Environmental stress effects on reproduction and ...

Environmental stress effects on reproduction and sexual dimorphism in the gynodioecious species Silene acaulis Quim Canelles1,2*, Sandra Saura-Mas2,3, María B. García4, Jesús Villellas4,5*, Lluís Brotons1,2, Francisco Lloret2, William F. Morris6 1

Centre Tecnològic Forestal de Catalunya (CTFC), Crta. de Sant Llorenç de Morunys, Km.2 25280 Solsona, Spain

2

Centre de Recerca Ecològica i Aplicacions Forestals (CREAF), Campus de Bellaterra (UAB) Edifici C 08193 Cerdanyola del

Vallès, Spain

3

Unitat d’Ecologia, Dept. Biologia Animal, Biologia Vegetal i Ecologia, Universitat Autònoma Barcelona, Edifici C, Campus

UAB, 08193, Cerdanyola del Vallès, Barcelona, Spain;

4

Instituto Pirenaico de Ecología (IPE-CSIC), Avda. Montañana, 1005. 50059 Zaragoza, Spain

5

Present address: School of Natural Sciences, Trinity College Dublin, College Green, Dublin 2, Ireland

6

Biology Department. Duke University, 125 Science Dr, Durham, NC 27708, USA.

Author for correspondence: E-mail: [email protected] Postal address: Centre Tecnològic Forestal de Catalunya, Crtra, de Sant Llorenç de Morunys km2 25280 Solsona, Spain

Abstract In gynodioecious species, hermaphrodite plants invest both in seed and pollen production, whereas female plants only produce fruits. For both sexes to coexist, such unbalanced investment is expected to translate in some kind of reproductive compensation, particularly under severe nutrient shortage or environmental stress. This study investigates 1) whether females of the gynodioecious boreo-alpine plant Silene acaulis show any overall compensation in fitness components; 2) the existence of sexual dimorphism in flowers that may favor different modes in each sex; 3) whether environmental severity strength these patterns. Flower size, fruit and seed production, and germination success were recorded in six populations of the Pyrenees and the Alps. To characterize environmental conditions of studied populations, we built a Species Distribution Model of the species. Fruiting success was significantly higher in female (59%) than in hermaphrodite plants (17%), and female organs (ovary and style) were larger in female than in hermaphrodite plants, but their flowes were otherwise smaller. Contrary to expectations, however, environment severity was not related with most of the biological studied traits, and only germination success was associated to environmental gradients. These results confirm that fruit production plays an important role as a compensatory strategy between sexes and, consequently, may favor the presence of both sexes in a gynodioecious species.

Keywords: Environmental stress, species distribution model, suitability, gynodioecy, reproduction, germination, floral traits, boreoalpine plant.

1. Introduction Gynodioecy is a breeding system found in some angiosperms in which separate plants producing female and hermaphrodite flowers coexist (Darwin, 1877). This polygamy modality evolved independently from hermaphrodites (Frohlich, 2003) in 71 families and 500 species (0.6% of all plant species; Dem’yanova, 1985; Kaul, 1988; Cuevas and Abarca, 2006). Gynodioecy may result from male sterility in females caused by genes located in the cell nucleus (nuclear male sterility; Lewis, 1941), or the mitochondria (cytoplasmic male sterility; Lewis, 1941) or more frequently by the interaction between nuclear and mitochondrial genes (nuclear-cytoplasmic male sterility; Charlesworth, 1981; Charlesworth and Laporte, 1998). Darwin (1877) already recognized the relevance of gynodioecy in an evolutionary context when he postulated that females were in disadvantage compared to hermaphrodites. Hermaphrodite plants contribute to the next generation through both ovules and pollen that fertilizes ovules on the same or other plants. In contrast, females only contribute to the next generation via ovules, and they always need to receive pollen from hermaphrodites (Richards, 1986). To persist in co-sexual populations, females might show some compensatory advantage (Lewis, 1941), such as higher seed production (Darwin, 1877; Lewis, 1941; Lloyd, 1976; Wolfe and Shmida, 1997; Thompson et al. 2002; Shykoff et al., 2003), differences in 2

lifetime reproduction through seeds (Morris and Doak, 1998) or higher pollen reception (Shykoff, 1992; Cuevas and Lopez, 2011; Arnan et al., 2014). This compensation can also arise through inbreeding depression in hermaphrodites that may reduce their fitness (Lloyd, 1976; Charlesworth, 1978; Ashman, 2006). Sexual dimorphism is frequent in dioecious species (Miller and Venable, 2003; Cuevas and López, 2011) and widely studied in terms of vegetative traits (Dawson and Geber, 1999; Obeso, 2002; Sánchez-Vilas et al., 2012). Regarding floral traits, hermaphrodites produce bigger flowers than females in most gynodioecious plant species studied (Delph, 1996). Three hypotheses have been proposed to explain this sexual size dimorphism (Delph 1996): a positive correlation between stamens presence and corolla aperture; the requirement of anthers to be protected by bigger petals and corolla; and, the most accepted hypothesis, the higher success of bigger flowers in hermaphrodites to attract pollinators and enhance male reproduction (Shykoff et al., 2003). Reproductive investment in females and hermaphrodites may respond differently to environmental severity (Delph, 2003). Females and hermaphrodites invest differently in reproduction (hermaphrodites have double reproduction pathway to invest), and consequently may have different plasticity in response to environmental stresses (Moreno and Bertiller, 2016; Sánchez-Vilas et al., 2016). In severe environments (in terms of temperature, precipitation, soil nutrients), hermaphrodites allocate more resources in pollen production than in ovules and fruits, most probably because it entails lower costs (Ashman, 2006). In addition, stressful environments make cross-pollination more unreliable, enhancing self-fertilisation and inbreeding depression (Shykoff, 1988; Ashman, 2006), which in turn reduce seed number and quality in hermaphrodite plants, while pollen production holds. Females do not show such pattern due the lack of sexual trade-off, but they also reduce reproductive investment as resources decrease (Darwin, 1877; Cuevas and López, 2011). Pollination is also altered in this context due to effects on pollinator species and reduced flower attraction, so that in severe environments pollination rate decrease in both sexes (Ashman, 2006). The effects of environmental severity have also been studied on specific floral traits, frequently showing that reduced corollas are advantageous under stressful conditions (Carroll et al., 2001; Case and Barret, 2004; Galen, 2005). For example, floral dimorphism was found to be correlated with climate variation in the gynodioecious Kallstroemia grandiflora (Cuevas and López 2011), where diameter of hermaphrodite flowers decreased with temperature and increased with precipitation, while female flowers remained unchanged. Silene acaulis (L.) Jacq. is a widespread gynodioecious plant in boreo-alpine ecosystems, and thus is exposed to a range of environmental conditions. Its reproductive system and patterns have been studied in relation to sex (Shykoff, 1988; Morris and Doak, 1998; Hermanutz and Innes, 1994), elevation range (Alatalo and Molau, 1995), and environmental severity (Philipp et al., 1990; Delph and Carroll, 2001; Ashman, 2006; Villellas et al., 2016) in different parts of its distribution range. Results show that the proportion of female individuals in the populations is positively correlated with environmental severity defined by precipitation, temperature and elevation (Alatalo and Molau, 1995; Delph and Carroll, 2001; Delph, 2003; Ashman, 2006). Shykoff (1988) and Morris and Doak (1998) found that fruit production is higher in females than in hermaphrodites. This can be interpreted as a reproductive compensation strategy that allows the coexistence of both sexes. Regarding fertilization, females and hermaphrodites seem to receive the same amount of pollen, but females become receptive earlier and develop a longer style (Philipp et al., 1990; Shykoff, 1992; Delph, 2003). The goal of this investigation 3

is to integrate and analyse reproductive patterns of S. acaulis in relation with environmental severity, across a broad latitudinal and altitudinal range. This study examines the relation among reproduction patterns, floral dimorphism and environment in an integrative way. To achieve this goal, variation in these reproductive traits in the gynodioecious species S. acaulis was analysed, spanning a broad spatial scale, driving the following questions: 1) Does S. acaulis present any kind of compensation between female and hermaphrodite plants as a consequence of differential reproductive investment, in terms of plant size, flower production, fruit production, seed production, or quality of the seeds? 2) Do S. acaulis flowers show any sexual dimorphism? 3) Is there any relationship between flower dimorphism, reproductive success, and environmental stress? Do hermaphrodites lose their female function in severe environments? Hypothesis here presented was that there is reproductive compensation in females as well as sexual dimorphism in floral traits in S. acaulis, and that both are related with environmental severity, so that hermaphrodites in severe conditions would invest more in male function, in terms of production and related flower traits, leading gynodioecy to dioecy.

2. Materials and methods 2. 1. Study species Silene acaulis (L.) Jacq. (family Caryophyllaceae) is a common cushion-plant with a single deep tap-root at the centre of each cushion, and which may live over 100 years (Jones and Richards, 1962; Morris and Doak, 1998). Flowers are pink and small (6-12mm), and occur singly. The breeding system is usually described as gynodioecy, due to nuclear-cytoplasmic sex determination (Lloyd, 1974; Delph and Carroll, 2001). Some cases of sex variation like gynomonoecy and tryoecy are documented (Jones and Richards, 1962; Shykoff, 1988; Philipp et al., 1990; Alatalo, 1997), but not found in the populations of this study. S. acaulis has an arctic and alpine distribution, occurring in the boreal and mountainous regions of Europe, Asia, North America and Greenland. The southern distribution area in Europe is along the Alps, Pyrenees, Cantabrian Mountains, Balkans and Carpathians. In these mountain ranges, S. acaulis occurs at high elevation sites, reaching 3700m, and is not found below 1000m. It lives in cold areas, where mean annual temperatures range from -8ºC to a maximum of 13ºC, and in regions of high rainfall, mostly over 1250mm/m2 (Jones and Richards, 1962).

2. 2. Population sampling This study was conducted in summer 2013 and 2014 in the Swiss Alps (three populations) and the Spanish Pyrenees (three populations), fulfilling a broad spatial range. Populations in the Pyrenees were located in Valle de Ordesa (“PyreneesA”: 42º 40' 23''N, 0º 00' 53'' E), Urdiceto lake (“PyreneesB”: 42º 40' 00'' N, 0ª 16' 44'' E) and Aísa mountains (“PyreneesC”: 42º 43' 05''N, 0º 33' 42''W), all of them in central Pyrenees. Populations in the Alps were located in Val Bercla, a 7 km valley situated in the central Alps of SE Switzerland (AlpsA: 46º 28' 31''N, 9º 34' 69'' E; AlpsB: 46º 29' 16''N, 9º 35' 15'' E; AlpsC: 46º 30' 14''N, 9º 35' 19'' E; Fig. 1). The six locations are above treeline, in rocky and flat sites (except PyreneesC, which 4

was located in a slope), isolated from livestock, and far from busy trails. Populations were chosen to fulfil a gradient of elevation and diverse climatic conditions (Table 1) using a bioclimatic model. All six populations were visited at least twice during summers of 2013 and 2014. The first sampling every year was carried in early summer, during the flowering season, to set up 2 m wide transects that included at least 100 plants per transect (101 in the smallest transect and 264 plants in the largest one). The sex of plants was recorded, and the number of flowers counted (except in in AlpsC, because by the sampling period most of the flowers had wilted). Three to five mature flowers from each of 10-15 different plants per sex were collected in each population outside transects in order not to interfere natural seed production in monitored plants. The following flower measurements (to the nearest 0.01 mm) were taken in the laboratory using callipers (Cuevas and Lopez, 2011): corolla width, entire largest petal length, ovary length and width (from which ovary volume was calculated) and style length. Last sampling every year was in late August, during the fruiting period. The number of fruits per plant were counted and three to five mature fruits from 10-15 different plants per sex were randomly collected in order to count seeds per fruit. Seeds from another set of three to five mature fruits from 10-15 different plants per sex were used for germination test, placing 150 randomly seeds per population and sex in Petri dishes with wet filter paper. Ten seeds per Petri dish were placed in a germination chamber on a 12-hour day light cycle at 22ºC (Jolls and Chenier 1989; Shykoff, 1998). Every two days the Petri dishes were watered and germinated seeds were counted and removed. To estimate flower and fruit production/area, cushion surface was calculated as an ellipse, using data of the length of the major axis (H), the perpendicular axis (h) and the estimated missing percentage to complete a perfect ellipse (MP) in every cushion. Plant size results from: 𝐴𝑟𝑒𝑎 = (1 − 𝑀𝑃) ∗ (𝜋 ∗ 𝐻/2 ∗ ℎ/2)

2. 3. Environmental suitability To evaluate the effects of environmental suitability on sexual dimorphism and reproductive output, a species distribution model (SDM) was built. Based on the principle of maximum entropy, SDMs use the optimum combination of a species occurrence and environmental variables to obtain an estimation of the suitability at each site based on the available environmental information analysed (Phillips, 2006). In this study, MaxEnt 3.3.3a. software was used for model calibration and projection. 4340 occurrences data of S. acaulis in Europe were obtained from Data Portal of the Global Biodiversity Information Facility (http://www.gbif.org/). Environmental suitability was characterized from bioclimatic data, but not from soil richness, presence of competitors or human/livestock impact because their effects on S. acaulis presence are low (Delph and Carroll, 2001) and limited spatial information is available on these factors. The 7 most representative bioclimatic variables data were chosen (Wollan et al., 2008) and downloaded from WorldClim (http://worldclim.org/) in 1 km pixel raster: annual mean temperature, isothermality (index of variability of temperature), temperature annual range (maximum temperature - minimum temperature), mean temperature of wettest season, mean temperature of the driest season, annual precipitation and precipitation seasonality (coefficient 5

of variation). MaxEnt constructs a model considering bioclimatic data in the occurrence points. Afterwards, the model is projected to the whole of Europe, creating an output raster map containing an estimation of suitability values for S. acaulis throughout Europe. Additionally, the effects of each bioclimatic variable over the presence of S. acaulis are evaluated to study the most restrictive bioclimatic conditions for this species. The model was run five times, taking the average of all as the final model. To validate the model quality, AUC-ROC (Area Under Curve – Receiving Operating Characteristic) was calculated using the 30% of occurrence data available (Huberty 1994). AUC values are between 0 and 1, where values similar to 0.5 are equivalent to a random prediction and values higher than 0.8 are considered an excellent prediction (Fielding and Bell, 1997). Finally, we used the generated projection to estimate an environmental suitability value for each of the six studied populations (Table 1). These values were later used to evaluate environmental effects in sexual dimorphism and reproductive production.

2. 4. Data analysis To determine how sex influences flower traits and the different reproductive patterns, several analyses were performed using the statistical package R (R Core Team, 2014). Linear Models were used to test differences between sexes in plant size (log-transformed), corolla width, petal length, ovary volume, style length, flower production, fruit production and seed per fruit (including plant size as a covariate for all these traits). Correlation between petal length and corolla width were tested with a Pearson correlation. In addition, the proportion of flowers successfully maturing into fruits was analysed using Generalized Linear Models (GLM; binomial distribution) and the number of successful and unsuccessful flowers in setting fruit as the response variable. Germination was also tested using a GLM (binomial distribution), with the number of seeds that germinated as the response variable. In all analyses, the two regions (Alps and Pyrenees) and the six populations were studied as fixed factors. Because none of studied traits showed significant differences according to region, this variable, but not population variable, was removed from final analyses. The consequences of environmental severity on reproduction and floral variables were analysed with a GLM (Poisson error distribution) with suitability, elevation and population as fixed factors.

3. Results 3. 1. Species distribution model Our SDM revealed a suitability index for the six studied populations that varies from 0.390 in the PyreneesC to a maximum of 0.528 in the AlpsB (Fig. 1; Table 1). Validation via ROC curve showed a good quality of adjustment model obtaining values of 0.886 for AUC that confirmed the model as optimal. MaxEnt also produces a heuristic estimation of the relative contribution of every environmental variable used in the model (Phillips, 2006). In this study the relative contribution of the main environmental variables to the model was: temperature annual range (28.8%); mean temperature of wettest quarter (26.5%); annual precipitation (21.4%); annual mean temperature (18.9%). The contribution of the other variables was low (4.3% in total). 6

According to the model, the most suitable areas for S. acaulis in Europe are those with annual temperatures from -5ºC to 5ºC, with an annual range of temperatures lower than 20ªC and with an annual precipitation higher than 1200mm/m2. 3. 2. Reproductive patterns Reproductive patterns were studied in terms of flower and fruit production per plant, seed production per fruit and seed germination success, accounting for plant sizes. Plant size did not differ significantly between sexes (Table 2). However, plant size was positively related to flower production (F544 = 166.6; p < 0.001) in both sexes (absence of interaction between plant size and sex: F543 = 83.7; p = 0.351), and to fruit production in female plants (F351 = 47.9; p < 0.001), but not in hermaphrodites (F262 = 10.1; p = 0.561). Flower number per plant was not significantly different between females and hermaphrodites (Fig. 2; Table 2). In contrast, females produced almost four times more fruits per plant than hermaphrodites (Fig. 2; Table 2). A significantly higher fruiting success was also found in flowers of females than hermaphrodites (Table 2). Plant size was correlated with fruiting success (p < 0,001), and differently for both sexes (significant interaction between plant size and sex; p < 0,001). Seeds per fruit and germination success did not show significant differences between sexes (Table 2). Seeds of S. acaulis began to germinate the fourth day after they were first exposed to light and water, and showed a final germination of 32% after 55 days. Most seeds germinated during the first 24 days (85% of germinated seeds).

3. 3. Sexual dimorphism in floral traits Female and hermaphrodite flowers differed in size (Fig.3), but there were no significant correlations between flower traits and plant size (corolla size: F318 = 118.9; p = 0.609; petal length: F355 = 35.7; p = 0.110; ovary volume: F355 = 51.37; p = 0.686; style length: F355 = 241.9; p = 0.781). Analysis showed that corolla width and petal length were significantly bigger in hermaphrodites than in females (Table 2), and were positively correlated across plants (Pearson correlation coefficient rho = 0.716, p < 0.001). Ovary volume was almost three times larger and styles much longer in females than in hermaphrodites (Table 2).

3. 4. Environmental suitability, reproduction and flower dimorphism Plant size was not correlated with suitability (Fig. 4) but was significantly bigger in AlpsA population (F610= 18.9; p = 0.008). Number of flowers and fruits per plant increased with plant size with a similar correlation in each population, although marginally significant interactions between plant size and location were found for flower production (F535 = 47.84; p = 0,061) and fruit production (F593= 17.06; p = 0,076). Number of flowers per plant was not significantly affected by suitability, although it showed a marginal tendency to decrease as suitability increased (Fig. 4). Fruit production was not affected by suitability either (Fig. 4). PyreneesB values for flower production and fruit production in females were higher than for all other populations (F539= 22.84; p < 0.001 for flower production; F610= 22.84; p < 0.001 for fruit production). In the same trend as other reproductive traits, fruiting success was not significantly related with suitability (Fig.4) and the 7

interaction between suitability and sex was not significant. Seed production was not significantly affected by suitability in either sex, although the trend was negative in females and positive in hermaphrodites (Fig. 4) and slightly bigger in AlpsA population (F605= 2.29; p = 0.041). Germination success was negatively correlated with suitability, regardless of sex (Fig. 4). Finally, suitability was not correlated with either corolla size, petal length, style or ovary volume. AlpsC and PyreneesB had bigger female ovaries than the other females, but it did not happen in the other populations (Fig. 4).

4. Discussion In this study three concrete hypotheses were tested in relation to the maintenance of the gynodioecious system of S. acaulis. The first hypothesis proposed that females might present a compensation for the lack of pollen production in terms of seed production that support the coexistence of two sexes. The second hypothesis predicted that S. acaulis flowers would be sexually dimorphic. The third hypothesis stated that, if compensation in reproductive patterns and floral dimorphism between sexes are related to environment leading hermaphrodites to invest more to a male function, then gynodioecy would decrease in favour of dioecy in a more severe environment. The SDM model revealed that cold temperatures throughout the year (around 0ºC) and low temperature variation were the main restrictive parameters for S. acaulis, as well as abundant annual precipitation (over 1200mm/m2 per year), in accordance to results in Dahl (1951) and Jones and Richards (1962). Also, moderate temperatures and abundant precipitation during the growth season were crucial to understand S. acaulis presence (Alatalo, 1997). Thus, high latitudes (arctic tundra, Norway and Iceland) and alpine areas (Alps, Apennines, Balkans, Carpathians and Pyrenees) were the most favourable for S. acaulis in Europe. The production of flowers per area was not different between females and hermaphrodites, but the fruiting success and fruit production per area was higher in females. Females and hermaphrodites received the same amount of pollen (Shykoff, 1992), but a longer flowering time (Philipp et al., 1990; Delph, 2003) as well as a larger style and bigger stigmatic surface in the female flowers may increase their efficiency in capturing pollen (Dulberger and Horovitz, 1984). In S. acaulis, female flowers have a more developed female reproductive system (ovary and style), which would benefit effective reception of pollen and enhance fruit production, as shown in this study. Once ovaries are fertilized, however, seed production was not significantly different between sexes, neither seed quality according to the germination test. Previous studies suggested that the total seed set ranged between 2.9-3.8 (Shykoff, 1988) and 4.4 higher (Morris and Doak, 1998) in females than in hermaphrodites. Results of this study proved that the main strategy in S. acaulis for female compensation, and thus for the maintenance of gynodioecy, was through higher fruiting success. Hermaphrodite flowers of S. acaulis were larger than female flowers, as shown for other gynodioecious species previously studied (Delph 1996; Miller and Vanable, 2003; Cuevas and Lopez, 2011). Plak (1957) explained this on the basis of the presence of stamens in hermaphrodite flowers, which involves a greater opening of its petals to accommodate stamens, leading to wider corollas. But in S. acaulis hermaphrodites not only the corolla was 8

wider, but petals were longer as well. Hence, the explanation of this sexual dimorphism was not likely related with stamens presence, but to enhance pollinator visits and then increase pollen directional movement (Delph, 1996). In contrast, only one or few visits might be sufficient to deliver enough pollen to fertilize the ovules in the case of female flowers. Moreover, female organs were larger in females than in hermaphrodites, which could result from saving nutrients in pollen production Shykoff (1988). Altogether these results suggest that hermaphrodite flowers prioritize pollen release (male function), which leads to some failure in setting fruits and seeds. But once hermaphrodite flowers are fertilized, they are as successful (in number of recruits per fruit) as pure females. Compensation for reproductive patterns and flower dimorphism were analysed in relation with suitability of each population, with unexpectedly diverse results. Most of the studied traits, such as plant size, flower and fruit production per plant, were unaffected by site suitability. Only the performance in PyreneesB and AlpsA populations were significantly different from the others, with higher production of flowers and fruits in PyreneesB and bigger plants and higher production of seeds per fruit in AlpsA. These results suggest important regional or local effects, and imply that differences in production may respond more to the particularities of each population (competition with other species, interaction with pollinators, etc.) than to overall climatic suitability. Seed production per fruit was not significantly affected by suitability either, but the trend was positive in hermaphrodites, in contrast with the negative tendency in females. These trends, although not significant, agreed with the prediction of Ashman (2006), who indicated that hermaphrodites could produce more seeds where maximum resources were available. But if conditions were not as favourable, then hermaphrodites produced fewer seeds, and reduce the importance of their female role. Germination success was the only trait significantly affected by suitability, showing a negative correlation. Germination success in S. acaulis is strongly related with seed size and seed competition for resources (Shykoff, 1992). In this study, number of seeds per fruit were considered, but not seed size or other seed traits that might foster germination. In any case, the species seems to compensate the unfavourable environmental conditions of some populations by producing more successful seeds in terms of germination. Which traits are involved in this strategy remains an open question, and more information, regarding the quality of seeds, would be necessary to better understand these reproductive patterns. Finally, sexual dimorphism in flowers was also analysed in relation with the suitability of every population, but no significant differences were found. In fact, S. acaulis showed little variation in flower size both within and between populations. The low overall influence of environmental suitability can be perhaps understood by attending to Silene acaulis life history and ecological strategies. Longevity is very high in S. acaulis plants may be extremely high (over 100 years and some individuals could reach over 300 years; Morris and Doak, 1998), so that plants have many opportunities to produce seeds during their lifetime. This, in turn, reduces selection on floral traits relative to shorter lived plants, which would explain the differences in the importance of environmental suitability on reproductive traits among taxa (Cuevas and López, 2011). The hypothesis that gynodioecy decreases in favour of dioecy in severe environments was not clearly supported by results here presented. However, it is worth mentioning that the six populations here studied showed a relatively high suitability for S. acaulis presence, despite the broad altitudinal and latitudinal span of this study and stress does not seem to play an important differential role between populations in the range of conditions considered. In any case, it is important to consider that stressful conditions might be more severe for boreoalpine 9

plants in the future, if global warming goes on (Engler et al., 2011, Pauli et al. 2012). According to the SDM model presented here, temperature is the main restrictive variable for the presence of S. acaulis, so populations in the southern limit of the distribution, such as the populations in the Pyrenees, will likely be in a more dramatic situation situation if the warming trend registered in the last decades (López-Moreno et al. 2010) continues. A continued monitoring of performance in female and hermaphrodite plants in gynodioecious taxa like S. acaulis is thus needed, to check whether the current global change has an effect on their reproductive traits and patterns.

5. Conclusions This study indicates that a higher fruiting success of female plants constitutes a compensation mechanism in the reproductive system of S. acaulis. Results also support the hypothesis that hermaphrodite flowers are bigger than female flowers in order to increase the male function, but are not as successful for the female function although, once flowers are fertilized, fruits and seeds produced by the two sexual forms are equally viable. Female organs are bigger in female than in hermaphrodite flowers in accordance with the higher fruit production in females. Finally, results do not indicate that environment suitability has any effect on most of the compensation strategies in reproduction and in flower dimorphism, suggesting that local considerations of suitability should be taken in account.

6. Acknowledgements This study was realized with the help and company of many people to whom we are pleased to mention. Thanks to Christian Rixen his help, suggestions and motivation in the design and execution of work; Pablo Tejero, Gerard Sapés, Samuel Pironon, Miquel Riba, Arnald Marcer and Esther Prat who have taken the time to help in certain aspects of the work; Pablo Tejero, María Leo, Samuel Pironon, Chelsea Little, Sofia Häggberg, Tomàs Miralles and Dani Riba for their company and help in the fieldwork. Funding was provided by projects CAMBIO (CGL2010-21642) and RECAMBIO (430/211) to MGB, and by Swedish Research Council (Ref: 2012-42619-94710-26). This study results from the collaboration in other projects with the SLF institute and CSIC-IPE, which we also would like to mention.

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References Alatalo, J.M., 1997. Gender lability in trioecious Silene acaulis (Caryophyllaceae). Nord. J. Bot. 17, 181-183. Alatalo, J.M., Molau, U., 1995. Effect of altitude on the sex ratio in populations of Silene acaulis (Caryophyllaceae). Nord. J. Bot.15, 251–256. Arnan, X., Escolà, A., Rodrigo, A., Bosc, J., 2014. Female reproductive success in gynodioecious Thymus vulgaris: pollen versus nutrient limitation and pollinator foraging behaviour. Bot. Jour. Linnean soc.175.3, 395-408. Ashman, T.L., 2006. The evolution of separate sexes: a focus on the ecological context. In: Harder, L.D., Barrett S.C.H. (Eds), Ecology and Evolution of Flowers, Oxford University Press, 419-465. Bell, G., 1985. On function of flowers. Proc. R. Soc. Lond. Ser. B. Biol. Sci. 224, 223- 265. Carroll, A.B., Pallardy, S.G., Galen, C., 2001. Drought stress, plant water status, and floral trait expression in fireweed Epilobium angustifolium(Onagraceae). Am. J. Bot. 88, 438–446. Case, A., Barret, S., 2004. Environmental stress and the evolution of dioecy: Wurmbeadioica (Colchicaceae) in Western Australia. Evol. Ecol. 18, 145-164. Charlesworth, D., 1981. A further study of the problem of the maintenance of females in a gynodioecious species. Heredity. 46, 27–39. Charlesworth D., Charlesworth B. 1978., A model for the evolution of dioecy and gynodioecy. Am. Nat.112, 975-997. Charlesworth D., Laporte V., 1998. The male-sterility polymorphism of Silene vulgaris: analysis of genetic data from two populations and comparison with Thymus vulgaris. Genetics. 150, 1267-1282. Cuevas, E., Abarca, C., 2006. Origen, mantenimiento y evolución del ginodioicismo. Bol. Soc. Bot. Mex. 78, 33-42. Cuevas, E., López, S., 2011. Sex ratio and sex-specific latitudinal variation in floral characteristics of gynodioecious Kallstroemia grandiflora (Zygophylaceae) in Mexico. Biotropica 43, 317-323. Dahl, E. 1951., On the relation between summer temperature and the distribution of alpine vascular plants in the lowlands of Fennoscandia. Oikos. 3.1, 22-52. Darwin, C., 1877. The different forms of flowers on plants of the same species. Murray, J. (Ed.), University of Chicago Press, Chicago. Dawson, T.E., Geber, M.A., 1999. Sexual dimorphism in physiology and morphology. In: Geber, M.A., Dawson, T.E., Delph, L.F. (Eds.), Gender and Sexual Dimorphism in Flowering Plants. Springer-Verlag, New York, USA, 175–215.

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Delph, L.F., 1996. Flower size dimorphism in plants with unisexual flowers. In: Lloyd, D.G., Barrett, S.C.H. (Eds.) Floral biology: studies on floral evolution in animal-pollinated plants, 217-237. Delph, L.F., 2003. Sexual dimorphism in gender plasticity and its consequences for breeding system evolution. Evol. & Devel. 5, 34-39. Delph, L.F., Carroll, S., 2001. Factors affecting relative seed fitness and female frequency in a gynodioecious species, Silene acaulis. Evo. Eco. Research. 3, 487-505. Dem’yanova A.E., 1985. Distribution of gynodioecy in flowering plants. Botaicheskii Zhurnal. 70, 1289-1301. Dulberger, R., Horovitz, A. 1984. Gender polymorphism in flowers of Silene vulgaris (Moench) Garcke (Caryophyllaceae). Biological Journal of the Linnean Society. 89, 101- 117. Engler R et al., 2011. 21st century climate change threatens mountain flora unequally across Europe. Global Change Biology. 17, 2330-2341 Fielding, A.H., Bell, J.F., 1997. A review of methods for the assessment of prediction errors in conservation presence/absence models. Environ. Conserv. 24, 38-49. Frohlich, M., 2003. An evolutionary scenario for the origin of flowers. Nature Reviews. 4, 559566. Galen, C., 2005. It never rains but then it pours: The diverse effects of water on flower integrity and function. In: Rekie, E.G., Bazzaz, F.A. (Eds.), Reproductive allocation in plants. 75–90. Hermanutz, L.A., Innes, D.J., 1994. Gender variation in Silene acaulis (Caryophyllaceae). Pl. Syst. Evol. 191, 69–81 Jolls, C. L., Chenier, T. C., 1989. Gynodioecy in Silene vulgaris (Caryophyllaceae): progeny success, experimental design, and maternal effects. American journal of botany. 9, 13601367. Jones, V., Richards, P. W., 1962. Biological flora of the British Isles. Journal of Ecology. 50-2, 475-487. Kaul, M.L.H., 1988. Male sterility in higher plants. Springer-Verlag, Germany. Lewis, D., 1941. Male sterility in natural populations of hermaphroditic plants. New Phytol. 40, 56–63. Lloyd, D.G., 1974. Theoretical sex ratios of dioecious and gynodioecious angiosperms. Heredity. 32, 11–34. Lloyd, D.G., 1976. The transmission of genes via pollen and ovules in gynodioecious angiosperms. Theor. Pop. Biol. 9, 299-316. López-Moreno, J.I., Vicente-Serrano, S.M., Moran-Tejeda, E., Zabalza, J., Lorenzo-Lacruz, J., García-Ruiz, J.M., 2010. Impact of climate evolution and land use changes on water yield in the Ebro basin. Hydrol Earth Syst Sci. 15, 311-322

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Miller, M., Venable, D.L., 2003. Floral morphometric and the evolution of sexual dimorphism in Lycium (Solanaceae). Evolution. 57, 74-86. Moreno, L., Bertiller, M. B., 2016. Variation of morphological and chemical traits in sexes of the dioecious perennial grass Poa ligularis in relation to shrub cover and aridity in Patagonian ecosystems. Population ecology. 58(1), 189-197. Morris, W., Doak, D., 1998. Life history of the long-lived gynodioecious cushion plant Silene acaulis (Caryophyllaceae), inferred from size-based population projection matrices. American journal of botany. 85(6), 784-793. Obeso, J.R., 2002. The cost of reproduction in plants. New Phytol. 155, 321–348. Pauli H., Gottfried M., Dullinger S., Abdaladze O., Akhalkatsi M., Alonso J.L.R, et al., 2012. Recent plant diversity changes on Europe's mountain summits. Science. 336, 353-355. Philipp, M., Bocher, J., Mattsson, 0., Woodell, S.R.J., 1990. A quantitative approach to the sexual reproductive biology and population structure in some arctic flowering plants: Dryas integrifolia, Silene acaulis and Ranunculus nivalis. Commission for Scientific Research in Greenland. 34, 1-60 Phillips, S.J., Anderson, R.P., Schapire, R.E., 2006. Maximum entropy modelling of species geographic distributions. Ecol. Model. 190, 231-259. Plack, A., 1957. Sexual dimorphism in Labiate. Nature. 180, 1218–1219. R Core Team, 2014. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. Richards, A. J., 1986. Plant breeding systems. London Schools, London. Sánchez-Vilas, J., Bermúdez, R., Retuerto, R., 2012. Soil water content and patterns of allocation to below - and above - ground biomass in the sexes of the subdioecious plant Honckenya peploides. Ann. Bot. 110, 839–848. Sánchez-Vilas, J., Campoy, G., Retuerto, R., 2016. Sex and heavy metals: Study of sexual dimorphism in response to soil pollution. Environ. Exp. Bot. 126, 68-75. Shykoff, J.A., 1988. Maintenance of gynodioecy in Silene acaulis (Caryophyllaceae): Stage specific fecundity and viability selection. Am. J. Bot. 75, 844-850. Shykoff, J.A., 1992. Sex polymorphism in Silene acaulis (Caryophyllaceae) and the possible role of selection in maintaining females. Am. J. Bot. 79, 138-143. Shykoff, J.A., Kolokotronis, S.O., Collin, C. L., López-Villavicencio, M., 2003. Effects of male sterility on reproductive traits in gynodioecious plants: a meta-analysis. Oecologia. 135(1), 1– 9. Thompson, J. D., Rolland, A., Prugnolle, F. 2002. Genetic variation for sexual dimorphism in flower size within and between populations of gynodioecious Thymus vulgaris. Jour. Evo. Bio. 15, 362–372.

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Villellas, J., Cardós, J. L., García, M.B., 2016. Contrasting Population Dynamics in the BoreoAlpine Silene acaulis (Caryophyllaceae) at Its Southern Distribution Limit. In: Annales Botanici Fennici. Finnish Zoological and Botanical Publishing Board. Wolfe, L. M., Shmida, A., 1997. The ecology of sex expression in a gynodioecious israeli desert shrub (Ochradenus baccatus). Ecology. 78(1), 101–110. Wollan, A. K. 2008. Modelling and predicting fungal distribution patterns using herbarium data. Journal of Biogeography. 35(12), 2298-2310.

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Figures and tables FIGURE 1. Species Distribution Model for Silene acaulis in Europe. Legend indicates the values of the suitability index across colours. Points represent the studied populations: 1) Pyrenees C; 2) PyreneesA; 3) PyreneesB; 4) AlpsA; 5) AlpsB; 6) AlpsC. [Figure size: 1 column]

FIGURE 2. Number of flowers per plant, percentage of fruiting success, and number of fruits per plant in females (Fem; dark grey) and hermaphrodites (Herm; light grey) in all studied populations of Silene acaulis. [Figure size: 1 column]

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FIGURE 3. Differences between females (Fem; dark grey) and hermaphrodites (Herm; light grey) in floral parameters: a) Corolla; b) Ovary volume; c) Petal length; d) Style length in all studied populations. The appearance of flowers and ovaries of each sex is drafted in e) and f). [Figure size: 1.5 column]

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FIGURE 4. Relationship between suitability and A) Flower production per plant; B) Fruit production per plant; C) Fruiting success; D) Seed production per fruit (different significance for hermaphrodites and females); E) Germination; F) Plant size; G) Corolla width; H) Petal length; I) Style length; and J) Ovary volume, analysed with Generalized and Linear Models. Symbols indicate females (dark grey circles) and hermaphrodites (light grey triangles). [Figure size: 2 columns]

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TABLE 1. Climatic characteristics of studied populations: elevation, annual mean temperature (Mean T.), isothermality (Isotherm.), temperature annual range (Range T.), mean temperature of wettest season (Wett. S. T.), mean temperature of season (Driest S. T), annual precipitation (Ann. P.), precipitation seasonality (Seas. P.) and suitability output from the Species Distribution Model (Suit. Index). Population

Elevation (m)

Mean T. (ºC)

Isotherm. (ºC)

Range T. (ºC)

Wett. S.T. (ºC)

Driest S.T. (ºC)

Ann. P. (mm/m2)

Seas. P. (mm/m2)

Suit. index

AlpA

2495

-1.0

2.6

19.6

5.5

-7.2

1559

20

0.457

AlpB

2260

0.4

2.7

20.6

7.1

-5.9

1400

24

0.528

AlpC

2050

2.3

2.8

22.2

9.4

-4.3

1221

29

0.519

PyreneesA

2700

5.0

3.2

22.1

1.2

7.6

1446

12

0.436

PyreneesB

2380

1.3

3.2

22.1

2.1

8.4

1403

12

0.451

PyreneesC

2020

3.6

3.6

23.4

4.8

10.8

1219

13

0.390

TABLE 2. Means and standard deviation (s.d.) for reproduction and flower traits. P-values for differences between sexes; significant p-values are indicated in bold letters. Z-score (Z) for Generalized Linear Models binomial distribution analysis or F-test (F) and degrees of freedom for Linear Model analysis are given. Female

Hermaphrodite

Mean

s.d.

Mean

s.d.

Statistic

p-value

Plant size (cm2)

260.7

333.8

269.5

349.1

F610 = 18.9

0.671

Flowers/plant (nº)

21.9

6.3

16.7

12.1

F539 = 48.8

0.453

Fruits/plant (nº)

21.5

3.2

5.1

3.2

F609 = 31.74