et al.-Self-incompatibility in Campanula rapunculoides Isoelectric focusing (IEF) gels revealed a highly abundant and polymorphic pistil protein with a similar (and high pH 87-9-5) isoelectric point to the Solanaceous S- glycoproteins, and F and F2 plants possessed protein bands that co-migrated with the parental bands. More importantly, 2-dimensional gels revealed that the abundant 32 kD protein is the same one that is polymorphic and that it has a high isoelectric point (Fig. 2). Furthermore, we have shown that these proteins have RNase activity (Fig. 3). In FIG. 1. Spatial and developmental regulation of 32 kD putative S-protein in C. rapunculoides. The approx. 32 kD protein is absent in short, we have identified a protein that co-segregates with anther (AN) and leaf (LE) tissues and it is not expressed in the pistils the S-phenotype whose molecular weight, relative abun- from green or purple buds (GB, PB). The protein first appears in pistils dance, temporal and spatial distribution, RNase activity, Downloaded from https://academic.oup.com/aob/article/85/suppl_1/211/102914 by guest on 29 September 2021 in the early male (EM) phase and increases in quantity through late male (LM), early female (EF) phase and is greatest in late female and allelic diversity are similar to those reported for S-locus (LF) pistils. glycoproteins in the Solanaceae. Currently, we are attempt- ing to isolate, amplify, clone and sequence S-alleles from C. rapunculoides. To date S-RNase based SI has been Dekker, 1982 for technique). Although preliminary, these identified in the Solanaceae (the most well studied), three types of evidence indicate that the molecular proper- Scrophulariaceae, Rosaceae, and now in the Campanula- ties of the 32 kD protein in C. rapunculoides are similar to ceae. those reported for the S-glycoprotein in the Solanaceae In order to further explore the heritability of SI and its (Stephenson et al., 1992). Recently, we found that the breakdown, we fully characterized the strength of SI in 32 kD RNase protein was localized or more abundant young and old flowers on 35 plants that were originally (depending upon the genotype) in the top half of the style. obtained from one natural population. From these plants,

FIG. 2. Two-dimensional gel of total pistillate protein in C. rapunculoides revealing presence of a highly abundant and polymorphic protein of approx. 32 kD molecular weight. There appear to be four alleles (isoforms) of the protein with isoelectric points between 8.7 and 9.3 (four arrows on right hand side). Smaller arrow on left identifies a marker protein at MW 37 kD and pl 5.2. Stephenson et al.-Self-incompatibility in Campanula rapunculoides 215

FIG. 3. PAGE-gel stained for RNase activity in 17 individuals of C. rapunculoides. These RNases fall within 30-35 kD. Each individual has between three and seven (usually six) RNase-bands, and the bands are highly polymorphic. Analysis of RNase-activity in IEF gels reveals that one to two of these bands have a low pl (i.e. are acidic), suggesting that individuals have from two to four high pl (i.e. basic) RNase proteins which are

the putative S-alleles. Downloaded from https://academic.oup.com/aob/article/85/suppl_1/211/102914 by guest on 29 September 2021 we selected 20 genotypes that represented the range of SI breakdown of self-incompatibility can provide reproductive phenotypes (weak SI, strong SI and typical breakdown assurance. If both self and outcross pollen are deposited plants) and randomly crossed them to produce 45 families. onto a stigma (as may commonly occur with geitonogamy) From each of these families we grew five progeny and, on the consequences will depend on the ability of the pistil to each progeny, we made a series of cross- and self- discriminate against self pollen and the presence of any pollinations on young (day 1 in the female phase) and old outcross pollen that shares one or both S-alleles with that (day 4 in the female phase) flowers. We counted the seeds in plant. In small populations with limited S-allele diversity the resulting fruits and determined the self compatibility even random mating patterns may result in some amount of index for young and old flowers and the breakdown in SI incompatible cross-pollen being deposited on stigmas (SCI of old flowers minus the SCI of young flowers). To (Goodell et al., 1997). If the pistil can discriminate against estimate the heritability of the strength of SI on young and incompatible/self pollen by reducing fertilization success, old flowers and the breakdown in SI, we used the slope seeds will be sired disproportionately, or exclusively by from a regression of the mean progeny values (SCI of outcross pollen. If, however, the breakdown of self- young flowers, SCI of old flowers, or SCI of old minus SCI incompatibility allows both self and outcross pollen to of young flowers) on the mid-parent values (Falconer, fertilize ovules (and there is not complete selective abortion 1989). Our findings reveal that there is significant narrow of selfed progeny) then the offspring will be the product of sense heritability for the magnitude of SI on young and old mixed mating. flowers and for the age dependent breakdown in SI In this study (Vogler and Stephenson, 2000), equal (Figs 4-6). Together with the phenotypic plasticity study, mixtures of self and cross pollen were deposited onto young these findings indicate that there is a genetic basis to and old pistils and the paternity of each progeny was scored variation in the expression of SI and that natural selection using isozyme markers. We found that the progeny from could act on this variation. Currently, experiments are young flowers could not be distinguished from expectations underway to determine the number of modifier genes of pure outcrossing but that the progeny from old flowers responsible for variations in the expression of SI and included a mixture of selfed and outcrossed seeds. However, whether the modifier genes are linked (including the pos- sibility of weak and strong S-alleles) or unlinked to the S-locus.

>s CONSEQUENCES OF VARIATION AND PLASTICITY IN SI ON THE BREEDING SYSTEM ' i 1 The studies reported above have revealed both environ- . .. mental and genetic variation in the strength of SI. In 0 another series of studies, we have investigated the con- I I. II .. sequences of this variation in SI on the breeding system of o C. rapunculoides. In the first study, we examined the .. . . interrelationships among the pollen load, the age dependent 0 breakdown in SI, and the breeding system. We reasoned II II I I I I that the immediate consequences of a breakdown of self- 00 01 02 03 04 05 06 07 08 09 1.0 compatibility will vary with the type of pollen deposited Mid-parental value of day 1 SCI index (i.e. self pollen alone or self pollen in competition with outcross pollen) and the ability of the pistil to discriminate FIG. 4. Heritability of day 1 self-compatibility index (SCI) in C. rapunculoides. The mean value of the progeny day I SCI is against self pollen (Stephenson and Winsor, 1986). If only regressed on the mid-parental day SCI index. The linear regression self pollen is available, as might occur in isolated plants or equation is y = 0-103989 + 0402380 x. The slope of the regression is in populations with low pollinator availability, the an estimate for the narrow sense heritability. 216 Stephenson et al.-Self incompatibility in Campanula rapunculoides

2

I- I - a .o 1 0 v -r 3, o U 1 C rJ2Co . I >1 Cd 0 5 a I . . a~ ben · .",...... - 00 I- a) . . .

I+ · i · 1 I

e Downloaded from https://academic.oup.com/aob/article/85/suppl_1/211/102914 by guest on 29 September 2021 I·I · Ia· I : . -. . :· : 00C 0 _- * I::a ,,e . .l . * .*: . I I I . I 0.0 01 0-2 03 04 0'5 0-0 0-5 1-0 Mid-parental value of breakdown in SI Mid-parental value of day 4 SCI index FIG. 6. Heritability of the breakdown in self-incompatibility (SI) from FlG. 5. Heritability of day 4 self-compatibility index (SCI) in C. day I to day 4 in C. rapunculoides. The mean value of the breakdown in rapunculoies.The mean value of the progeny day 4 SCI is regressed on progeny degree of SI from day I to day 4 is regressed on the mid- the mid-parental day 4 SCI index. The linear regression equation is parental value of the breakdown. The linear regression equation is y = 0-172100 + 0.383241 x. The slope of the regression is an estimate y = 0 129248 + 0.375793 x. The slope of the regression is an estimate for the narrow sense heritability. for the narrow sense heritability. the proportion of selfed seeds in the old flowers was respectively) using plants with the typical breakdown significantly less than that expected based upon seed set phenotype. In 1997, the weaker S phenotype set signifi- following pure self and pure cross pollinations. It appears cantly more seeds across the three treatments and in both that self pollen is discriminated against, even on old pistils. years there was a significant effect of floral position on the Consequently, the breakdown in S in C. rapunculoides outcrossing rate. In the first year, the outcrossing rate enhances the potential for selfing in the absence of cross increased from the lowest to the highest positions on the pollen but does not substantially reduce the potential for inflorescences and, in year 2, the outcrossing rate was outcrossing when cross and self pollen are both present on a highest in the middle of the inflorescence (at the peak stage stigma (Vogler and Stephenson, 2000). of flowering). This study reveals that the breeding system In the second study, we experimentally varied pollinator (outcrossing rate) of individual plants can vary with an availability (access of the pollinators to the plants). Other environmental condition (pollinator availability) that is investigators have found that pollinator availability in known from many studies to vary by year and location. In natural populations of plants can vary within years, short, plasticity in the S system of C. rapunculoides between years and among populations (see Stephenson translates into plasticity in the breeding system. et al., 1995 for review). Consequently, this study examines In the third study, the consequences of variation in SI on an environmentally relevant variable and it differs from our progeny vigour (inbreeding depression) were examined in a previous studies in that it relies on the foraging of natural multigenerational study (Vogler, Filmore and Stephenson, pollinators and on natural patterns of pollen deposition 1999). Controlled crosses created a range of inbreeding (mixed and pure pollen loads, variations in the ages of coefficients (0, 025, 0.50, 075) in families derived from flowers at the time of pollen deposition etc). Clones of strong and weak SI phenotypes. A sample of the progeny 20 plants with alternative alleles at the GPI locus [18 with from each of these families was scored for survivorship one allele, two with the alternative allele (the focal plants)] were grown in 2 m x 2 m x 2 m shadecloth cages in the (percentage germination, survivorship to the rosette stage field and exposed to pollinators for I h d-', 24 h every third and survivorship to flowering) and reproductive output day, or open pollinated. The three experimental treatments (flower number and seeds per fruit). We found that fitness were created by rolling up the sides of the cages for the declined significantly over the range of inbreeding coeffic- appropriate amount of time each day. We harvested the ients, that the decline in fitness was less severe for families mature fruits, counted the seeds, and scored the paternity derived from weak SI phenotypes than for families derived (self or outcross) of a sample of approx. 100 seeds from from strong S phenotypes (perhaps indicating a prior each focal plant. The clones for this study were created by history of inbreeding and a purging of some of the genetic repeatedly dividing the rootstalks and then synchronizing load), and that inbred maternal plants had a significant the flowering schedule by providing an appropriate cold non-genetic effect on the performance of their progeny - treatment. Our findings from the summers of 1997 and 1998 even when we compared progeny with the same (0 and 05) indicate that the outcrossing rate varied significantly with coefficient of inbreeding (Vogler et al., 1999). Our ongoing treatment (open pollination cages > I h cages > cages studies of the progeny from these same families indicates open every third day in both years; 1997 outcrossing that inbreeding also adversely affects the male function of rate = 077 open cages, 0-70 h cages, 035 open every C. rapunculoides (pollen production, pollen viability, speed third day; 1998 outcrossing rate = 055, 050, 040, of germination and pollen tube growth rates). Stephenson et al.-Self-incompatibility in Campanula rapunculoides 217 DISCUSSION self-pollination (before, during and after opportunities for outcrossing), and the relative performance (speed of Perhaps no area of plant population biology has received germination and pollen tube growth rates) of self and more theoretical and empirical attention than the evolution cross pollen in the pistils of flowers. of breeding systems. Fisher (1941) argued that genetic Our greenhouse studies of inbreeding depression in modifiers that promote self-fertilization without reducing C. rapunculoides(Vogler et al., 1999) reveal that the fitness cross-fertilization through the male (pollen) function of a selfed seed (f = 0.5) is, on average, only 10% of that of should increase due to the transmission of genes via pollen an outcrossed seed when one considers only survivorship to to both selfed and outcrossed progeny. Theoretical studies flowering and reproductive output through the female that assume independence between the level of inbreeding function. It is reasonable to assume that inbreeding depression and modifiers of the breeding system (selfing depression would be greater under field conditions than rate) predict that the viability of outbred offspring must under greenhouse conditions (e.g. Wolfe, 1993; Carr and Downloaded from https://academic.oup.com/aob/article/85/suppl_1/211/102914 by guest on 29 September 2021 exceed the viability of the inbred offspring two-fold in order Dudash, 1995). Moreover, our preliminary evidence to counteract this transmission advantage (e.g. Kimura, indicates that selfing also has an adverse effect on the 1959; Nagylaki, 1976; Maynard Smith, 1977; Charlesworth, male function of Campanula which would further reduce 1980b; and others). Pollen discounting (reductions in the value of selfed progeny. Even though we (Vogler et al., outcrossing through the male function due to enhanced 1999) found significant variation in the level of inbreeding selfing) and seed discounting (reductions in the production depression among families, the transmission advantage of of outcrossed seed due to enhanced selling) promote selfed seeds is unlikely to outweigh the disadvantages of outcrossing by reducing the minimum levels of inbreeding selfing (inbreeding depression, pollen and seed discounting) depression necessary to maintain outcrossing (see Nagylaki, when seed production is not limited by the availability of 1976; Holsinger et al., 1984; Lloyd, 1992; Harder and outcross pollen. Wilson, 1998). Most of the models that assume indepen- Campanula rapunculoides has at least three mechanisms dence between the level of inbreeding depression and that avoid selling and promote outcrossing. First, C. modifiers of the breeding system sellingg rate) exclude the rapunculoides is protandrous-the temporal separation of possibility of mixed mating systems (stable mixes of the male and female phases of each flower decreases the selling and outcrossing over evolutionary time) (but see opportunities for autogamy and pollinator mediated Uyenoyama, 1986; Holsinger, 1986 for special exceptions). transfer of self pollen within flowers. Secondly, the com- Models that permit the level of inbreeding depression to bination of protandry, acropetal inflorescence development change with the breeding system also rarely permit the and the foraging behaviour of the bumblebees (the upward evolution of mixed mating systems (Maynard Smith, 1977; movement of bees from female to male phase flowers within Lande and Schemske, 1985; Campbell, 1986; Damgaard, each inflorescence) reduces the amount of self pollen that is Couvet and Loeschcke, 1992). In short, there was a gap transferred within inflorescences. These mechanisms not between what is (mixed mating systems are not uncommon only avoid the adverse effects of selfing on progeny vigour in nature) and what ought to be (theoretically). but they also avoid pollen and seed discounting. Never- To bridge this gap, Holsinger (1988) and Charlesworth theless, geitonogamy undoubtedly occurs when the polli- and Charlesworth (1990) speculated that the failure of nators make the occasional downward movement within an simple models to predict the range of mating patterns inflorescence or move between inflorescences of the same observed in nature may reflect a violation of the assumption genotype. Thirdly, when self pollen is deposited onto a of independence between the genetic modifiers of the stigma of a young flower, an S-RNase based SI system breeding system and the causes of inbreeding depression prevents or greatly reduces the number of self-fertilizations. (viability loci). A series of theoretical studies investigating When a mixture of self and cross pollen is deposited onto the nature and magnitude of genetic associations that the stigmas of older flowers, the cross pollen is significantly evolve between loci influencing offspring viability and more likely to achieve fertilization than the self pollen due modifiers of SI (e.g. Uyenoyama, 1988) and self-fertilization to differences in the growth rates of self and cross pollen (Uyenoyama and Waller, 1991a,b,c) conclude that inbreed- tubes. [It should be noted, however, that while SI systems ing depression should not be defined at the level of the can avoid inbreeding depression and seed discounting, the population but at the level of families (individual plants and pollen deposited onto self stigmas is unavailable for out- their offspring) and that the associations between the loci crossing (i.e. it is discounted).] Together, these mechanisms determining inbreeding depression and modifiers of the (protandry, protandry/foraging behaviour, SI) promote breeding system (such as SI) together determine evolution- outcrossing and avoid the adverse consequences of selfing ary shifts in the breeding system. Another group of theor- when outcross pollen does not limit seed production. eticians have recently attempted to explain the range of However, when seed production is limited by the mating systems found in nature by including some com- availability of cross pollen on the stigmas (as would occur ponent of reproductive assurance in their models for the when pollinators are scarce, or when the size of the evolution and maintenance of mating systems (e.g. Schoen Campanula population is small, or when founding popu- and Lloyd, 1984; Lloyd, 1992; Holsinger, 1996; Harder and lations consist of only a few S-alleles) plasticity in the SI Wilson, 1998). These studies focus attention on the various system of C. rapunculoides allows selfing to occur. Our types of self-pollination (autogamy, pollinator mediated studies reveal that the SCI increases (SI breaks down) within flower pollen transfer, geitonogamy), the timing of with floral age (e.g. Richardson and Stephenson, 1989; 218 Stephenson et al.-Self incompatibility in Campanula rapunculoides Stephenson et al., 1992; Vogler et al., 1998, 1999). Consequ- ACKNOWLEDGEMENTS ently, if a flower is not outcrossed when it is young, it will We thank Christine Difolco, Kevin Filmore, Chandreyee permit at least some self-fertilization rather than abscising Das, Cortney Springstead, Lisa Copper, Tom Nagel, and without fruit set. Our studies also reveal that the amount of Christina Klescz for field, greenhouse and lab assistance, self seed that can be produced on old flowers increases when Tony Omeis and his staff at the Buckhout Greenhouse, and are developing on a raceme (when pollination of few fruits Bob Oberheim and his staff at The Pennsylvania State the earlier flowers on a raceme has been inadequate). University Agricultural Experiment Station at Rock Finally, when pollinators are scarce (when pollen has not Springs, PA, USA. This research was supported by NSF been removed from male phase flowers and when cross grant DEB 95-27739 to A.G.S. pollen has not been deposited onto the stigma), the fully reflexed stigmas of old female phase flowers are in close

proximity to the left over pollen and may autonomously self Downloaded from https://academic.oup.com/aob/article/85/suppl_1/211/102914 by guest on 29 September 2021 pollinate. In short, self-fertility in C. rapunculoides is LITERATURE CITED delayed until after most opportunities for outcrossing Ascher PD. 1984. Self-incompatibility in Petunia. Monographs on have occurred. Consequently, it has, in the words of Theoretical and Applied Genetics 9: 92-110. Becarra and Lloyd (1992), a breeding system that is the Ascher PD, Peloquin SJ. 1966. Effect of floral aging on the growth of best of both worlds-the advantages of outcrossing when compatible and incompatible pollen tubes in Lilium longiflorum. American Journal of' Botany 53: 99-102. outcross pollen is available and the higher seed set of a Baker HG. 1955. Self-compatibility and establishment after 'long- selfer when outcross pollen is unavailable. distance' dispersal. Evolution 47: 125--135. 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