Sex-Allocation Plasticity in Hermaphrodites of Sexually Dimorphic Fragaria Virginiana (Rosaceae)

231 Sex-allocation plasticity in hermaphrodites of sexually dimorphic Fragaria virginiana (Rosaceae)

Eric J. Bishop, Rachel B. Spigler, and Tia-Lynn Ashman

Abstract: Sex-allocation plasticity is thought to play an important role in the evolution of separate sexes in plants. Ac- cordingly, much attention has been paid to environmentally induced variation in fruit and seed production in sexually dimorphic species, but we know little about whether this variation arises as a direct response to environmental variation or is instead an indirect consequence of changes in plant size. In this study, we characterize sex-allocation plasticity across a resource gradient for several reproductive traits in hermaphrodites of gyno(sub)dioecious Fragaria virginiana Duch. We find significant plasticity, on average, for flower number, proportion fruit set, ovule number, proportion seed set, and runner number in response to resource variation. Plasticity of most traits examined tended to be at least partially independent of variation in plant size, suggesting that it is not simply an indirect consequence of plant allometry. Moreover, we find genetic variation for plasticity of key reproductive traits. Comparisons of relative plasticities among traits reveal that F. virginiana hermaphrodites are more likely to adjust female investment via changes in fruit and seed set than ovule number, and most likely to adjust male investment via flower number rather than anther number or pollen per anther, although there is genotypic variation for plasticity in pollen per anther. Evidence of within-population variation can logically be ex- tended to suggest that variation in hermaphrodite sex-expression seen among natural populations of F. virginiana may be due, at least in part, to sex-allocation plasticity. Key words: dioecy, gynodioecy, phenotypic plasticity, sex allocation, strawberry, subdioecy. Re´sume´ : On croit que la plasticite´ sexuelle joue un roˆle important dans l’e´volution de la se´paration des sexes chez les plantes. Conse´quemment, on a accorde´ beaucoup d’attention aux variations induites par le milieu dans la production de fruits et de graines chez les espe`ces sexuellement dimorphiques, mais nous connaissons peu de choses a` savoir si cette variation constitue une reáction directe au milieu ou plutoˆt une conse´quence indirecte de la modification des dimensions de la plante. Les auteurs ont caracte´rise´ la plasticite´ de l’allocation sexuelle le long d’un gradient de ressources portant sur des caracte`res reproductifs chez les plants hermaphrodites du Fragaria virginiana Duch. gyno(sub)dioı¨que. Ils ont observe´ une plasticite´ significative, en moyenne, pour le nombre de fleurs, la proportion des mises a` fruit, le nombre d’ovules, la proportion des mises a` graine, et le nombre de stolons, en reáction a` une variation des ressources. La plasticite´ des caracte`- res observe´s a tendance a` eˆtre au moins partiellement inde´pendante en grosseur des plants, ce qui sugge`re qu’il ne s’agit pas simplement d’une conse´quence indirecte de l’allome´trie. De plus, ils ont observe´ une variation geńe´tique pour la plasticite´ de caracte`res reproductifs cle´s. Des comparaisons de plasticite´s relatives entre les caracte`res re´ve`lent que les hermaphrodites du F. virginiana augmenteront plus vraisemblablement l’investissement femelle via des modifications dans la mise a` fruit et a` graine, que dans le nombre d’ovules, et ajusteront l’investissement maˆle via le nombre de fleurs plutoˆt que le nombre d’anthe`res ou la quantite´ de pollen par anthe`re, bien qu’il n’y ait pas de variation geńotypique pour la variation de pollen par anthe`re. On peut logiquement e´tendre la preuve de variation au sein de la population et sugge´rer que la variation dans l’expression sexuelle hermaphrodite observeé entre les populations naturelles du F. virginiana pourrait eˆtre due, au moins en partie, a` la plasticite´ de l’allocation sexuelle. Mots-cle´s:dioı¨que, gynodioı¨que, plasticite´ pheńotypique, allocation sexuelle, fraise, subdioećie. [Traduit par la Re´daction]

Introduction important phenomenon influencing the evolution of dimor- Sex-allocation plasticity, the ability of a hermaphrodite to phic sexual systems in plants (reviewed in Delph 2003; change allocation to male and female sex functions in re- Delph and Wolf 2005). Sex-allocation plasticity is thought sponse to changes in the environment, is thought to be an to influence both the likelihood that female individuals establish in hermaphroditic populations (i.e., the evolution of gynodioecy), as well as whether hermaphrodites are main- Received 29 July 2009. Published on the NRC Research Press tained once females are abundant and males exist (i.e., sub- Web site at botany.nrc.ca on 26 February 2010. dioecy). For example, in low resource environments, if E.J. Bishop, R.B. Spigler,1 and T.-L. Ashman.2 Department of hermaphrodites preferentially reduce investment in female Biological Sciences, University of Pittsburgh, Pittsburgh, PA function to maintain or increase male function, females 15260-3929, USA; Pymatuning Laboratory of Ecology, could more easily achieve the seed fertility advantage re- Linesville, PA 16424, USA. quired to invade and establish (Delph 1990, 2003; Dorken 1Corresponding author (e-mail: [email protected]). and Mitchard 2008). In resource rich environments, how- 2Corresponding author (e-mail: [email protected]). ever, sexually plastic hermaphrodites that take advantage of

Botany 88: 231–240 (2010) doi:10.1139/B10-005 Published by NRC Research Press 232 Botany Vol. 88, 2010 reduced trade-offs between male and female function and in- 1985; Sutherland 1987; Ashman and Penet 2007). Because crease investment in female function will reduce females’ genetic, functional, and (or) developmental constraints vary seed-fertility advantage (Dorken and Mitchard 2008), and among traits (reviewed in DeWitt et al. 1998; van Kleunen ultimately, this will be reflected in the equilibrium fre- and Fischer 2005) and trait plasticities as well (Waitt and quency of females. Observations of higher female frequen- Levin 1993; Pigliucci et al. 2003), some traits may be less cies and greater sex specialization in gynodioecious canalized than others. While previous work has revealed populations under resource-poor environments (Delph 1990; phenotypic and genetic correlations among reproductive Costich 1995; Wolfe and Shmida 1997; Ashman 1999a; traits in hermaphrodites (reviewed in Ashman 2003), we do Vaughton and Ramsey 2002; Delph 2003; Barr 2004; Case not know whether plasticities of these traits are integrated. and Barrett 2004; also see citations in Ashman 2006) are Given the predicted differences in fitness gain curves in re- consistent with these hypotheses. lation to resource availability and (or) plant size for male Yet in spite of the importance of the role that plasticity and female function, we might expect female-function traits, may play in sexual system evolution, we still know rela- in general, to be more plastic than male-function traits, as tively little about the mechanisms underlying variation in has been shown in other studies (Vogler et al. 1999; Sarkis- hermaphrodite sex allocation across natural resource gra- sian et al. 2001; Wolfe and Mazer 2005), but we do not dients. It is often assumed that this variation is due to plasti- know whether specific components of sex expression are city, but it may simply reflect past divergent selection on more important than others as conduits to variable sex ex- sex allocation across environments (Ashman 2006; Case pression. and Ashman 2007). Even if the variation in sex expression In this study, we characterize plasticity of multiple com- seen across environments is due to plasticity, we do not ponents of sex-expression in hermaphrodites of gyno(sub)- know whether such variation represents a direct response to dioecious Fragaria virginiana Duch. in response to resource variation or whether it is merely an indirect conse- variation in resource availability. Fragaria virginiana quence of changes in plant size with resource availability. presents an ideal system in which to study sex-allocation Such a distinction is important because it can inform on plasticity for several reasons. First, it is clonal, thus a given whether sex-allocation plasticity in response to resource genotype is prone to encounter different environments in a availability is a separate phenomenon from well-described heterogeneous habitat. Second, this clonality lends itself to allometric plant responses or size-dependent sex allocation experimental replication of genotypes in a controlled design. (de Jong and Klinkhamer 1989; Klinkhamer et al. 1997; Sar- Third, previous research suggests that plasticity of at least kissian et al. 2001; Zhang and Jiang 2002; Paquin and Aars- one trait, proportion fruit set, occurs in hermaphrodites and sen 2004; Ashman 2006). Theories based on both is associated with among-population variation in sex ratio mechanisms (direct vs. size-mediated) have similar predic- (i.e., the frequency of females) (Ashman 2006). Specifically, tions: individuals at low resources or small size should in- we asked whether reproductive traits are phenotypically vest more heavily in male than female function because of plastic in hermaphroditic F. virginiana, and whether there is the high cost of fruits relative to pollen, whereas individuals genetic variation for plasticity of these traits. In addition, we at high resources or large plant sizes should invest more in compared trait plasticities to determine whether certain traits female function. This latter prediction arises because of di- are more plastic than others. In particular, we predicted fe- minishing fitness returns associated with increased invest- male function traits would be more plastic than male ones. ment in male function with increasing resources (or size), in Lastly, we evaluated the extent to which trait plasticities are contrast to increasing fitness returns associated with in- phenotypically integrated by estimating correlations among creased investment in female function. Several experimental trait plasticities. The results of this study will not only in- studies involving species with dimorphic sexual systems form on the basis of phenotypic variation in sex expression have demonstrated changes in components of sex expression in hermaphrodites of a gyno(sub)dioecious species but also with changes in resource environment (Delph and Lloyd provide a foundation for addressing more mechanistic ques- 1991; McArthur et al. 1992; Diggle 1993; Dorken and Bar- tions concerning the relationship between sex-allocation rett 2004; Ashman 2006; Dorken and Mitchard 2008; plasticity and sexual system evolution. Dorken and Pannell 2008; Pannell et al. 2008) and others have done so with respect to changes in plant size (Ashman Materials and methods et al. 2001; Sarkissian et al. 2001), but to our knowledge none have experimentally teased these two apart. Study system Most studies examining variation in hermaphrodite sex Fragaria virginiana (Rosaceae), the Virginian wild straw- expression in natural populations have focused on fruit and berry, is a clonal perennial herb native to eastern North seed production, presumably because of the importance of America (Staudt 1989), where it commonly inhabits mead- relative seed fertility between females and hermaphrodites ows, old fields, and road and forest edges. Reproduction oc- for female invasion. However, hermaphrodites may alter curs sexually by seed and asexually by creeping stolons or their sex expression through adjusting a variety of reproduc- ‘‘runners’’. The sexual system of F. virginiana may be con- tive traits. For instance, hermaphrodites may adjust invest- sidered gynodioecious to subdioecious (sensu Sakai and ment in female function via ovule production, proportion Weller 1999), depending on whether populations contain fe- seed set, or proportion fruit set, and male investment may males and hermaphrodites or contain females, hermaphro- be altered via the number of anthers produced in each dites and males, respectively. Recent genetic analysis flower, pollen production, or total flower number, often revealed that sexual phenotype is determined by two closely seen as an important component of male function (e.g., Bell linked gene regions with limited recombination that account

Published by NRC Research Press Bishop et al. 233 for the presence of three genders (females, hermaphrodites, we are confident that this design created the intended simu- and males), as well as the occasional neuter in subdioecious lated resource gradient based on measurements of plant size populations (Spigler et al. 2008). While female fruit-setting at the end of the experiment. Specifically, mean plant size, ability is relatively consistent and high and some pollen- estimated as the product of leaf number and leaf size (fol- bearing individuals do not produce fruit under any condi- lowing Ashman 1999a), was significantly different among tions (i.e., males), the proportion of fruit set among her- all treatments, increasing in the expected direction from RE maphrodites is highly variable (0.05–0.80), (Staudt 1989; 1 to RE 4 as shown in Fig. 1 (P < 0.0001 for all pairwise Ashman 2003, 2006; Spigler et al. 2008). Female frequen- comparisons). cies in northwestern Pennsylvanian wild populations range from approximately 0.15 to almost 0.50 (Ashman 1999a; Plant traits T.-L. Ashman and R. Spigler, unpublished data). Flowering Plants began to flower in early May 2007. For each plant, occurs between late April to mid-June (Ashman 1999a). we collected one bud at the secondary position (as in Ash- Hermaphrodites are self-compatible and have a mixed mat- man and Hitchens 2000) after the first flower opened and ing system (Rohde and Ashman 2010), and there is no evi- stored them in vials of 70% EtOH until processing. For dence of inbreeding depression at the seed formation stage each bud, we counted the number of anthers and ovules, col- (T.-L. Ashman, unpublished data). lected two anthers, and counted pollen using a particle coun- ter (Elzone II 5390 V1.03, Norcross, Georgia, USA) Plant material and cultivation following Ashman and Hitchens (2000). We estimated pol- We selected nine hermaphrodite genotypes for this study len per anther using these data. that originated from a wild population located in Crawford To ensure full potential seed fertilization, we supple- County, Pennsylvania (‘‘PR’’ in Ashman 1999a). Prior to mented natural pollination with hand-pollinations three this study, genotypes were serially cloned in the greenhouse times per week. All supplemental pollen came from potted for several years to eliminate environmental maternal ef- wild strawberry plants that originated from the same source fects. In Summer 2006, we generated 20 clones (plantlets) population as the study plants. Upon fruit maturation, we per genotype. We initially rooted each plantlet in a 95 mL collected between one to five fruits per plant, depending on pot containing a 2:1 Fafard No. 4 soil – sand mixture. Once availability, in vials of 70% EtOH and later dissected them they established roots, we transplanted half of the clones of to enumerate developed, fertilized ovules, i.e., seeds, which each genotype into 280 mL pots containing the same soil are easily distinguishable by size under a dissecting scope mixture. The other half remained in the 95 mL pots. In No- from undeveloped, presumably unfertilized ovules. How- vember 2006, we moved the study plants to Pymatuning ever, we note that we cannot distinguish between unfertil- Laboratory of Ecology (Crawford Co., Penn.; 41834’N, ized and early-aborted fertilized ovules. For each plant, we 80827’W), where they overwintered. recorded total flower and fruit number and calculated pro- In April 2007 we established a gradient of resource avail- portion fruit set as the number of fruits divided by the num- ability via a combination of pot sizes and fertilizer regimes ber of flowers produced per plant. We calculated proportion at Pymatuning Laboratory of Ecology. One half of the seed set as the number of seeds divided by the total number clones in the smaller pots did not receive any fertilizer and of ovules per fruit. When more than one fruit per genotype represented the lowest resource environment (hereinafter, re- was scored, we calculated seed set as the average across ferred to as ‘‘resource environment 1’’ or ‘‘RE 1’’). The fruits. We also recorded runner number, a measure of asex- other half of the small pots each received approximately ual reproduction, at the end of flowering. 180 mg of Nutricote 13–13–13 N–P–K fertilizer (Sun Gro Plasticity is most often measured as the difference be- Horticulture, Bellevue, Washington, USA) (‘‘resource envi- tween trait means across environments. However, because ronment 2’’ or ‘‘RE 2’’). For one half of clones in the larger our design incorporates more than two environments, this pot size, we did not apply any fertilizer, creating the next calculation is not as straightforward. Therefore, we esti- highest resource environment (‘‘resource environment 3’’ or mated plasticity as the coefficient of variation (CV) for ‘‘RE 3’’), and the remaining clones in large pots received each trait (e.g., Schlichting and Levin 1984; Wolfe and *360 mg of Nutricote each to establish the highest resource Mazer 2005; Caruso 2006). For each trait we first calculated environment (‘‘resource environment 4’’ or ‘‘RE 4’’). Each the within-resource environment mean for each genotype genotype had five replicate clones in each of the four re- and then we calculated the mean and standard deviation source environments. We randomly assigned one clone of across resource environments for each genotype-trait combi- each resource environment – genotype combination to one nation and used these to estimate the CV. By using the CV of five blocks and within each block, randomly assigned as an estimate of plasticity, we can compare the plasticity of their placement. We fertilized all plants again in May to different traits and genotypes irrespective of their means, maintain plant health: RE 1 and 2 received *60 mg of Nu- i.e., we can examine the relative amounts of variation (i.e., tricote each, and RE 3 and 4 plants received *180 mg. plasticity) for each trait and genotype across all environ- Thus, the total amount of fertilizer applied in the spring to ments. each treatment was: 60 mg for RE 1; 240 mg for RE 2; 180 mg for RE 3; and 540 mg for RE 4. We also watered Statistical analyses plants daily throughout the duration of the experiment. Be- All analyses were performed using SAS version 9.1 (SAS cause small pots tend to dry out more quickly than larger Institute Inc. Cary, North Carolina, USA). We performed pots, water and nutrient availability were likely correlated. two analyses to determine whether the examined traits ex- Thus although we cannot identify the exact resource cue, hibited plastic variation across the resource gradient. First,

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Fig. 1. Reaction norms for plant size, illustrating the response of therefore, we proceeded by obtaining size-adjusted residuals nine hermaphrodite Fragaria virginiana genotypes to the experi- using within-treatment regressions and adding these resid- mental resource gradient created using four resource environments uals to the appropriate within-treatment size-adjusted mean. (RE). Different symbols and corresponding lines refer to the nine Block effects were also removed where appropriate. We unique genotypes and the mean response across genotypes. The subsequently examined the effect of genotype, environment, mean response is indicated by the thicker line (red colouration, web and their interaction on the size-adjusted trait values for version only). each trait (e.g., Hoverman and Relyea 2008) in PROC 600 MIXED as described above. We compared plasticity among traits using ANOVA 500 (PROC GLM). Because coefficient of variation is a relative measure, comparisons among trait plasticities are feasible 400 because variation in the scale of trait values is taken into account. Plasticity values were log transformed to improve 300 normality. We used Tukey’s HSD test for post hoc comparisons to evaluate which trait plasticities were significantly Plant size 200 different in magnitude. 100 We used correlation analysis (PROC CORR) to evaluate the degree of phenotypic integration among trait plasticities, 0 estimated as the coefficient of variation (CV). CV for anther RE 1 RE 2 RE 3 RE 4 number was log transformed to fit a normal distribution. We did not examine correlations among phenotypic trait means Resource environment in this study. Readers interested in phenotypic and genetic correlations among trait means in this species should refer we used a mixed model ANOVA (PROC MIXED) to evalu- to previous work (Ashman 1999a, 2003). ate the effect of genotype (random effect), resource environment (fixed effect), and their interaction (random effect) on all reproductive traits. Block was also included as a fixed ef- Results fect in initial models and left in the model where its effect Resource environment significantly affected all traits ex- was significant. We log transformed flower number, runner cept for anther number per flower and pollen per anther, in- number, and anther number and arcsine square-root trans- dicating that most reproductive traits are plastic (Table 1A). formed seed set to conform to ANOVA assumptions of nor- In each of the cases where the main effect of environment mality. Proportion fruit-set data could not be normalized, so was significant, overall mean trait values across genotypes we used a mixed generalized linear model (PROC GLIM- increased along the resource gradient from RE 1 to 2 to 3 MIX) and treated proportion fruit set as a probability follow- to 4 (Figs. 2A–2G). For pollen production, only some geno- ing a binomial distribution. Significance of random effects types responded plastically to resource availability (signifi- in all models were evaluated in a hierarchical fashion by re- cant genotype-by-environment interaction, hereinafter moving one effect at a time and performing a likelihood ra- ‘‘GÂE’’; Table 1A). There was also significant genetic var- tio test using –2 REML Log Likelihood scores of the iation for plasticities of flower number and proportion fruit competing models (Littell et al. 1996). The difference be- set (significant GÂE; Table 1A). Not surprisingly, genotype tween these likelihood scores approximately follows a c2 significantly affected all traits examined (Table 1A). distribution and was tested with 1 df (Littell et al. 1996). In Results based on size-adjusted trait values can inform on all of these analyses, a significant main effect of environ- whether traits are plastic in direct response to resource avail- ment indicates that a trait is plastic, whereas a significant ability or whether the effect of the resource environment is genotype effect reflects genetic variation in the trait. A sig- mediated through variation in plant size only. When traits nificant genotype-by-environment interaction indicates that were adjusted for variation in plant size, the main effect of genotypes vary in their response to resource environment, resource environment remained significant for proportion which can be interpreted as broad-sense genetic variation in fruit set, proportion seed set, and runner number plasticity for a given trait. (Table 1B). In addition, the effect of the resource environ- In a second analysis, we used size-adjusted trait values to ment became significant for pollen production and anther determine whether resource environment resulted in differ- number (Table 1B). Thus, for all reproductive traits except ences in trait values beyond those attributable to general flower number and ovule number, plasticity of those traits, plasticity in plant size. We calculated size-adjusted values on average, is not merely an indirect consequence of re- by performing an ANCOVA on each trait with resource en- source-based changes in plant size. However, for some gen- vironment as the main effect and plant size as the covariate otypes, flower number did vary in direct response to and obtained size-adjusted treatment means (LSMEANS in resource availability (GÂE; Table 1B). Similar results were PROC GLM). We examined the interaction between re- found for size-adjusted values of fruit number and pollen per source environment and plant size to evaluate whether the anther (Table 1B). As in the initial analyses, genotype sig- assumption of a common allometric relationship between nificantly affected all size-adjusted traits examined size and the response variable among resource environments (Table 1B; Fig. 2), indicating broad sense genetic variation (i.e., a non-significant interaction) was met (McCoy et al. for all traits examined. 2006). This assumption was met for all traits examined, We detected significant variation among traits in their de-

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gree of plasticity (F = 34.74, P < 0.0001). Runner number was the most plastic of all traits (Fig. 3), and was signifi- 0.05; *,

£ cantly more plastic than flower number, pollen production, P ovule production, and anther number. Among sexual traits, female function traits tended to be more plastic than male function traits, as predicted. Proportion fruit set was the most plastic sexual trait, significantly more so than ovule 16.5*** 0.6 number or anther number, the least plastic traits. Most correlations among trait plasticities were strong

= 3,162. Only significant random (Table 2), although there was no general pattern in the di- < 0.0001, **, 0.0001 < c size.

P rection of the relationships among plasticities. Plasticity for flower number and plasticity for proportion fruit set and proportion seed set were negatively correlated, indicating that = 3,122; ******** 18.2*** 0.0 8.1** 3.4* ** 0.1 a a c b c a a b individuals highly plastic for flower number were relatively canalized for these two female function traits (Table 2). Equally strong was the positive correlation between plasticity for proportion fruit set and plasticity for proportion

= 3,19–24.5; seed set (Table 2), suggesting that these female function trait a plasticities are positively integrated. E) on flower number, proportion fruit set, ovule number per Â Discussion Our results show that plasticity exists for most reproduc-

B tive traits in hermaphrodites of F. virginiana and that plasti-

-values for these statistics are indicated (***, city is genetically variable for many of these. Importantly, P by accounting for general plasticity in plant size in response E’’.

Â to resource variation, we were able to disentangle size-

for E. dependent responses from those directly attributable to re- F source availability and demonstrate that plasticity of most traits examined was not the sole consequence of plant size variation. This result suggests that sex-allocation plasticity

0.09.1** 33.0*** 45.8***is 6.02 7.93 a phenomenon distinct from size-dependent sex allocation and (or) plant allometry (Klinkhamer et al. 1997; Zhang and Jiang 2002) or at least that the relationship between sex expression and size is itself plastic (Vitt et al. = 0.002). 2003) and deserves more attention to understand sexual P

value is presented instead of system evolution. Finally, our comparison among plastici- 2

c ties for several traits revealed that fruit-setting ability is ****** 12.1** 5.7** 67.2*** 142.5*** 2.13 3.12 *** 1.1 49.9*** 5.04 = 5.90, a a c ** 0.3 73.9*** 4.18 *** 0.1 68.5*** 1.30

c b c a the most plastic and anther number the least plastic among

[4,165] the sexual traits examined. F Significant plasticity for fruit set was also seen in a previous study in F. virginiana in which plants were grown in

values are given for both random effects, ‘‘G’’ and ‘‘G a greenhouse versus a field environment (Ashman 2006), but 2 c

tests of significance of each random effect are equal to one. was not seen in another study in which resources were ma- 2

c nipulated in experimental arrays (Case and Ashman 2007). Differences among the latter study and the current one are likely related to differences in the pollination environments, range of variation in resource availability across treatments, and (or) diversity of hermaphrodite genotypes included in A 701.8*** 16.65 the studies. This study goes beyond previous ones on plastic responses in strawberry (Ashman et al. 2001; Ashman 2006) by examining a wider range of resource levels and traits, as well as specifically evaluating relationships among trait {

, plasticities. Many studies on sex-allocation plasticity in species with sexually dimorphic breeding systems have focused solely on fruit and seed production (reviewed in Delph and Wolf 2005). However, hermaphrodites may alter sex expres- 0.10). Degrees of freedom for ‘‘E’’ vary according to whether random terms were left in the model and are indicated by subscripts: Results from ANOVA examining the effect of genotype (G), resource environment (E), and their interaction (G -statistics are given for ‘‘E’’ (a fixed effect) and sion through a variety of mechanisms. Our study reveals that £ F

P hermaphrodites of F. virginiana can alter total seed produc-

There was a significant effect of block for fruit set in analysis A ( tion in response to variation in resource availability via plas- Note: *Fruit set was analyzed in A using PROC GENMOD (SAS version 9.1). A { TraitFlower number 85.8*** G 12.50 E GxE G E GxE Proportion fruit set* Proportion seed set 79.7*** 5.24 Pollen per anther 52.2*** 1.92 Ovule numberAnther numberRunner number 87.5*** 41.6*** 47.6*** 7.42 0.62 56.25 0.05 < flower, proportion seed set, anther number per flower, pollen per anther, and runner number before (A) and after (B) accounting for variation in plant Table 1. terms were left in the model. Degrees of freedom for ticity in flower number, proportion fruit set and proportion

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Fig. 2. Reaction norms for (A) flower number per plant, (B) proportion fruit set, (C) ovule number per flower, (D) proportion seed set, (E) anther number per flower, (F) pollen per anther, and (G) runner number for nine genotypes of Fragaria virginiana hermaphrodites across four resource environments. See legend for explanation of the different symbols, their corresponding lines refer to unique genotypes, and the mean response across genotypes. The mean response is indicated by the thicker lines (red colouration, web version only). 30 1.0 AB 25 0.8 20 0.6 15 0.4 10

Flower number

Proportion fruit set 0.2 5

0 0.0 RE 1 RE 2 RE 3 RE 4 RE 1 RE 2 RE 3 RE 4 Resource environment Resource environment 110 1.0 100 CD 0.8 90 80 0.6

70 0.4

Ovule number 60

Proportion seed set 0.2 50

40 0.0 RE 1 RE 2 RE 3 RE 4 RE 1 RE 2 RE 3 RE 4 Resource environment Resource environment

28 16000 EF14000 26 12000

24 10000 8000 22 6000

Anther number

Pollen per anther 4000 20 2000 18 0 RE 1 RE 2 RE 3 RE 4 RE 1 RE 2 RE 3 RE 4 Resource environment Resource environment 8 7 G o76613 o17 6 o254 5 o410 4 o432 3 o435 o456 Runner number 2 o516 1 o564 0 Mean RE 1 RE 2 RE 3 RE 4 Resource environment

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Table 2. Correlations among plasticities (CV) for reproductive traits of F. virginiana hermaphrodites.

Flower Ovule Anther Pollen per Runner number Fruit set number Seed set number anther number Flower number –0.73 0.46 –0.66 –0.24 –0.18 0.00 Fruit set –0.19 0.78 0.53 0.10 –0.02 Ovule number –0.06 0.16 –0.03 –0.34 Seed set 0.08 0.49 –0.18 Anther number –0.34 –0.39 Pollen per anther –0.40 Runner number Note: Correlation coefficients in bold font are significant (P £ 0.05). No correlations remained significant after a sequential Bonferroni correction.

Fig. 3. Mean plasticity (CV) (±1 SE) for reproductive traits of Fra- types showed the greatest diversity of responses for pollen garia virginiana hermaphrodites. Significant differences among production per anther; in addition to canalized phenotypes, traits are indicated by different lower-case letters. some genotypes increased pollen production per anther in 0.7 response to increased resource availability whereas others a decreased it. Together, the variation in plasticity among gen- ab 0.6 otypes suggests that individuals may respond to selection on 0.5 sex-allocation plasticity in F. virginiana, if broad sense ge- ab netic variation is at least modestly representative of narrow (CV) 0.4 b sense genetic variation, as has been shown for mean values of reproductive traits in previous studies of F. virginiana 0.3 b (Ashman 1999b, 2003). Whether plasticity is adaptive, how- 0.2 ever, and whether certain responses or strategies confer a Plasticity c greater fitness advantage remains to be determined. Indeed, 0.1 d plasticity for reproductive traits that influence sex-allocation could instead be maladaptive, representing a genotype’s in- 0.0 r n n r e r e e r r ability to maintain an optimal phenotype under a stressful r io io e r n h l e e r nRNe FSrt SSrt FNe POe t OVu b ANh e n b t w ll n v t b u o t o e o b a m n or harsh environment rather than an opportunistic response m p e p s l o O m R o s o F m P r u A u r t r d u e n u n P i P e n p n u e in a resource-rich environment (Smith-Gill 1983; Dorn et fr s Trait al. 2000; Alpert and Simms 2002; van Kleunen and Fischer 2005). Thus an important next step is to evaluate how plas- seed set. Moreover, by examining a wide range of traits we ticity of reproductive traits influences fitness — both male were able to evaluate plasticity in male function as well and and female components — across environments in address the degree of phenotypic integration among trait F. virginiana using selection gradient analysis (Lande and plasticities. In particular, we found that plasticities for pro- Arnold 1983; Weis and Gorman 1990; Scheiner 2006), as portion fruit and seed set were positively integrated, but has been done in studies on plasticity related to shade avoid- that these two were negatively correlated with plasticity for ance (e.g., Dorn et al. 2000; Weijschede´ et al. 2006) and flower number, indicating strong trade-offs between clonal foraging (van Kleunen and Fischer 2001). plasticities of these male- and female-function traits. Such While hermaphrodites were able to alter sex expression trade-offs may constrain the seed fertility of hermaphrodites through a variety of mechanisms, certain traits were signifi- in resource-rich habitats. Whether positive integration cantly more plastic than others (Fig. 3). In general, plasticity among trait plasticities represents a common genetic or de- for asexual reproduction was high and was greater than that velopmental basis or is the result of selection to maintain for sexual traits. This result is consistent with evidence from an integrated phenotype, as suggested by Waitt and Levin previous studies suggesting floral or inflorescence traits are (1993) in a study on floral trait plasticities in Phlox, is unless plastic than vegetative traits (Schlichting and Levin known. Experimental work, such as artificial selection 1986; Frazee and Marquis 1994; van Kleunen et al. 2002; studies, could serve to disentangle the possible mechanisms Caruso 2006). However, in our study, plasticities of fruit set responsible for phenotypic integration of trait plasticities and seed set were statistically indistinguishable from plasti- (e.g., Brakefield 2003). city in runner number, suggesting that some sexual traits can Our data revealed striking contrasts among genotypes in exhibit relatively high plasticity (Fig. 3). Among sexual their response to resource availability for flower number, traits, we predicted that traits related to female function fruit set, and pollen production per anther (Fig. 2). For in- would be more plastic than those related to male function. stance, genotypes o17, o435, and o76613 were extremely Although small sample size likely reduced our power to de- plastic with respect to flower number, in contrast to the tect significant differences among all pair-wise trait compar- highly canalized responses of genotypes o410, o516, and isons, female traits, with the exception of ovule number, o456 across the resource gradient. Consistent with a trade- were more plastic than male traits. Variation in the magni- off between plasticity for flower number and fruit set, this tude of plasticity among these traits can be due to different pattern switched for fruit set: o435 and o76613 showed can- genetic, physiological, and (or) developmental constraints alized responses whereas o456 was highly plastic. Geno- (reviewed in DeWitt et al. 1998; van Kleunen and Fischer

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2005). While we do not know the precise mechanism under- This is contribution number 236 to the Pymatuning Labora- lying the variation in this study, we suggest that develop- tory of Ecology. mental constraints might explain why other traits were less plastic than proportion fruit and seed set. For example, the References degree to which a trait can respond to the environment may Alpert, P., and Simms, E.L. 2002. The relative advantages of plas- be a function of the time needed between sensory of the re- ticity and fixity in different environments: when is it good for a source environment and production of the trait (DeWitt et al. plant to adjust? Evol. Ecol. 16(3): 285–297. doi:10.1023/ 1998). Since fruit and seed maturation occur latest in the A:1019684612767. season, these traits may be more plastic because there has Ashman, T.-L. 1999a. Determinants of sex allocation in a gyno- been sufficient time between sensory and initiation of a re- dioecious wild strawberry: implications for the evolution of source-based response or because there has been a longer dioecy and sexual dimorphism. J. Evol. Biol. 12(4): 648–661. period of resource accumulation for fruit and seed produc- doi:10.1046/j.1420-9101.1999.00059.x. tion than for anther or ovule number, the latter two of which Ashman, T.-L. 1999b. Quantitative genetics of floral traits in a gy- are determined in the fall when flower bud primordia are nodioecious wild strawberry Fragaria virginiana: implications formed. Similar developmental timing could also explain in for the independent evolution of female and hermaphrodite part why plasticity for proportion fruit and seed set are floral phenotypes. Heredity, 83(6): 733–741. doi:10.1046/j. highly correlated. 1365-2540.1999.00639.x. PMID:10651918. In conclusion, these results demonstrate that hermaphro- Ashman, T.-L. 2003. Constraints on the evolution of males and sexual dimorphism: field estimates of genetic architecture of re- dites of F. virginiana are capable of altering investment in productive traits in three populations of gynodioecious Fragaria male and female function in response to varying resource virginiana. Evolution, 57(9): 2012–2025. PMID:14575323. availability, and that they may do this by varying proportion Ashman, T.-L. 2006. The evolution of separate sexes: a focus on fruit and seed set rather than ovule number and by varying the ecological context. In The Ecology and Evolution of Flow- flower number and pollen production rather than anther ers. Edited by L.D. Harder and S.C.H. Barrett. Oxford Univer- number. We note that the results from this study are based sity Press, New York, N.Y. pp. 204–222. on hermaphrodites from a single population, and thus we do Ashman, T.-L., and Hitchens, M.S. 2000. Dissecting the causes of not know whether the degree of plasticity seen in this study variation in intra-inflorescence allocation in a sexually poly- is representative of F. virginiana populations on the whole. morphic species, Fragaria virginiana (Rosaceae). Am. J. Bot. Future work will evaluate sex-allocation plasticity in a con- 87(2): 197–204. doi:10.2307/2656906. PMID:10675306. trolled pollination and resource environment across multiple Ashman, T.-L., and Penet, L. 2007. Direct and indirect effects of a populations to better characterize the degree of sexual plas- sex-biased antagonist on male and female fertility: consequences ticity among hermaphrodites. Whether sex allocation plasti- for reproductive trait evolution in a gender-dimorphic plant. Am. city as found in this study is an important factor influencing Nat. 169(5): 595–608. doi:10.1086/513150. PMID:17427131. sexual system evolution will depend on the frequency of Ashman, T.-L., Pacyna, J., Diefenderfer, C., and Leftwich, T. 2001. plastic hermaphrodite individuals in natural populations and Size-dependent sex allocation in a gynodioecious wild straw- whether sex-allocation plasticity confers a fitness advantage berry: The effects of sex morph and inflorescence architecture. over fixed-sex (male or female) individuals and (or) canal- Int. J. Plant Sci. 162(2): 327–334. doi:10.1086/319571. Barr, C.M. 2004. Soil moisture and sex ratio in a plant with nu- ized hermaphrodites. Ultimately, we expect a connection be- clear–cytoplasmic sex inheritance. Proc. R. Soc. Lond. B Biol. tween the frequency of plastic hermaphrodites and the sex Sci. 271(1551): 1935–1939. doi:10.1098/rspb.2004.2820. ratio in natural populations of this subdioecious species as Bell, G. 1985. On the function of flowers. Proc. R. Soc. Lond. has been seen for other species with sexually dimorphic B Biol. Sci. 224(1235): 223–265. doi:10.1098/rspb.1985.0031. breeding systems (e.g., Wolfe and Shmida 1997), unless Brakefield, P.M. 2003. Artificial selection and the development of plastic hermaphrodites are maintained due to other features ecologically relevant phenotypes. Ecology, 84(7): 1661–1671. of population dynamics such as colonization (Pannell 1997). doi:10.1890/0012-9658(2003)084[1661:ASATDO]2.0.CO;2. Understanding this pattern in the context of recent work on Caruso, C.M. 2006. Plasticity of inflorescence traits in Lobelia si- the genetics of sex determination in F. virginiana (Spigler et philitica (Lobeliaceae) in response to soil water availability. al. 2008), e.g., whether plasticity is related to genotype at Am. J. Bot. 93(4): 531–538. doi:10.3732/ajb.93.4.531. the sex loci, can further reveal how plasticity in sex expres- Case, A.L., and Ashman, T.-L. 2007. An experimental test of the sion is related to the evolution of dioecy at the genetic level. effects of resources and sex ratio on maternal fitness and phenotypic selection in gynodioecious Fragaria virginiana. Evolution, Acknowledgements 61(8): 1900–1911. doi:10.1111/j.1558-5646.2007.00148.x. PMID:17683432. The authors wish to thank R. Botham, C.L. Collin, Case, A.L., and Barrett, S.C.H. 2004. Environmental stress and the S. Good, E. Korns, R. Pileggi, H. Tam, and E. York for field evolution of dioecy: Wurmbea dioica (Colchicaceae) in Western and greenhouse assistance, the staff at Pymatuning Labora- Australia. Evol. Ecol. 18(2): 145–164. doi:10.1023/B:EVEC. tory of Ecology for logistical assistance, and R. Relyea for 0000021152.34483.77. statistical advice. Ideas presented in the manuscript bene- Costich, D.E. 1995. Gender specialization across a climatic gradi- fited from discussions with M. Dorken, J. Pannell, and ent — experimental comparison of monoecious and dioecious L. Delph. This manuscript was improved by comments Ecballium. Ecology, 76(4): 1036–1050. doi:10.2307/1940914. from L. Galloway, J. Auld, two anonymous reviewers, and de Jong, T.J., and Klinkhamer, P.G.L. 1989. Size-dependency of the editor. This work was supported by the National Science sex-allocation in hermaphroditic, monocarpic plants. Funct. Foundation (DEB 0449488 and REU supplements) to T.L.A. Ecol. 3(2): 201–206. doi:10.2307/2389301.

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