Female reproductive success in gynodioecious Thymus vulgaris: pollen versus nutrient limitation and pollinator foraging behaviour

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1 bs_bs_banner Botanical Journal of the Linnean Society, 2014, 175, With 3 figures Female reproductive success in gynodioecious Thymus vulgaris: pollen versus nutrient limitation and pollinator foraging behaviour XVIER RNN 1,2 *, NN ESCOLÀ 3, NSELM RODRIGO 1,3 and JORDI BOSCH 1 1 CREF, Cerdanyola del Vallès 08193, Catalonia, Spain 2 Faculty of Biology, TU Darmstadt, Schnittspahnstrasse 3, D Darmstadt, Germany 3 Universitat utònoma Barcelona, Cerdanyola del Vallès 08193, Catalonia, Spain Received 4 ugust 2013; revised 10 January 2014; accepted for publication 28 March 2014 Gynodioecy is a dimorphic breeding system in which female individuals coexist with hermaphroditic individuals in the same population. Females only contribute to the next generation via ovules, and many studies have shown that they are usually less attractive than hermaphrodites to pollinators. Several mechanisms have been proposed to explain how females manage to persist in populations despite these disadvantages. The resource reallocation hypothesis (RRH) states that females channel resources not invested in pollen production and floral advertisement towards the production of more and/or larger seeds. We investigated pollination patterns and tested the RRH in a population of Thymus vulgaris. We measured flower display, flower size, nectar production, visitation rates, pollinator constancy and flower lifespan in the two morphs. In addition, we measured experimentally the effects of pollen and resource addition on female reproductive success (fruit set, seed set, seed weight) of the two morphs. Despite lower investment in floral advertisement, female individuals were no less attractive to pollinators than hermaphrodites on a per flower basis. Other measures of pollinator behaviour (number of flowers visited per plant, morph preference and morph constancy) also showed that pollinators did not discriminate against female flowers. In addition, stigma receptivity was longer in female flowers. ccordingly, and contrary to most studies on gynodioecious species, reproductive success of females was not pollen limited. Instead, seed production was pollen limited in hermaphrodites, suggesting low levels of cross-pollination in hermaphrodites. Seed production was resource limited in hermaphrodites, but not in females, thus providing support for the RRH The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175, DDITIONL KEYWORDS: floral display inbreeding depression hypothesis pollinator constancy resource reallocation hypothesis visitation rates. INTRODUCTION Gynodioecy is a dimorphic breeding system in which male-sterile individuals (females) coexist with hermaphroditic individuals in the same population (Dufay & Billard, 2012). Gynodioecy occurs in > 500 species from 71 plant families, representing 0.6% of the angiosperms (Kaul, 1988). It is often regarded as a transitional stage from hermaphroditism to dioecy, or vice versa (Delph, 2009; lonso & Herrera, 2011). However, some gynodioecious lineages (notably Lamiaceae and Thymus L. in particular) have no known dioecious species, and appear to maintain stable *Corresponding author. x.arnan@creaf.uab.es gynodioecious populations, often with high frequencies of female individuals (Cuevas & barca, 2006). Darwin recognized the evolutionary importance of gynodioecy when he acknowledged that females are at a disadvantage compared with hermaphrodites because they do not produce pollen, and thus only contribute to the next generation via ovules (Darwin, 1877). In addition, female flowers are often less attractive than hermaphroditic flowers to pollinators (e.g. Bell, 1985; Stanton, Snow & Handel, 1986; shman & Stanton, 1991; Klinkhamer, de Jong & Wesselingh, 1991; Eckhart, 1992; Delph & Lively, 1992; Williams, Kuchenreuther & Drew, 2000), which has been attributed to the smaller flower size and lack of pollen production in females (Delph, 1996; 395

2 396 X. RNN ET L. Eckhart, 1999; Shykoff et al., 2003). Therefore, females are more prone to pollen limitation, because of their lower attractiveness to pollinators and their inability to self-pollinate (shman & Stanton, 1991; sikainen & Mutikainen, 2005). For male sterility mutations to persist in a population, it is necessary that females have higher female fitness than do hermaphrodites. Several non-exclusive mechanisms have been proposed to explain how females attain this reproductive advantage and avoid being wiped out by natural selection (Shykoff et al., 2003; Dufay & Billard, 2012). Females may have some reproductive advantage in situations of high levels of selfpollination and inbreeding depression in hermaphrodites ( inbreeding depression hypothesis ; Lloyd, 1975; Richards, 1986; Charlesworth & Charlesworth, 1987; Cuevas & barca, 2006). lternatively, females may be able to channel resources not invested in pollen production towards the production of a greater number of seeds and/or seeds of larger size than hermaphrodites ( resource reallocation hypothesis ; Darwin, 1877; Lewis, 1941; Lloyd, 1976; Charlesworth & Charlesworth, 1978; Richards, 1986; Eckhart, 1992; Sakai et al., 1997). ccording to this hypothesis, hermaphrodites would be more likely than females to suffer from resource limitation. Reproductive advantage of females could also be achieved through greater adherence of pollen to stigmatic surfaces (ssouad & Valdeyron, 1975; Shykoff, 1992) or greater flower longevity in females relative to hermaphrodites (shman & Stanton, 1991; Petterson, 1992). Finally, females might be less affected than hermaphrodites by seed predation and/or herbivory (Dufay & Billard, 2012). Pollination is a key process in most of the abovementioned mechanisms. For correct functioning of the system, it is necessary that the two sexual phenotypes have some degree of coincidence in the pollinator species they attract and that individual pollinators show some lack of morph constancy, alternating visits to the two morphs during their foraging bouts. t least a priori, not all pollinators would be expected to meet these two requirements because different species collect different floral resources (pollen, nectar or both) (Proctor, Yeo & Lack, 1996) and therefore might favour one morph over the other. Given some degree of pollinator sharing, the reproductive success of the female morph will depend on pollinator visitation rates (number of pollinators attracted per unit time), the number of flowers visited per individual plant and the degree of morph constancy (or lack thereof). These three variables ultimately depend on floral display (number of open flowers per individual), flower size, rewards offered (shman & Stanton, 1991) and the proportion of individuals of each morph in the population. In this study, we investigate pollination and test the resource reallocation hypothesis in a population of the gynodioecious species Thymus vulgaris L. We study visitation rates and pollinator foraging behaviour, and measure flower lifespan and the effect of pollen and nutrient addition on female reproductive success of the two morphs. We address the following questions: (1) re there differences between the two morphs in floral display, nectar production and flower lifespan? (2) Have the two morphs similar pollinator spectra? To what extent are pollinators constant to a particular morph or do they alternate visits between morphs? (3) Do female flowers receive fewer visits? Is reproductive success of females pollen limited? (4) Is nutrient limitation greater in hermaphrodites than in females as predicted by the resource reallocation hypothesis? MTERIL ND METHODS STUDY SPECIES Thyme, Thymus vulgaris L., is a gynodioecious, perennial shrub in Lamiaceae, indigenous to the Mediterranean regions of eastern Spain, southern France and northern Italy. The species is dichogamous and requires insect vectors for pollination. It typically grows cm tall and vegetative size is similar in the two morphs. The population sex ratio is usually biased towards females (range, 5% 95%; mean, 60%; Valdeyron, Dommée & Vernet, 1977; Dommée, ssouad & Valdeyron, 1978). Flowering occurs in March to June. Flowers, which range from deep purple to pinkish white, are arranged in glomeruli. Hermaphroditic individuals are self-compatible (Valdeyron et al., 1977) and sex determination is nuclearcytoplasmic (ssouad et al., 1978; Thompson, 2002). Flowers contain four ovules, each of which may develop into a nutlet (a seed covered by a stony layer), which mature approximately 3 weeks after pollination. STUDY RE This study was carried out in a Mediterranean scrubland in El Garraf Natural Park (Barcelona, northeastern Iberian Peninsula; UTM coordinates: , ) at 340 m above sea level and 1700 m from the coastline. The vegetation in the scrubland is dominated by Quercus coccifera L., Pistacia lentiscus L., Rosmarinus officinalis L. and Thymus vulgaris. Field work was conducted from March to June in , encompassing the whole flowering period of T. vulgaris, in an area of c. 2 ha containing several hundred individuals. SEX RTIO ND FLORL DVERTISEMENT The proportion of individuals of each morph (sex ratio) may strongly influence pollinator foraging deci-

3 FEMLE DVNTGE IN GYNODIOECIOUS THYME 397 sions. In 2009, we selected m 2 plots across the study area, in which we counted the number of female and hermaphrodite individuals, thus obtaining a measure of morph density and the ratio of female to hermaphrodite individuals. Plots were separated from each other by at least 10 m. Because data were not normally distributed, differences in density between morphs were analysed with a Wilcoxon paired test. Morph counts were not repeated in consecutive years because T. vulgaris is a perennial shrub. t peak bloom, we randomly selected 30 hermaphrodite and 28 female individuals and counted the number of open flowers. Differences between morphs in the number of open flowers per individual were analysed with Student s t-test. Within a species, large flowers tend to produce more pollen and nectar than do small ones (e.g. Kaczorowski, Gardener & Holtsford, 2005; Gómez et al., 2008) and pollinators have been shown to preferentially visit large flowers (e.g. Martin, 2004; Ishii & Harder, 2006; Gómez et al., 2008). To measure corolla size, in 2009, we selected 15 hermaphrodite and 15 female individuals and collected one flower from each. We then removed the calyx and the reproductive column, and measured the maximum corolla height, width and depth. Differences between morphs in corolla height, width and depth were analysed with Student s t-test. In addition to floral display and flower size, the amount and quality of nectar/pollen rewards produced per flower may also be regarded as an investment in attractiveness to pollinators. In 2010, we measured nectar production on nine hermaphrodite and eight female individuals (between two and 12 flowers per individual; total number of flowers measured: 36 hermaphrodite and 50 female flowers). Using cheesecloth bags, we enclosed branches with mature flower buds and, 2 days later, extracted the nectar accumulated in individual flowers with 0.25-μl microcapillary tubes. We also measured sugar concentration with field refractometers. From these data, we calculated the sugar content per flower (Dafni, 1992). Differences between morphs in nectar volume and sugar content (both square root transformed) were analysed with general linear mixed models (GLMMs), with morph as a fixed factor and individual as a random factor. STIGM RECEPTIVITY Female plants could compensate for low visitation rates by extending their stigma receptivity. In 2011, we monitored stigma receptivity in 15 hermaphrodite and 15 female individuals, in which we selected two flowers from two different branches. One of the two branches was bagged to prevent visitation and thus to provide an estimate of maximum flower lifespan. The other branch was left unbagged to provide a measure of lifespan under natural conditions. During a 15-day period, we described the appearance of the stigma observed under a magnifying glass. We assumed that the stigma was receptive from the day it unfolded until spots of necrosis appeared on its surface. Differences between morphs and treatments (bagged and unbagged) in the number of days the stigma was apparently receptive were analysed with repeatedmeasures analysis of variance (NOV), using treatment as the within-subjects factor and morph as the main factor. POLLINTOR SPECTR ND VISITTION RTES Every 2 3 days throughout peak bloom (5 weeks) in 2008, we surveyed pollinators visiting the flowers of female and hermaphrodite T. vulgaris individuals. To perform these surveys, we tagged individual plants (41 hermaphrodites and 26 females) across the study area and counted the number of open flowers. Then, we spent several 4-min intervals observing these plants and noted all pollinators landing on and visiting the flowers. These surveys were conducted from 10:00 to 18:00 h and, for most individuals, we conducted three or four surveys distributed throughout the day. We use the term contact to denote an individual pollinator landing on an individual plant, and the term visit to denote a pollinator individual visiting a flower (thus a contact may involve one or more visits). We conducted a total of min surveys (155 on hermaphrodites and 104 on females). The total observation time was > 17 h. From these surveys, we obtained the pollinator assemblage of each morph (frequency of contacts by each pollinator species) and contact rate (measured as the number of contacts per individual and hour and as the number of contacts per 1000 open flowers and hour). Differences between morphs in contact rate were analysed with the Mann Whitney test because data were not normally distributed. To measure pollinator assemblage overlap between morphs, we used a proportional similarity index (PSI; Natalis & Wesselingh, 2012): PSI = Pfi Phi n i= 1 where P fi and P hi are the proportions of contacts made by pollinator species i to females and hermaphrodites, respectively. Values close to unity indicate high similarity between morphs in pollinator assemblage. POLLINTOR FORGING BEHVIOUR During peak bloom in 2009, we monitored 286 foraging bouts of eight pollinator species (three bees, two hover-flies, two butterflies and one wasp) visiting

4 398 X. RNN ET L. T. vulgaris. The total observation time was > 20 h. Pollinators were followed until the observer lost sight of them. For each bout, we recorded the sex (female or hermaphrodite) and the number of flowers visited on each consecutively contacted individual plant. The mean number of individual T. vulgaris contacted per bout was nine and ranged from two to 40. Differences between morphs in the number of flowers visited per individual (log transformed) were analysed with GLMM, one for each pollinator species, with morph as a fixed factor. Because several measures were taken on each bout, bout was included as a random factor. For each bout, we used the proportion of contacts to hermaphrodite and female individuals as a measure of morph preference. We only considered bouts with at least five plants visited (four interplant flights). Differences in preference for a particular morph among pollinator species were analysed with one-way NOV followed by a post-hoc honestly significant difference (HSD) Tukey test. Data were arcsine transformed to improve homoscedasticity. Then, for each pollinator species, we used goodness-of-fit tests to analyse whether morph preference was different from the expected contact frequencies if pollinators moved randomly between plants based on morph frequency. Successful pollination of female individuals is dependent on some lack of morph constancy, defined as the tendency to visit individuals of the same morph in consecutive contacts (Waser, 1986). For each pollinator species, we calculated the constancy index (C) of Gegear & Thomson (2004): C = ( c e) ( c+ e 2ce) where c represents the observed proportion of transitions between same-morph individuals (constant transitions) and e is the expected proportion of transitions between same morph individuals based on contact frequency to a particular morph (morph preference as defined above). If p is the proportion of approaches to, for example, hermaphrodites, then e = p 2 +(1 p) 2 (Pohl, Van Wyk & Campbell, 2011). This index ranges between 1 (complete constancy), 0 (random visitation) and 1 (complete inconstancy). Pollinators attracted to a female individual might respond to the lack of pollen by favouring the hermaphroditic morph in subsequent contacts. To explore this possibility, we used goodness-of-fit tests to compare the observed frequency of female-to-female (FF) and female-to-hermaphrodite (FH) transitions against the expected frequencies based on morph preference. FEMLE REPRODUCTIVE SUCCESS In 2011, we conducted a pollen/nutrient addition experiment to examine whether female reproductive success was pollen/nutrient limited. The factorial design had two fixed factors: pollination and resources (nutrients + a small amount of water). Both factors had two levels [supplementary pollination (P+) versus natural pollination (P ), and resource addition (R+) versus no resource addition (R )]. We selected 60 female and 60 hermaphrodite individuals. Within each morph, 20 individuals were left untreated (R P ) and served as an external control (group EC). In each of these plants, we marked two flowers in a given glomerulus. Twenty other individuals of each morph received resource addition (group R+). In these plants, we marked two flowers in each of two glomeruli on different branches. The two flowers of one glomerulus were cross-pollinated by hand (P+) and the two flowers of the other glomerulus were left untreated and exposed to natural pollination (P ). The remaining 20 individuals of each morph did not receive resource addition (group R ). On these individuals, we also selected two flowers in each of two glomeruli, one of which received pollen addition (P+) and the other did not (P ). Flowers that did not receive pollen addition (R P ) were used as an internal control (IC) for group R. Resource addition started on anthesis of the first flowers. Every 6 days from the beginning of flowering (pril) until nutlets started to mature (4 weeks later), plants assigned to the R+ treatment received a 7: 5: 6 NPK fertilizer (COMPO) at the manufacturer s recommended dose (7 ml l 1 ) in 250 ml of water per plant. Flowers assigned to the P+ treatment were cross-pollinated by rubbing a mature, fully pollenloaded anther over the stigma in the early morning of the day on which the stigma unfolded. The distance between pollen recipient and pollen donor plants was m. Calices were collected 3 weeks after the end of flowering and the number of nutlets per flower was counted in the laboratory. Each nutlet was weighed to the nearest 1 μg with an MX5 utomated-s Microbalance (Mettler Toledo). We encountered a proportion of nutlets (0.1) that were not fully developed. However, the total number of nutlets and the number of fully developed nutlets per flower were highly correlated, and we only worked with the latter. We used three measures of reproductive success: (1) flower success rate: the proportion of flowers producing at least one nutlet (analogous to fruit set in most studies); (2) nutlet production: the number of nutlets (one to four) produced per successful flower (analogous to seed set in most studies); and (3) nutlet weight. Differences between morphs in the effect of pollen and resource addition on the three reproductive success variables were analysed with GLMM. Flower success rate was analysed with a GLMM with a binomial distribution error and Logit link function, with setting (yes/no) as the dependent variable.

5 FEMLE DVNTGE IN GYNODIOECIOUS THYME 399 Nutlet production was analysed with a GLMM with a Poisson distribution error. Lastly, nutlet weight was analysed using a GLMM with a Gaussian distribution error. Nutlet weight was log-transformed to improve homoscedasticity. ll three models included the same fixed factors: morph (female, hermaphrodite), morph resource addition (R+, R ), morph pollen addition (P+, P ) and morph resource addition pollen addition. In addition, all models included individual as a random factor to account for the fact that several measures were taken on the same individual, and flower was also included as a random factor in the analysis of nutlet weight. We also tested for a potential trade-off between nutlet production and weight, and whether it was modulated by morph, pollination and resource addition. We used a GLMM with nutlet weight as the dependent variable, and nutlet production and its interactions with morph, pollen addition, resource addition, morph and pollen addition, morph and resource addition and morph, pollen addition and resource addition as fixed factors. Individual was again included as a random factor. In those models in which the interaction was significant, we conducted multiple comparisons to test differences among the different combinations of treatments. For this purpose, we created a new variable with as many levels as possible combinations of levels of the original factors, which was then the only fixed factor included in the previous model. Within each morph, comparison of the two controls (R P flowers of groups EC and R ) allowed us to assess the potential reallocation of resources within an experimental glomerulus (Zimmerman & Pyke, 1988; Wesselingh, 2007). Higher values of reproductive success in external than in internal controls would indicate resource reallocation from control to treated flowers. For each response variable, the comparison of the two controls was performed with the same analyses as explained above, but including group (R P flowers of group EC versus R P flowers of group R ) as fixed factor. ll means and standard errors presented in the figures are back-transformed values. ll statistical analyses were conducted with the R software system, version (R Development Core Team, 2010). RESULTS SEX RTIO, FLORL REWRDS ND STIGM RECEPTIVITY Female density (mean ± SE: 0.40 ± 0.06 individuals m 2 ) was higher (Wilcoxon matched pairs test, Z = 2.13, P = 0.03, N = 12) than hermaphrodite density (0.29 ± 0.06 individuals m 2 ), so that female frequency in the population was 58%. The number of flowers per individual was similar in the two morphs (Table 1). Overall, flower density was 149 flowers m 2 for hermaphrodites and 225 flowers m 2 for females. Hermaphroditic flowers were larger than female flowers for all three measures of flower size (Table 1). However, we could not detect significant differences between morphs in the volume of nectar and sugar content per flower (Table 1). There were differences between morphs (repeatedmeasures NOV; F 1,53 = 8.0, P = 0.007) and treatments (F 1,35 = 166.2, P < 0.001) in the number of days the stigma remained receptive. pparent stigma receptivity was 1 day longer in females than in her- Table 1. Comparison of floral traits, stigma longevity and pollinator contact rates in hermaphrodite and female Thymus vulgaris individuals. Significant comparisons in bold Floral trait Hermaphrodites (mean ± SE) Females (mean ± SE) Test Statistic P d.f. Floral display (flowers/ 561 ± ± 125 Student s t individual) Corolla depth (mm) 4.68 ± ± 0.56 Student s t Corolla height (mm) 6.49 ± ± 0.80 Student s t Corolla width (mm) 5.05 ± ± 0.75 Student s t Nectar volume (μl nectar/ ± ± 0.01 GLMM flowers) Sugar production (mg/ ± ± 0.15 GLMM flowers) Contact rate (pollinators/ 26.4 ± ± 4.2 Mann Whitney U , 114 individual h 1 ) Contact rate (pollinators/1000 flowers h 1 ) 35.4 ± ± 13.2 Mann Whitney U , 106

6 400 X. RNN ET L. Table 2. Number of flowers (mean ± SE) visited per plant by eight pollinator species in hermaphrodite and female Thymus vulgaris and outputs of general linear mixed models (GLMMs). Values of the morph constancy index for each species are also shown. Sample sizes in parentheses. Significant comparisons in bold Pollinator species Functional group Hermaphrodite Female Estimate (± SE) t P Constancy index Bombus terrestris Bee 22.7 ± 2.8 (128) 23.6 ± 3.8 (86) 0.11 ± pis mellifera Bee 18.7 ± 2.1 (216) 19.8 ± 2.3 (249) 0.08 ± Lasioglossum spp.* Bee 6.8 ± 1.3 (58) 6.5 ± 0.7 (115) 0.04 ± Eupeodes corollae Hoverfly 4.2 ± 1.3 (43) 5.4 ± 0.9 (51) 0.19 ± Chrysotoxum spp. Hoverfly 3.0 ± 0.9 (27) 6.8 ± 2.8 (43) 0.43 ± Pseudophilotes Butterfly 1.4 ± 0.2 (23) 2.0 ± 0.3 (54) 0.12 ± panoptes Cynthia cardui Butterfly 14.4 ± 4.5 (41) 2.7 ± 0.6 (20) 0.68 ± Polistes dominulus Wasp 3.9 ± 1.9 (23) 10.2 ± 1.9 (52) 0.83 ± *Mostly Lasioglossum transitorium planulum, but also some L. bimaculatum, L. griseolum, L. soror and L. subhirtum. C. cautum and C. intermedium.c maphrodites (mean ± SE: 4.0 ± 0.4 and 3.0 ± 0.3, respectively) and 4 days longer in bagged (nonpollinated) than unbagged flowers (5.4 ± 0.3 and 1.4 ± 0.1 days, respectively). The interaction between the two factors was not significant (F 1,53 = 1.5, P = 0.223). POLLINTORS We recorded 274 individual pollinators visiting hermaphrodites and 142 visiting females (ppendix 1). The pollinator assemblages of the two morphs were similar (PSI = 0.76). Bees (Bombus terrestris, pis mellifera, Lasioglossum spp. and Hylaeus hyalinatus) were the most frequent pollinators to both morphs. However, the pollinator assemblage of females was more diverse than that of hermaphrodites, because a single species (B. terrestris) accounted for almost one-third of the interactions in hermaphrodites. The contact rate measured as the number of pollinators attracted per individual and unit time was higher in hermaphrodite than in female individuals (Table 1). However, these differences disappeared when the contact rate was measured as the number of pollinators attracted per 1000 flowers and unit time (Table 1). We analysed the number of flowers visited per contact by eight pollinator species (Table 2). In both morphs, the bees B. terrestris and. mellifera were the species that visited more flowers per contact. Five species (including B. terrestris and. mellifera) visited similar numbers of flowers on the two morphs. However, the hoverfly, Chrysotoxum sp., and the paper wasp, Polistes dominulus, visited more flowers on female individuals, and the butterfly, Cynthia cardui, visited more flowers on hermaphrodite individuals. We followed 286 foraging bouts by the same pollinator species. s many as 79.3 ± 1.2% of these bouts included only T. vulgaris individuals (range: 70% in B. terrestris and C. cardui to 96% in the butterfly Pseudophilotes panoptes). Most bouts (86%) contained visits to both morphs. We found differences in morph preference between pollinator species (one-way NOV, F 7,96 = 4.5, P = ; Fig. 1). Most pollinator species contacted more females than hermophrodites, but this was expected based on the higher density of female individuals. Only in Lasioglossum spp. was contact frequency to females significantly higher than expected if pollinators moved randomly among T. vulgaris individuals (χ 2 = 4.0, P = 0.045, d.f. = 1) (Fig. 1). However, C. cardui showed a significant preference for hermaphrodites (χ 2 = 18.7, P < , d.f. = 1) (Fig. 1). We also found differences between species in morph constancy. ll species made more homomorphic than heteromorphic flights (Fig. 2). However, the constancy index was closer to zero (random visitation) than to unity (complete constancy) for all species, except the hoverfly Eupeodes corollae (C = 0.53; Table 2). pis mellifera and B. terrestris showed rather low morph constancy. It is important to note that, in most species, about 20% of the interplant flights were hermaphrodite-to-female (HF) (Fig. 2), thus promoting pollen transfer to females. Finally, none of the eight pollinator species analysed showed frequencies of FF and FH transitions significantly different from those expected based on morph preference (all goodness-of-fit tests P > 0.07). Thus, pollinators did not discriminate against females after visiting a female individual.

7 FEMLE DVNTGE IN GYNODIOECIOUS THYME 401 a* (13) ab (15) b (40) b (6) b (6) b (8) b (5) b* (11) Hermaphrodite Female Figure 1 Mean ± SE proportion of contacts to hermaphrodite and female Thymus vulgaris morphs by eight pollinator species. Numbers in parentheses indicate sample sizes (number of individual pollinators monitored). Different letters indicate significant differences in morph preference among pollinator species (honestly significant difference (HSD) Tukey test, P < 0.05). sterisks indicate significant differences from contact frequencies expected if pollinators moved randomly between morphs according to morph abundance. Interplant flights (%) (67) (116) (51) (52) (44) (304) (141) (90) HH FF HF FH 0 Species Figure 2 Proportion of homomorphic and heteromorphic interplant flights departing from a female (F) and a hermaphrodite (H) Thymus vulgaris by eight pollinator species. Numbers in parentheses indicate sample sizes. FEMLE REPRODUCTIVE SUCCESS The GLMMs revealed no significant differences between the two controls (P R of group EC versus P R of group R ) for any of the three variables measured in any of the two morphs (Table 3), indicating no reallocation of resources from P to P+ flowers. From these untreated flowers, we found that

8 402 X. RNN ET L. Table 3. Output of general linear mixed model (GLMM) on the effects of pollen and resource addition on female reproductive success of female and hermaphrodite Thymus vulgaris. The comparison between internal and external controls is also shown. R+, resource addition; R, no resource addition; P+, pollen addition; P, no pollen addition; IC, internal control (R P ) of group R plants. Significant values in bold Flower success rate Main effects Estimate SE Z P Morph (hermaphrodite) Female R Hermaphrodite R Female P Hermaphrodite P Female:R+ P Hermaphrodite:R+ P Controls comparison IC Morph (hermaphrodite) Hermaphrodite IC Nutlet production* Main effects Estimate SE Z P Morph (hermaphrodite) Female R Hermaphrodite R Female P Hermaphrodite P Female:R+ P Hermaphrodite:R+ P Controls comparison IC Morph (hermaphrodite) Hermaphrodite IC Nutlet weight Main effects Estimate SE t P Morph (hermaphrodite) Female R Hermaphrodite R Female P Hermaphrodite P Female:R+ P Hermaphrodite:R+ P Controls comparison IC Morph (hermaphrodite) Hermaphrodite IC *Number of nutlets per successful flower.

9 FEMLE DVNTGE IN GYNODIOECIOUS THYME 403 ) Flower success rate (%) BC C B B BC B BC Female Hermaphrodite B) Nutlet set Female Hermaphrodite 10 0 EC P-R- P+R- P-R+ P+R+ 0 EC P-R- P+R- P-R+ P+R+ Treatment Treatment C) 0.2 B Nutlet weight (mg) BC BC C BCBC BC Female Hermaphrodite EC P-R- P+R- P-R+ P+R+ Treatment Figure 3 Flower success rate (), mean (± SE) nutlet production (B) and mean (± SE) nutlet weight (C) in female and hermaphrodite Thymus vulgaris subjected to pollen and resource addition treatments. R+, resource addition; R, no resource addition; P+, pollen addition; P, no pollen addition; EC, external control (R P ). Different letters indicate significant differences among treatments according to general linear mixed model (GLMM) multiple contrasts. External control results are shown for comparison. the flower success rate was clearly higher in females (68.8%) than in hermaphrodites (43.7%) (Table 3). Nutlet production (nutlets per successful flower) was similar in the two morphs (Table 3; mean ± SE: 2.1 ± 0.2 in females and 1.7 ± 0.2 in hermaphrodites). However, nutlet weight was higher in hermaphrodites (Table 3; mean ± SE: 0.15 ± 0.01 and 0.12 ± 0.01 mg, respectively). We found a significant effect of the interaction between morph, resource addition and pollen addition on the proportion of successful flowers. Hermaphrodites showed an increase when resources or pollen were supplemented, although differences between the control (P R ) and P+R+ flowers were not significant (Fig. 3, Table 3). Conversely, resource and pollen addition did not modify the proportion of successful flowers in females (Fig. 3, Table 3). We did not detect any effects of pollen and resource addition on nutlet production in either of the morphs (Table 3, Fig. 3B). Lastly, nutlet weight decreased with pollen addition, but not with resource addition or with resource + pollen addition in hermaphrodites (Fig. 3C, Table 3). In females, nutlet weight increased only with pollen + resource addition (Fig. 3C, Table 3). We found a significant negative relationship between nutlet weight and nutlet production (t = 2.27, P = , d.f. = 110), and this relationship was not dependent on morph, pollen addition or nutrient addition (all interactions, P > 0.05).

10 404 X. RNN ET L. DISCUSSION FLORL DISPLY ND POLLINTOR TTRCTION In gynodioecious species, females are predicted to be more prone to pollen limitation because they are usually less attractive to pollinators, cannot resort to self-pollination and pollen carryover from flower to flower diminishes as the number of flowers visited per contact increases. In agreement with other studies on T. vulgaris (ssouad et al., 1978; Thompson & Tarayre, 2000), females and hermaphrodites in our population produced similar numbers of flowers per individual. lso in agreement with other studies (Thompson, Rolland & Prugnolle, 2002), females in our population produced smaller flowers and no pollen, thus investing less than hermaphrodites in floral advertisement. Nevertheless, the pollinator composition of females and hermaphrodites was similar and, more importantly, although hermaphrodites attracted more pollinators per individual, females attracted as many pollinators as hermaphrodites on a per flower basis. This is an unexpected result because many studies have shown that pollinators prefer large, pollen-producing, hermaphroditic flowers to female flowers (Bell, 1985; shman & Stanton, 1991; Delph & Lively, 1992; shman, Shivitz & Swetz, 2000; Case & shman, 2009). In our population, only one pollinator species, C. cardui, showed a significant preference for hermaphrodites. The rest of the species studied either showed no significant preferences or favoured the female morph (Lasioglossum spp.). On contacting a female individual, some pollinators might be discouraged by the lack of pollen and visit fewer flowers on these individuals. However, most visitors, including three bee species that were amongst the most frequent and presumably most effective pollinators, visited similar numbers of flowers on female and hermaphroditic individuals. One hoverfly and one wasp species even visited more flowers per contact on female individuals. Thus, our first conclusion is that, despite their lower investment per flower, females manage to lure and maintain pollinators at similar levels to hermaphrodites. Pollinators attracted to female individuals might also respond to the lack of pollen by favouring the hermaphroditic morph in subsequent contacts. gain, our results show that pollinators did not respond in this way. Most of the pollinator species monitored showed low values of morph constancy, indicating close to random intermorph transitions. This is important because, assuming low levels of pollen carryover, female pollination relies on a lack of morph constancy. Most foraging bouts monitored included heteromorphic flights, and c. 20% of all flights recorded were from hermaphrodites to females. These results again indicate that most pollinators do not discriminate against female individuals, and corroborate our previous conclusion that female flowers are no less attractive than hermaphroditic flowers. INCRESED STIGM RECEPTIVITY IN FEMLES lthough female flowers received contact rates similar to those of hermaphroditic flowers, they still might experience lower pollen deposition because pollinators do not acquire new pollen when visiting female flowers and the deposition of carryover pollen tapers off with consecutive visits (Thomson & Plowright, 1980; Thomson, 1986). Females could compensate for this potential lower pollen deposition by extending stigma receptivity. In naturally pollinated flowers, apparent stigma receptivity was 1 day longer in females than in hermaphrodites. These results corroborate previous studies, in which pollen grains were found to adhere more easily to the stigma surface of females than of hermaphrodites (ssouad & Valdeyron, 1975), further enhancing pollen deposition in females. REPRODUCTIVE OUTPUT UNDER OPEN POLLINTION ll the above evidence suggests that, even with the lower investment in floral advertisement, reproductive success in females might not be pollen limited. In addition, and in agreement with Couvet, Henry & Gouyon (1985), the proportion of successful control flowers was higher in females than in hermaphrodites. Some gynodioecious species show a similar pattern, but others do not (reviews in Shykoff et al., 2003 and Dufay & Billard, 2012). t the same time, nutlet production did not differ between morphs in our population. In most, but not all, T. vulgaris populations analysed in previous studies, nutlet production was higher in females than in hermaphrodites (tlan et al., 1992; Manicacci et al., 1998). crude estimate of nutlet yield (nutlets produced per individual) in our population (computed from the mean number of open flowers at peak bloom, mean flower success rate and mean nutlet production) suggests higher yields in females than in hermaphrodites (811 and 383 nutlets, respectively). In general, nutlet yields in other T. vulgaris populations have also been found to be higher in females (ssouad et al., 1978; Couvet, Bonnemaison & Gouyon, 1986; Thompson & Tarayre, 2000). Nutlet weight in naturally pollinated flowers was higher in hermaphrodites. The vast majority of studies on other gynodioecious species show similar seed weight in the two morphs (review in Dufay & Billard, 2012) or a higher seed weight in females (Puterbaugh, Wied & Galen, 1997; Shykoff et al., 2003; Zhang et al.,

11 FEMLE DVNTGE IN GYNODIOECIOUS THYME ), but at least two studies show higher seed weight in hermaphrodites (Vaarama & Jaaskeläinen, 1967; Ramsey & Vaughton, 2002). We have found no previous measures of nutlet size in T. vulgaris, but several studies have shown higher germination rates (usually associated with higher seed weight; McGinley, Temme & Geber, 1987) in nutlets produced by females (ssouad et al., 1978; Manicacci et al., 1998; Thompson & Tarayre, 2000). Importantly, the results from the above-mentioned studies were obtained in outcrossed individuals, and thus cannot be attributed to selfing in hermaphrodites. Thus, the two T. vulgaris morphs in our population show different nutlet yield patterns, with females producing more nutlets and hermaphrodites producing larger nutlets. This difference might be explained by selective abortion of selfed embryos in hermaphrodites (Stephenson, 1981), as suggested by the increased flower success rate together with decreased nutlet weight in hermaphroditic flowers supplemented with outcrossed pollen. However, previous studies on T. vulgaris have concluded that sexual selection and subsequent specialization in male function is more than twice as important as maternal choice in explaining the low flower success rate of hermaphrodite individuals (Couvet et al., 1985). REPRODUCTIVE OUTPUT UNDER POLLEN ND RESOURCE DDITION The absence of pollen limitation in females of our population was definitively corroborated in the pollen addition experiments. Females showed no evidence of pollen limitation in any of the three reproductive success variables analysed. This result is remarkable in view of the high female frequency in our population, and is again counter to most studies on gynodioecious species, which show evidence of pollen limitation in females (Dufay & Billard, 2012, and references therein; but see Delph & Lively, 1992; sikainen & Mutikainen, 2005). t the same time, reproductive success in female flowers under resource addition did not differ from that of controls. The combination of no pollen limitation and no resource limitation indicates that our female population is at equilibrium sensu Haig & Westoby (1988), with fitness being limited simultaneously by both factors (shman et al., 2004). This conclusion is further supported by the fact that females only increased reproductive success (in terms of increased nutlet size) when both pollen and resources were supplemented. s opposed to females, and in contrast with most other gynodioecious species (Dufay & Billard, 2012), hermaphrodites in our population showed signs of pollen limitation. That is, flowers supplemented with outcrossed pollen (P+R ) had a higher probability of success than control (P R ) flowers. This result is consistent with hermaphrodites receiving adequate levels of self (geitonogamous)-pollination, but insufficient cross-pollination. If so, pollen limitation in hermaphrodites would be based on pollen quality, rather than quantity (shman et al., 2004). This conclusion is congruent with the high number (c. 20) of flowers per individual consecutively visited by the two most frequent and presumably effective pollinators of hermaphrodites (B. terrestris and. mellifera). In addition to promoting selfing, high levels of geitonogamy may, through stigma clogging, hinder fertilization by subsequently deposited outcrossed pollen (Holland & Chamberlain, 2007). In addition to increased flower success rate, the addition of outcrossed pollen resulted in a decrease in nutlet weight in hermaphrodites. This result suggests some level of within-glomerulus resource reallocation. In contrast, we found no evidence of resource reallocation between glomeruli, as reflected by the lack of differences between EC (external control) and P R flowers of group R in any of the three variables measured. Hermaphrodites also showed evidence of resource limitation on flower success rate, with resourcesupplemented flowers (P R+) (although differences between P+R+ and P R failed to reach significance, P < 0.08) having higher success than control flowers. In combination with the finding of no resource limitation in females, this result supports the resource reallocation hypothesis (Eckhart, 1992; Sakai et al., 1997; Dufay & Billard, 2012), whereby resources not invested in floral advertisement and male function can be channelled towards fruit and seed development in females. In addition, nutlet weight recovered control levels in R+P and R+P+ flowers, corroborating within-inflorescence resource reallocation in R P+ glomeruli. CONCLUDING REMRKS The overall aim of this study was to combine information on pollinator foraging behaviour and experimental manipulation of pollen and nutrient resources to understand the mechanisms by which females of T. vulgaris manage to persist in gynodioecious populations. Our results provide evidence for the hypothesis of resource reallocation, because seed production was resource limited in hermaphrodites but not in females. Importantly, decreased investment by females in floral advertisement did not negatively affect pollinator attraction, and, as a result, females showed no signs of pollen limitation. In most other gynodiecious species, females have been found to have a reproductive advantage in spite of their lower pollinator attraction (Dufay & Billard, 2012). In our study, pollinator attraction (on a per flower basis) was not lower in females. Extended

12 406 X. RNN ET L. stigma receptivity also appears to contribute to the absence of pollen limitation in females. Our results also provide indirect evidence suggesting high levels of self-pollination, but insufficient levels of cross-pollination, in hermaphrodites, resulting in significant pollen limitation in this morph. If so, this hermaphrodite disadvantage would be expected to diminish in populations with low female frequencies or in populations with pollinators visiting few flowers per individual and thus incurring low levels of geitonogamy (in populations with low. mellifera and B. terrestris visitation). Selfed progeny have been shown to be less fit than outcrossed progeny in T. vulgaris (ssouad et al., 1978; Bonnemaison, Dommée & Jacquard, 1979; Perrot, Dommée & Jacquard, 1982; Thompson & Tarayre, 2000), and selfing rates have been shown to be high in some populations (Thompson, 2002). This could be interpreted as evidence in favour of the inbreeding depression hypothesis (Sun & Ganders, 1986). However, a better performance of seeds from female parents compared with seeds from hermaphroditic parents has been found in experiments using outcrossed pollen, so that the reproductive advantage of females occurs even in the absence of inbreeding depression (Thompson & Tarayre, 2000). In addition, selfing has been shown to be significant only in populations with high female frequency (Thompson, 2002). Finally, biparental inbreeding (as a result of mating with related individuals) has been found to be higher in females than in hermaphrodites (Thompson & Tarayre, 2000). For these reasons, Thompson (2002) concluded that selfing is probably not the major force driving the evolution of female frequencies, but may be important in maintaining high female frequencies, such as in our population. Our results were obtained on a single population. n important question is whether our population is representative of other T. vulgaris populations. Most of the traits measured in our study (main pollinator functional group, flower size, floral display, nutlet yield and, possibly, nutlet weight) show patterns similar to populations from southern France or (sex ratio, nutlet production) fall within the range of results obtained in these populations. However, the similar levels of pollinator attraction and the fact that seed production was pollen limited in hermaphrodites, but not in females, is counter to the results obtained in most studies on other gynodioecious species. The mechanisms by which T. vulgaris females achieve high levels of pollinator attraction despite their lower investment remain unexplained and deserve future consideration. CKNOWLEDGEMENTS We thank the Diputació de Barcelona for allowing us to work in El Garraf Natural Park. We are very grateful to H. Barril (CREF) for her help during different phases of the study.. M. Martín González and C. Primante participated in pollinator surveys, and. Torné helped with nectar measurements. We also thank J. M. Gómez (University of Granada) for valuable insights during the design of the experiments, and R.. Wesselingh and an anonymous reviewer for their constructive comments. M.. Marcos (University of licante) and. Pauly (Royal Belgian Institute of Natural Sciences) kindly identified some pollinator specimens. This study was supported by the Spanish MICINN (Ministerio de Ciencia e Innovación), projects CICYT CGL , CICYT CGL and CONSOLIDER CSD REFERENCES lonso C, Herrera CM Back-and-forth hermaphroditism: phylogenetic context of reproductive system evolution in subdioecious Daphne laureola. 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