RESEARCH PAPER Seed germination and seedling allogamy in Rosmarinus officinalis: the costs of inbreeding P. Garcia-Fayos1 , M. C. Castellanos2 & J. G. Segarra-Moragues3 1 Centro de Investigaciones sobre Desertificacion, (CIDE-CSIC-UV-GV), Moncada, Valencia, Spain 2 School of Life Sciences, University of Sussex, Falmer, Brighton, UK 3 Departamento Biolog ıa Vegetal, Facultad de Ciencias Biologicas, Universitat de Valencia, Burjassot, Valencia, Spain
Keywords ABSTRACT Allogamy; geitonogamy; male-sterile flowers; pollination; reproductive biology; seed • Self-pollination by geitonogamy is likely in self-compatible plants that simultaneously germination. expose a large number of flowers to pollinators. However, progeny of these plants is often highly allogamous. Although mechanisms to increase cross-pollination have Correspondence been identified and studied, their relative importance has rarely been addressed simul- P. Garcia-Fayos, Departament of Plant Ecology taneously in plant populations. Centro de Investigaciones sobre • We used Rosmarinus officinalis to explore factors that influence the probability of self- Desertificacion (CIDE-CSIC-UV-GV), C/ fertilisation due to geitonogamy or that purge its consequences, focusing on their Carretera de Moncada-Naquera Km 4.5, effects on seed germination and allogamy rate. We experimentally tested the effect of Apartado Oficial, E-46113 Moncada, Valencia, geitonogamy on the proportion of filled seeds and how it influences germination rate. Spain. During two field seasons, we studied how life history and flowering traits of individu- E-mail: [email protected] als influence seed germination and allogamy rates of their progeny in wild populations at the extremes of the altitudinal range. The traits considered were plant size, popula- Editor tion density, duration of the flowering season, number of open flowers, flowering syn- C. Smit chrony among individuals within populations and proportion of male-sterile flowers. • We found that most seeds obtained experimentally from self-pollination were appar- Received: 9 March 2017; Accepted: 20 ently healthy but empty, and that the proportion of filled seeds drove the differences in December 2017 germination rate between self- and cross-pollination experiments. Plants from wild populations consistently had low germination rate and high rate of allogamy, as deter- doi:10.1111/plb.12686 mined with microsatellites. Germination rate related positively to the length of the flow- ering season, flowering synchrony and the ratio of male-sterile flowers, whereas the rate of allogamous seedlings was positively related only to the ratio of male-sterile flowers. • Rosemary plants purge most of the inbreeding caused by its pollination system by aborting the seeds. This study showed that the rates of seed germination and allogamy of the seedlings depend on a complex combination of factors that vary in space and time. Male sterility of flowers, length of the flowering season and flowering synchrony of individuals within populations all favour high rates of cross-pollination, therefore increasing germination and allogamy rates. Flowering traits appear to be highly plastic and respond to local and seasonal conditions.
However, most self-compatible plants produce outcrossed INTRODUCTION progeny at high rates (Goodwillie et al. 2005) using multiple Self-compatible plants are faced with the challenge of reducing strategies that promote outcrossing or counteract the negative the potentially negative effects of pollen discounting and impacts of self-fertilisation. Self-compatible plants, for exam- inbreeding depression caused by self-pollination (Barrett ple, may have mechanisms to reduce the chance of self-pollina- 2002). Inbreeding is particularly likely in self-compatible tion by separating male and female functions in time insect-pollinated plants that bloom massively, where many (dichogamy) or space (herkogamy) (Barrett 2002). Also the flowers of the same plant or closely related neighbours are patterns of flowering phenology of self-compatible plants influ- exposed simultaneously to pollinators (Harder & Barrett 1995; ence the amount of self-pollination and the resulting outcross- Harder et al. 2004). When encountering a high amount of con- ing rates within populations (Kameyama & Kudo 2009). The centrated rewards, insect pollinators tend to remain within the duration of the flowering period, the number of open flowers same plant and potentially perform autogamous and geitonog- at the same time, and the synchrony of individuals also influ- amous pollinations (de Jong et al. 1993). For many plant ence the probability of geitonogamy (de Jong et al. 1993; Snow species this selfing component of mating may represent a non- et al. 1996; Kudo 2006). Flowering synchronously enhances the adaptive cost associated with large floral displays needed to attractiveness of populations to pollinators, but large flowering attract pollinators carrying pollen from and to other individu- displays of individuals can also have the disadvantage of caus- als (Barrett 2003). ing more sequential pollinator visits within the same plant
Plant Biology 20 (2018) 627–635 © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands 627 Inbreeding in Rosmarinus officinalis Garcia-Fayos, Castellanos & Segarra-Moragues thus increasing selfing rates due to geitonogamy (Vrieling flowers in individual plants (Roiz & Dulberger 1988). Although et al. 1999; Mitchell et al. 2004). Thus, reducing the number gynodioecy in the Lamiaceae has been frequently reported of flowers simultaneously exposed to pollinators but being (Owens & Ubera 1992), unstable gynodioecy, the incomplete highly synchronous with other individuals in the population and variable production of male-sterile flowers in a plant could effectively reduce selfing rates (de Jong et al. 1993; individual, is much less frequent. It has been reported so far for Snow et al. 1996). In addition, extending the flowering per- some Origanum L. and Teucrium L. species, as well as R. offici- iod could allow individual plants to increase the chances of nalis (Rodr ıguez-Riano~ & Dafni 2007; and references therein). outcrossing (Klinkhamer & de Jong 1990; Elzinga et al. In R. officinalis, populations range from those consisting of all 2007). Moreover, if the flowers have been self-pollinated, individuals with 100% pure hermaphrodite flowers to a early inbreeding depression may cause plants to purge mixture of plants with variable proportions of male-sterile embryos produced by self-fertilisation, therefore producing flowers (Herrera 1987a; Roiz & Dulberger 1988; Ubera-Jim enez high rates of outcrossed progeny (Stephenson 1981; Wiens & Hidalgo-Fern andez 1992); in addition, the proportion of et al. 1987; Husband & Schemske 1996; Snow et al. 1996; male-sterile flowers in individual rosemary plants varies Byers & Waller 1999). The extent and mode in which plants throughout the breeding season (Ubera-Jim enez & Hidalgo- use one or several of these mechanisms simultaneously varies Fern andez 1992). across species, among populations of the same species, In this study we aimed to explore factors that potentially during the life cycle of a plant or even along the breeding reduce the probability of self-fertilisation due to geitonogamy season (Barrett 2002; Eckert et al. 2006). or that purge its consequences in R. officinalis plants, focusing Persistent self-pollination because of geitonogamy in combi- on their effect on seed germination and allogamy rate of the nation with strong inbreeding depression after selfing repre- seedlings. Specifically, (i) we experimentally compared the rates sents a disadvantage of hermaphroditism. In some plant of viable seeds and germination between geitonogamous and lineages this could favour the spread of male-sterile flower cross-pollination treatments; (ii) in six rosemary populations mutants into populations in a well-recognised evolutionary with contrasting environmental characteristics we measured pathway to dioecy from gynodioecy (Thomson & Brunet 1990; plant density and plant size, and monitored through two Spigler & Ashman 2012). The advantage of male-sterile flowers breeding seasons the flowering phenology of individual plants, over hermaphrodite flowers commonly involves one or more the number of flowers opened, the flowering synchrony of fecundity-related components (higher fecundity and/or lower individuals within populations and the proportion of male- inbreeding depression) that allow them to compensate for the sterile flowers; and (iii) we then related these variables to the loss of the male reproductive function (Jordano 1993; Wolfe & germination rate of seeds collected from the same individuals Shmida 1997; Dufay & Baillard 2012). and also to the rate of allogamy of seedlings produced by those Here we focus on a widespread Mediterranean plant to seeds, estimated with microsatellite analysis. explore the relative importance of factors that potentially reduce selfing in natural populations. Rosmarinus officinalis L. (rosemary, Lamiaceae) is a shrub that grows in dense popula- MATERIAL AND METHODS tions and can produce hundreds or thousands of flowers simul- Study species taneously throughout the breeding season. Flowers are self-compatible and attract a wide array of non-specialist insect Rosmarinus officinalis is a common plant in scrublands and pollinators looking for nectar and pollen rewards (Herrera open woodlands in the western Mediterranean Basin. It grows 1987a, 2004). Because flowers are strongly protandrous, spon- under very diverse climate and soil conditions and exhibits taneous autogamy seems to be unimportant (Herrera 1987a; extraordinary morphological and biochemical variation (e.g. Hidalgo-Fern andez & Ubera 2001). In rosemary populations, growth habit, leaf size, flower size and colour, volatile com- less than one third of the open-pollinated flowers produce seed pounds). Such phenotypic variability has been proposed to (Herrera 1986, 1987a,b; Hidalgo-Fern andez & Ubera 2001), derive from the high phenotypic plasticity of plants and from and there is evidence that seed set is sensitive to inbreeding the high genetic diversity within and among populations depression (Hidalgo-Fern andez & Ubera 2001). Although there (Rossello et al. 2006; Morales 2010; Mateu-Andr es et al. 2013). are no data available on the effect of inbreeding depression on Significant levels of phenotypic plasticity have been long the quality of seeds, Madeiras et al. (2009) related the consis- described in this species (Maffei et al. 1993; Munn e-Bosch & tently low germination rates in this species to the high rates of Alegre 2000; Alarcon et al. 2006; Zunzunegui et al. 2011) and a empty seeds they found. All these features suggest a mixed mat- recent population genetics study in 18 wild rosemary popula- ing in rosemary plants, with insect-mediated geitonogamy tions in eastern Spain showed high levels of genetic diversity causing high rates of self-fertilisation but, at the same time, and low genetic differentiation between populations, even with a strong post-zygotic purging of inbred embryos that pro- across the entire altitudinal distribution of the species and in duce many apparently healthy but empty seeds. Consistently, a different soil types in a 6000-km2 region (Segarra-Moragues recent study on population genetics showed high levels of et al. 2016). genetic diversity in 18 wild populations (Segarra-Moragues The flowering period in R. officinalis fluctuates from a few et al. 2016), supporting the possibility that inbreeding depres- weeks in spring at the uppermost limit of its altitudinal range sion acts at early stages of seed development. Thus, it can be (around 1200 m a.s.l.) and far from the sea to almost 7 months expected that most, if not all, the seedlings that rosemary plants from autumn to late spring at the sea level (Herrera 1986; produce in the field originated from cross-pollination. Arroyo 1990). This flowering period is the consequence of Another important feature in R. officinalis is the variable pro- equally long flowering periods of individuals and not of the duction of male-sterile flowers intermixed with hermaphrodite sum of short flowering periods of different individuals within
628 Plant Biology 20 (2018) 627–635 © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands Garcia-Fayos, Castellanos & Segarra-Moragues Inbreeding in Rosmarinus officinalis populations during the season (Herrera 1986), and its length is the density of rosemary plants by counting the number of indi- related to the duration of favourable temperature and moisture viduals in three 20 9 20 m plots in each population. conditions (Petadinou et al. 1995 and references therein). In March 2010 we labelled 25 individuals per population The flowers have four ovules, each potentially producing an that were at least 5 m apart from each other and determined independent single-seeded nutlet that remains enclosed by the their size, estimated as the volume of a cone. Leaves from all calyx until maturation. Nutlets can develop even if the seed individuals were sampled and dried in silica gel until DNA aborts, and seed-full or seed-less nutlets are indistinguishable extraction. Individuals were genotyped using six R. officinalis in appearance but different in weight (Hidalgo-Fern andez & polymorphic microsatellite loci (Segarra-Moragues & Gleiser Ubera 2001). For convenience, we refer to ripe nutlets as seeds 2009; see Segarra-Moragues et al. 2016 for detailed genotyping in this paper. methods). These genotypes were used to estimate allogamy rates of the progeny of mother plants (see below). From September 2010 to June 2012 we visited all labelled Consequences of geitonogamy on rates of germination and plants biweekly. On each visit we counted all open flowers filled seeds and recorded each flower as hermaphrodite or male-sterile In September 2010 we performed an experiment to test the depending on the presence of aborted stamens. Four to consequences of geitonogamy on the rate of seed germination 5 weeks after a plant started flowering we began to collect, and the relationship between germination and proportion of on each visit, up to 20 calyces containing mature seeds in filled seeds. We used five 2-year-old potted clones obtained each monitored plant. This period was considered to be the from cuttings of each of three plants (R-10, R-13 and R-15) minimum time required for seed maturation according to from the population of Porta-Coeli (Table S1). All receptive previous field observations. Flower counts and calyx collec- flowers in each plant were hand-pollinated with pollen from tion continued for as long as the flowering and fruiting sea- one of two sources: pollen from the same plant (geitonoga- son continued in each population. Because we visited the mous self-pollination) or a mixture of pollen from flowers populations biweekly, it was possible that plants we recorded from the two other plants (cross-pollination). Hand-pollina- as flowering for the first or last time in a visit really started tion was applied to the flowers for 6 weeks in order to obtain or finished flowering sometime within the prior 15 days or enough seeds. Plants were kept in a greenhouse inaccessible to following to the visit. To prevent this from causing underes- potential pollinators and herbivores and were homogeneously timation of the number of days flowering, we added to all and regularly watered during the experiment. plants 7 days before and after the first and last flowering The obtained seeds per individual and pollination treatment date. We did not systematically census pollinators in this were germinated in plastic Petri dishes over moist filter paper in study, but we observed that Apis mellifera L. was the most a growth chamber (12 h light at 20 °C/12 h darkness at 10 °C). common and abundant pollinator in all populations. The Petri dishes contained 7–25 seeds and were checked for ger- From these data, for each individual plant and breeding sea- mination every 2 days for 28 days. After this time, seeds that son we then calculated the number of flowering days, the total failed to germinate were dissected under a microscope. Empty number of flowers produced (i.e. the sum of all flowers seeds, seeds without an embryo and seeds with a dried embryo counted), the maximum number of flowers open in a day (i.e. or cotyledons were considered as aborted, whereas filled seeds the maximum number counted in any census day), the female- with healthy embryos were considered viable. We did not study ness ratio (i.e. the ratio of male-sterile to total number of flow- the effects of the treatments on seed set as they have been docu- ers produced) and the degree of flowering synchrony with the mented previously (see Introduction). other monitored individuals in the population, using the Aug- spurger’s Synchrony rate index (Augspurger 1983). All these variables were selected because of their potential effect on the Influence of flowering traits and population characteristics on rate of geitonogamy of the plants. seed germination and allogamy of seedlings The collected calyces from each plant and date were taken to During the breeding seasons of 2010–2011 and 2011–2012 we the laboratory where the seeds were separated and stored in monitored several flowering traits, together with seed germina- paper bags in darkness at constant room temperature until the tion and allogamy rate of the seedlings obtained from these seed germination trials. All the seeds of each individual seeds, in 150 individuals in six wild populations of R. officinalis throughout the season were combined for germination experi- in eastern Spain (Table S1). In order to maximise differences in ments. In total we collected 11,576 seeds in the 2010–2011 environmental variables that potentially influence flowering season and 19,171 seeds in the 2011–2012 season. For the patterns, populations were located from near sea level to inland 2010–2011 season, this varied from 730 seeds in Ll ıria to 4263 areas at the highest altitudinal limit of the species (Table S1). in Porta-Coeli, and, in the 2011–2012 season, from 1100 seeds Populations were at least 8 km apart from each other and as far in Pedralba to 6638 in Porta-Coeli. For seeds collected along as 75 km. All populations had the same range of hillslope angle the 2010–2011 season, germination was carried out in plastic (25–15°) and aspect (southeast to southwest), and grew on cal- Petri dishes as in the pollination experiment. The method was careous soils under Mediterranean climate. The sampled popu- changed the following year because seedlings obtained in Petri lations have experienced the same type of land use during the dishes were often too small to successfully extract and amplify last centuries, including extensive grazing, charcoal and honey DNA. Therefore, germination for 2011–2012 seeds was carried production and hunting. The last forest fires occurred in 1980 out in cultivation trays with a mixture of Perlite, coconut fibre in Porta-Coeli and in 1988 in Pedralba, whereas there were no and peat in a greenhouse (9.5–25 °C; data from temperature recorded fires for the remaining localities at least in the sensors inside the greenhouse) until seedlings were of sufficient 40 years prior to our observations. In March 2010 we assessed size for DNA extraction.
Plant Biology 20 (2018) 627–635 © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands 629 Inbreeding in Rosmarinus officinalis Garcia-Fayos, Castellanos & Segarra-Moragues
Outcrossing rates of seedlings produced by each plant and density we did not include population because all individuals season were calculated from microsatellite analysis. We com- in a population had shared values. For those models where pared the genotypes at six polymorphic microsatellite loci population was not included we fitted linear models with the between mother plants and their corresponding offspring. A lm command (R package stats, version 3.3.2). Plant size, seedling was considered as generated by outcrossing when at number of flowers and maximum number of open flowers least one of the microsatellite alleles was not shared between were log-transformed before analyses. the seedling and its mother plant. In the case of a complete Prior to the analyses, we tested the variables included in the match between them we considered autogamy was possible. fixed term of the models and their interaction for multi-colli- We then obtained a minimum allogamy rate for each mother nearity using the vif command (car package, version 2.1–4in plant and breeding season as the proportion of allogamous R) and dropped those terms with variance inflation values of seedlings produced by the plant in relation to all seedlings pro- 2.5 or higher. The final model with the random and fixed terms duced in the germination trials. In total, we genotyped 229 was analysed with the glmmPQL command with binomial dis- seedlings in the 2010–2011 season and 2071 seedlings in the tribution of errors and logit link. 2011–2012 season. To estimate the contribution of the fixed and random terms in the mixed models to the total R2 value we used the r.squar- edGLMM command (MuMIn package, version 1.15.6) that Statistical analysis returned values of the marginal R2 (variance explained by the Logistic generalised linear mixed models (GLMM) via PQL fixed factor) and conditional R2 (variance explained by both were fitted to data of the hand-pollination experiment with the fixed and random factors; Nakagawa & Schielzeth 2013). glmmPQL command (MASS package version 7.3–45) in R (R Graphical model validation was used in all analyses to check Core Team 2017) with the binomial family distribution and for homoscedasticity and normality of residuals (Zuur et al. cloglog link because of the high proportion of zeros (Zuur et al. 2009). 2009). The seeds per plant that were filled and germinated rela- tive to the collected seeds and also the seeds that germinated relative to the seeds that were filled were used as dependent RESULTS variables. Pollination treatment was used as fixed factor and Consequences of geitonogamy on rates of germination and plant individual as random factor. filled seeds We applied GLMM to the field data to assess the influence of the flowering variables on the proportion of seeds that Seeds from the cross-pollination treatment were filled and ger- germinated and the rate of allogamy of the seedlings per minated with a higher probability than seeds obtained from plant. Plant identity was nested within population and used geitonogamy (Table 1). The model for the rate of seed germi- as random term in the models because of the clustering nation accounted for 57% of the variance (conditional R2) and structure of the design (Zuur et al. 2009). The interactions an important part of this variance was explained by the fixed between breeding season and all flowering characteristics term (marginal R2 = 41%); plant identity playing a minor role. were included in the fixed terms in order to test for the The model showed that seeds from cross-pollination had influence of the flowering variables for each season. We did 1.99 Æ 0.23 times higher probability of germinating than seeds not include altitude as a fixed factor because it was used in from geitonogamy (t = 8.739, P < 0.001). Concerning the filled the experimental design to increase the variation in flowering seeds, most of the variance was explained by the treatment traits of the plants. Therefore, including altitude as a variable (marginal R2 = 38% and conditional R2 = 47%), indicating that in the fixed term of the model would have a twofold influ- plant identity had a little effect too. The probability of seeds ence because it would affect both the response variable and being filled was 1.72 Æ 0.20 times higher for cross-pollination variables in the fixed term. To avoid this, prior to building than for geitonogamy (t = 8.542, P < 0.001). When we analysed the fixed term, we checked for the effect of altitude on flow- the rate of seed germination relative to the number of seeds ering variables using linear mixed models with the lme com- that were filled, the model accounted for 13% of the deviance, mand (nlme package, version 3.1–128 implemented in R) but now pollination treatment and plant identity contributed including altitude and season as fixed factors and population in a similar amount to the variation in seed germination (mar- as random factor. In the models for plant size and plant ginal R2 = 7%), indicating that seeds from cross-pollination density we did not include season since these variables were had 0.619 Æ 0.25 times higher probability of germinating than measured once during the two seasons, and in the model for seeds from geitonogamy (t = 2.438, P = 0.016).
Table 1. Number of seeds obtained from each plant in the hand-pollination experiment and number (and average Æ SE) of filled seeds relative to all seeds or that germinated relative to all seed. In italics, germinated seeds relative to filled seeds. Values of germination for plant R-15 were not tested because of the low number of filled seeds.
geitonogamous-pollination cross-pollination plant seeds filled germinated seeds filled germinated
R-10 27 10 (0.37 Æ 0.09) 3 (0.11 Æ 0.06) (0.30 Æ 0.14) 82 49 (0.60 Æ 0.05) 42 (0.51 Æ 0.05) (0.86 Æ 0.05) R-13 212 32 (0.15 Æ 0.02) 28 (0.13 Æ 0.02) (0.87 Æ 0.06) 62 45 (0.72 Æ 0.05) 42 (0.68 Æ 0.06) (0.93 Æ 0.04) R-15 21 1 (0.05 Æ 0.04) 1 (0.05 Æ 0.04) 29 9 (0.31 Æ 0.09) 5 (0.17 Æ 0.07)
630 Plant Biology 20 (2018) 627–635 © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands Garcia-Fayos, Castellanos & Segarra-Moragues Inbreeding in Rosmarinus officinalis
Influence of flowering traits and population characteristics on seed germination and allogamy of seedlings 0.10 (2) 0.12 (11) 0.06 (4) 0.01 (9) 0.19 (5) 0.10 (6) 0.20 (21) 0.03 (23) 0.02 (15) Æ Æ Æ Æ Æ Æ Æ Æ Tables S1 and 2 show the values of the studied variables for Æ each population and season of study. We found that altitude negatively influenced plant density and that plant size was affected by population but not by altitude, suggesting that soil conditions in populations and time from the last perturbation were more important than altitude in determining plant size 0.02 (18) 0.80 0.00 (24) 0.67 0.02 (16) 0.94 0.02 (12) n.a. (0) 0.02 (12) 0.98 0.01 (17) 0.76 0.02 (9) n.a. (0) 0.05 (7) 0.80 0.02 (23) 0.93 0.03 (23) 0.87 0.02 (17) 0.90 (Table S2). Analyses of flowering variables showed that the 0.03 (5) n.a. (0) Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ range of altitudes in our design had a wide influence, singly or Æ in interaction with season, but also that population still explained part of the variation in all the variables (Table S2). The number of days that plants bloomed in the season decreased with altitude and was shorter in the second season of study, the drier (Figures S1 and S2, Tables 2 and S2). Flowering 0.01 (25) 0.22 0.02 (25) 0.01 0.04 (25) 0.34 0.03 (25) 0.09 0.06 (24) 0.07 0.01 (25) 0.02 0.01 (25) 0.14 0.05 (25) 0.17 0.04 (25) 0.12 0.04 (25) 0.35 0.04 (25) 0.15 synchrony within populations was higher in the second than 0.05 (25) 0.20 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ the first season, but altitude had no effect on it (Tables 2 and Æ S2). The total number of flowers that plants produced in a sea- son and the number of flowers open on a census day increased with altitude but only in the first season of the study. In the second season, the large reduction in the number of flowers on plants of the highest populations vanished this effect (Tables 2 and S2). The proportion of male-sterile flowers that a plant 0.02 (25) 0.07 0.00 (25) 0.05 0.02 (25) 0.21 0.02 (25) 0.06 0.03 (24) 0.63 0.02 (25) 0.02 0.02 (25) 0.08 0.02 (25) 0.35 0.01 (25) 0.19 0.01 (25) 0.29 0.02 (25) 0.29 produced in a season varied between seasons and was nega- 0.02 (25) 0.44 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ tively affected by altitude (Tables 2 and S2). During the first Æ study season all plants in all populations had some male-sterile flowers (Tables 2 and S4), but in the second season 33% and 66% of individuals in Remedio and Manzaneruela, respectively, the two populations at highest altitudes, had only hermaphro- dite flowers. Seed germination rates were very low in all studied popula- tions (Table 2), reaching the maximum in the Porta-Coeli pop- ulation, and the minimum in Remedio, the highest altitude 194.5 (25) 0.88 121.9 (25) 0.97 84.5 (25) 0.81 285.7 (25) 0.84 18.7 (24) 0.67 37.1 (25) 0.85 144.1 (25) 0.88 29.9 (25) 0.69 38.3 (25) 0.86 56.1 (25) 0.91 35.9 (25) 0.84 15.3 (25) 0.65 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ and less dense population. Æ For the initial model of seed germination, variance inflation analyses indicated that the number of flowers that plants pro- duced in a season and the maximum number of flowers open on a census day were highly correlated, so we decided to retain during the two study seasons. Numbers in brackets indicate sample sizes. in the model the latter variable because of its higher potential relevance for geitonogamy. Variance inflation analyses also indicated that all the interactions among the variables were highly collinear and therefore were also removed from the R. officinalis 371.3 (25) 1624.9 236.4 (25) 573.1 183.6 (25) 488.1 693.2 (25) 1976.1 51.1 (24) 93.4 72.5 (25) 121.3 236.5 (25) 820.6 99.1 (25) 136.2 113.9 (25) 205.3 201.8 (25) 318.4 138.6 (25) 179.5 45.4 (25) 109.6 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ model. The resulting GLMM for seed germination explained Æ 23% of the variation (Table S3), mostly accounted for the fixed factor, suggesting that plant identity and population make a low contribution to the variation in seed germination. All the variables in the fixed factor, except the maximum number of flowers open on a census day, influenced the germination rate of plants in one or both seasons (Table S3). The number of 8.1 (25) 4050.2 4.6 (25) 1227.9 1.4 (25) 1152.2 9.4 (25) 1205.2 3.3 (25) 3722.1 7.5 (24) 219.0 3.0 (25) 215.9 2.4 (25) 1406.5 10.2 (25) 406.2 3.3 (25) 595.2 4.7 (25) 649.8 days that plants flowered and flowering synchrony positively 11.8 (25) 283.4 SE of reproductive variables of Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ affected seed germination in both seasons. Plant density and Æ the proportion of male-sterile flowers also positively affected Æ seed germination but did so only in the second season. Finally, plant size had a negative influence on the germination rate in the first season (Table S3). Because the data available for modelling the seedling allo-
gamy rate was very limited for the first season, we used only Population average 2012 78.8 2011 129.9 2012 158.1 2012 59.6 2011 152.6 2011 64.2 2012 71.3 2012 50.9 2011 58.6 2011 124.1 2012 113.0 2011 128.2 – – – – – – – – – – – the data for the second season (Table 2). We found that in this – ~ breeding season, the allogamy rate of seedlings of each plant net ıria 2011 2010 2011 2011 2010 2010 2011 2011 2010 2010 2011 2010 Saca Porta-Coeli Remedio Manzaneruela Pedralba Table 2. was very high in almost all populations, with most values population/seasonLl days flowering total number of flowers maximum number of flowers flowering synchrony femaleness germination allogamy
Plant Biology 20 (2018) 627–635 © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands 631 Inbreeding in Rosmarinus officinalis Garcia-Fayos, Castellanos & Segarra-Moragues
Despite the severe reduction in seed germination, plants pro- duce many seeds after geitonogamous self-pollination and a few of these germinated and developed into seedlings, support- ing a mixed mating system in R. officinalis (Goodwillie et al. 2005). In addition to geitonogamy, another source of the reduction in seed germination of seeds collected in the field could be empty seeds resulting from biparental inbreeding or mating between relatives. Neighbouring rosemary shrubs are likely close relatives, because this species lacks mechanisms for long-distance seed dispersal and the single-seeded dry indehis- cent fruits (schizocarps) or entire calyces containing fruits are gravity-dispersed, below or close to maternal plants (Bouman
Probability of allogamous seedling & Meeuse 1992). Furthermore, when moistened or wetted, the pericarp mucilage hydrates, glueing the fruits to the soil surface and preventing or reducing both secondary dispersal and pre- dation by ants (Engelbrecht & Garc ıa-Fayos 2012). Accord- 0.0 0.2 0.4 0.6 0.8 1.0 ingly, high inbreeding coefficients have been found in these 0.0 0.2 0.4 0.6 0.8 populations (Segarra-Moragues et al. 2016). The field observa- Femaleness ratio of plants tions strongly suggest that the conspicuous floral display of rosemary plants within populations successfully attracts polli- Fig. 1. Influence of femaleness ratio (proportion of female flowers in rela- nators but also causes high rates of geitonogamy, as predicted tion to total number of flowers) of R. officinalis on the probability of plants producing allogamous seedlings. (Charlesworth & Charlesworth 1987). However, experimental and observational results also indicate that plants purge most of the inbreeding caused by selfing by aborting seeds or reduc- >80% (Table 2). Variance inflation analyses for the allogamy ing their germination probability, as found in other species model indicated a high correlation between the number of (Wiens et al. 1987; Husband & Schemske 1996). Although this flowers that plants produce in a season and the maximum can explain the reported high proportions of empty seeds in number of flowers open on a census day and thus, we retained this species (Clemente et al. 2007; Madeiras et al. 2009), we the latter variable. The interactions among all the variables cannot discard the influence of other potential factors inducing were also removed from the fixed term because of their high seed abortion, e.g. environmental stress (Stephenson 1981). variance inflation values. The final model for the allogamy rate Previous experiments with rosemary clones showed a decrease of seedlings explained 68% of the variance and both, plant in the rate of filled seeds from 68% to 49% after reducing water identity and population, accounted for a substantial propor- availability by one third, independently of other controlled fac- tion of the variance (13%; Table S4). Femaleness ratio was the tors (pollen amount and source, plant size, etc.; Garcer a& only significant variable included in the fixed factor and indi- Garc ıa-Fayos unpublished). cates that plants with >50% male-sterile flowers during the sea- In addition to the self-pollination consequences of geitono- son guarantee that almost none of the seedlings produced is a gamy because of the highly attractive floral display of R. offici- consequence of self-pollination (Figure 1). nalis to pollinators, our study also confirms that several flowering characteristics are correlated with cross-pollination (Klinkhamer & de Jong 1990; Ison et al. 2014). Plant density DISCUSSION and degree of flowering synchrony both had a positive influ- A reduction in germination rate as a consequence of geitono- ence on the proportion of seeds per plant that actually germi- gamy seems to be the rule in the mass flowering shrub R. offici- nated. The magnitude of the floral display, represented by nalis. This study shows that the impact of inbreeding plant size and the number of flowers exposed simultaneously depression, measured here as the rates of seed germination and to pollinators, both of which could increase geitonogamy and allogamy of the seedlings, is a function of a complex combina- attractiveness to pollinators (de Jong et al. 1993; Albert et al. tion of factors that vary in space and time. 2008), had no effect on the rates of germination and allogamy We confirmed experimentally that geitonogamy strongly in plants, except for plant size, which negatively influenced reduced the probability of seed germination in R. officinalis, seed germination in the first season (Table 2). This suggests and that this reduction was mainly the consequence of the sim- that the potential increase in geitonogamy in plants with large ilarly high rates of empty seeds caused by geitonogamy. Our floral display is partially compensated by the simultaneous experimental results are based on a few individuals of a single influence of other flowering characteristics of this display. This population and could have been affected by a particular confirms that size and other characteristics of the floral display inbreeding level of that population; however, analyses based on cannot be fully understood by examining them in isolation 11 microsatellite loci showed that the source population did (Harder et al. 2004). not differ in the inbreeding coefficient from 18 other popula- Other flowering characteristics, such as the length of the tions of rosemary in eastern Spain (Segarra-Moragues et al. flowering period and the proportion of male-sterile flowers, 2016). Furthermore, previous pollination experiments with played a role in reducing the rate of geitonogamous pollina- individuals of a population not included in this study, similarly tion (Table S4). The length of the flowering period co-acted yielded a strong reduction in the proportion of filled seeds after with other flowering characteristics to reduce geitonogamy. geitonogamy (Garcer a & Garc ıa-Fayos, unpublished). Several studies have shown that extending the flowering
632 Plant Biology 20 (2018) 627–635 © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands Garcia-Fayos, Castellanos & Segarra-Moragues Inbreeding in Rosmarinus officinalis period is a good strategy to reduce geitonogamy only if each Damm 1996), and suggest abnormal development of flowers individual open a few flowers per day and has a high flower- (Richards 1997, pp. 320) rather than genetically controlled ing synchrony with other individuals in the neighbourhood variation. (de Jong et al. 1993; Kunin 1993; Brys et al. 2008), but this Our study provides a case on how several factors interact was not observed in our study. We found a negative rela- to prevent or purge the consequences of geitonogamy. Seed- tionship between the number of days that rosemary plants lings produced by R. officinalis plants in natural conditions flowered and flowering synchrony in both reproductive sea- were mostly allogamous, and it was strongly and positively sons (rp = À0.540, P < 0.001 and rp = À0.201, P = 0.030). influenced by the proportion of male-sterile flowers within However, the length of the flowering period is very plastic individuals. This high allogamy rate is, at least partially, the and influenced by environment, which can also influence consequence of plants aborting most of the inbred seeds or other flowering traits. So altitude strongly reduced the num- reducing their germination. Among the flowering traits pro- ber of days that plants flowered in their respective popula- moting cross-pollination in rosemary, long flowering spans tions in both seasons (Tables 2 and S2), but at the same and high flowering synchrony among individuals in popula- time, the length of the flowering season of plants in the tions emerged as the most important factors at both highest and lowest altitude populations overlapped when altitudes. Plant density also played a role and apparently clones from these plants were grown in a common garden counteracted the potential negative effects of intense flower (Castellanos & Garc ıa-Fayos, unpublished). displays attracting pollinators. However, all these flowering The proportion of male-sterile flowers in R. officinalis characteristics are highly plastic and further experimental showed large temporal variation during the breeding season studies are needed to test their role as adaptations of rose- (see Figure S2), confirming the findings of Ubera-Jim enez mary to reduce selfing. Rosemary plants can grow and & Hidalgo-Fern andez (1992), but it was negatively affected reproduce in a variety of environmental conditions that, by altitude (Tables 2 and S2). We thus confirm the positive combined with high levels of gene flow (Segarra-Moragues influence of the femaleness ratio on seed germination rates. et al. 2016), can explain the high levels of genetic variation At the same time, the femaleness ratio was the only trait of populations in this species. influencing the outcrossing rate of seedlings. After discount- ing the effects of flowering characteristics and population ACKNOWLEDGEMENTS on seed germination rate, the ratio of male-sterile flowers still has a positive influence on the outcrossing rate. These We thank Santiago Donat who performed most of the flow- findings are coherent with the idea that the joint presence ering counts and collected most of the seeds, Elena Garcer a of male-sterile and hermaphrodite flowers within individu- who examined the effect of water shortage on the rate of als will reduce the chance of geitonogamous pollination seed abortion, and Maite Castellanos who cared for the compared to individuals producing only hermaphrodite common garden experiment. Without their collaboration flowers (Albert et al. 2008; Dufay & Baillard 2012) and this work would have been impossible. Santiago Donat, strongly suggests that femaleness plays a central role in Jordi Chofre, Yolanda Carrion, Mar ıa Jos e Molina, Juan mediating in the conflict between attracting pollinators and Garc ıa-Fayos, Miguel Angel Navarro, Lara Bab ı, Carlos reducing geitonogamy in R. officinalis. Olaya, Iv an P erez, Maite Castellanos, Paula Cass a, Eva We found that 50% of male-sterile flowers is enough to S anchez and Luc ıa Tortajada aided with selection and sam- guarantee that almost all the progeny of R. officinalis origi- pling of populations. Jordi Chofre and Meike Engelbrecht nate from cross-pollination (Fig. 1). This is coherent with helped with pollination experiments. Miguel Angel Navarro the suggestion that geitonogamy could be a driver towards and Santiago Donat (2010–2011), and Maite Castellanos gynodioecy. This is true for self-compatible plants incurring (2011–2012) helped with germination experiments. We are high flowering cost to attract pollinators and simultaneously also grateful to the anonymous reviewer whose comments suffering strong inbreeding depression because of self-polli- considerably improved a previous version of the manuscript. nation (de Jong & Geritz 2002; Sinclair et al. 2013). How- Financial support was provided by CGL2009-07262/BOS ever, femaleness in rosemary does not seem to be a long- project from the Spanish Ministry of Science and Innovation term stable strategy since no wild plants of this species have (MICINN) and PROMETEO/2016/021 project from the been described as female. In our study we only found one Valencia Autonomous Government. J.G.S.-M. was supported plant (out of 150 individuals) that behaved exclusively as a by a ‘Ramon y Cajal’ post-doctoral contract from MICINN. female; however, only in one season. Within individual M.C.C. was supported by a JAE-Doc CSIC post-doctoral instability in male-sterility may be the result of genetically scholarship. controlled gender variation interacting with the environment (Wolfe & Shmida 1997; McCauley & Bailey 2009; Harkess & SUPPORTING INFORMATION Leebens-Mack 2017). However, Hidalgo-Fern andez et al. (1999) found that rosemary with some male-sterile flowers Additional Supporting Information may be found online in the had higher mitochondrial genome variability than plants supporting information tab for this article: with all hermaphrodite flowers, suggesting some kind of Figure S1. Temporal flowering pattern of R. officinalis dur- genetic control as in other Lamiaceae (Belhassen et al. ing 2 years of study in a low (Lliria) and a high (Manzaneruela) 1991). But the strong and persistent negative effect of alti- altitude population. Each line represents an individual plant; tude on the proportion of male-sterile flowers in our study lines for two individuals in each population were drawn differ- is coherent with the finding that warmer temperatures ently to highlight within and between season variability in favour expression of female flowers (Koelewijn & Van flowering pattern.
Plant Biology 20 (2018) 627–635 © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands 633 Inbreeding in Rosmarinus officinalis Garcia-Fayos, Castellanos & Segarra-Moragues
Figure S2. Temporal patterns in femaleness ratio (pro- cients). Plant size, number of flowers and maximum portion of male-sterile flowers) of R. officinalis during number of flowers open were log-transformed (log10). 2 years of study in a low (Lliria) and a high (Manzaneru- The symbol # denotes terms that were not included in ela) altitude population. Each line style represents a dif- the model. ferent plant. Table S3. Synthesis of GLMM for the effect of plant Table S1. Name, location and main characteristics of size, plant density and flowering characteristics (fixed the studied R. officinalis populations. Climate data are term) on the germination rate of seeds of R. officinalis. year averages from the nearest meteorological stations for Individual identities were included as a random term the 1961–1990 period (taken from P erez 1994). nested to populations. Significant partial coefficients are Table S2. Synthesis of GLMM for the effect of popula- in bold. tion (random term), altitude, season of study and their Table S4. Synthesis of GLMM for the effect of plant interaction (fixed terms) on flowering variables of R. offi- size, plant density and flowering characteristics (fixed term) cinalis. The first two columns indicate the proportion of on the allogamy rate of seedlings of R. officinalis. The model variance explained by random and fixed terms. The col- was fitted with data for the breeding season 2011–2012 only umns for altitude, season and their interaction indicate (see text for details). Individual identities were included as a their respective coefficients (ÆSE) in the model and random term nested to populations. Significant partial coeffi- significance (bold numbers indicate significant coeffi- cients are in bold.
REFERENCES monspeliensis and Rosmarinus officinalis, in relation Hidalgo-Fernandez P.J., Ubera J.L. (2001) Inbreeding to time since fire. Plant Ecology, 190, 175–188. depression in Rosmarinus officinalis L. International Alarcon J.J., Morales M.A., Fern andez M.T., S anchez- Dufay M., Baillard E. (2012) How much better are Journal of Developmental Biology, 45, S43–S44. Blanco M.J. (2006) Effect of water and salt stresses females? The occurrence of female advantage, its Hidalgo-Fern andez P.J., P erez-Vicente R., Maldonado on growth, water relations and gas exchange in Ros- proximal causes and its variation within and among J.M., Ubera-Jim enez J.L. (1999) Mitochondrial DNA marinus officinalis. Journal of Horticultural Science & gynodioecious species. Annals of Botany, 109, 505– polymorphism and gynodioecy in a natural popula- Biotechnology, 81, 845–853. 519. tion of Rosmarinus officinalis. Israel Journal of Plant Albert M.J., Iriondo J.M., Escudero A., Torres E. Eckert C.G., Samis K.E., Dart S. (2006) Reproductive Sciences, 47,77–83. (2008) Dissecting components of flowering pattern: assurance and the evolution of uniparental repro- Husband B.C., Schemske D.W. (1996) Evolution of the size effects on female fitness. Botanical Journal of the duction in flowering plants. In: Harder L. D., Bar- magnitude and timing of inbreeding depression in Linnean Society, 156, 227–236. rett S. C. H. (Eds), Ecology and evolution of plants. Evolution, 50,54–70. Arroyo J. (1990) Ritmos clim aticos y de floracion en flowers. Oxford University Press, Oxford, UK, pp Ison J.L., Wagenius S., Reitz D., Ashley M.V. (2014) matorrales del SW de Espana.~ Lagascalia, 16,25–50. 183–203. Mating between Echinacea angustifolia (Asteraceae) Augspurger C.K. (1983) Phenology, flowering syn- Elzinga J.A., Atlan A., Biere A., Gigord L., Weis A.E., individuals increases with their flowering synchrony chrony, and fruit-set of 6 neotropical shrubs. Bernasconi G. (2007) Time after time: flowering and spatial proximity. American Journal of Botany, Biotropica, 15, 257–267. phenology and biotic interactions. Trends in Ecology 101, 180–189. Barrett S.C. (2002) The evolution of plant sexual diver- & Evolution, 22, 432–439. de Jong T.J., Geritz S.A.H. (2002) The role of geitono- sity. Nature Reviews Genetics, 3, 274–284. Engelbrecht M., Garc ıa-Fayos P. (2012) Mucilage gamy in the gradual evolution towards dioecy in Barrett S.C. (2003) Mating strategies in flowering secretion by seeds doubles the chance to escape ant cosexual plants. Selection, 2, 133–146. plants: the outcrossing–selfing paradigm and removal. Plant Ecology, 213,1167–1175. de Jong T.J., Waser N.M., Klinkhamer P.G. (1993) Gei- beyond. Philosophical Transactions of the Royal Soci- Goodwillie C., Kalisz S., Eckert C.G. (2005) The evolu- tonogamy: the neglected side of selfing. Trends in ety B: Biological Sciences, 358, 991–1004. tionary enigma of mixed mating systems in plants: Ecology & Evolution, 8, 321–325. Belhassen E., Dommee B., Atlan A., Gouyon P.H., occurrence, theoretical explanations, and empirical Jordano P. (1993) Pollination biology of Prunus maha- Pomente D., Assouad M.W., Couvet D. (1991) evidence. Annual Review of Ecology, Evolution, and leb L.: deferred consequences of gender variation for Complex determination of male sterility in Thymus Systematics, 36,47–79. fecundity and seed size. Biological Journal of the Lin- vulgaris L.: genetic and molecular analysis. Theoreti- Harder L.D., Barrett S.C. (1995) Mating cost of large nean Society, 50,65–84. cal and Applied Genetics, 82, 137–143. floral displays in hermaphrodite plants. Nature, 373, Kameyama Y., Kudo G. (2009) Flowering phenology Bouman F., Meeuse A.D.J. (1992) Dispersal in Labia- 512–515. influences seed production and outcrossing rate in tae. In: Harley R. M., Reynolds T. (Eds), Advances in Harder L.D., Jordan C.Y., Gross W., Routley M.B. populations of an alpine snowbed shrub, Phyllodoce labiatae science. Royal Botanical Gardens, Kew, Lon- (2004) Beyond floricentrism: the pollination func- aleutica: effects of pollinators and self-incompatibil- don, UK, pp 193–202. tion of inflorescences. Plant Species Biology, 19, 137– ity. Annals of Botany, 103, 1385–1394. Brys R., Jacquemyn H., Hermy M. (2008) Pollination 148. Klinkhamer P.G., de Jong T.J. (1990) Effects of plant efficiency and reproductive patterns in relation to Harkess A., Leebens-Mack J. (2017) A century of sex size, plant density and sex differential nectar reward local plant density, population size, and floral dis- determination in flowering plants. Journal of Hered- on pollinator visitation in the protandrous Echium play in the rewarding Listera ovata (Orchidaceae). ity, 108,69–77. vulgare (Boraginaceae). Oikos, 57, 399–405. Botanical Journal of the Linnean Society, 157, 713– Herrera J. (1986) Flowering and fruiting phenology in Koelewijn H.P., Van Damm J.M.M. (1996) Gender 721. the coastal shrublands of Donana,~ south Spain. variation, partial male sterility and labile sex expres- Byers D.L., Waller D.M. (1999) Do plant populations Vegetatio, 68,91–98. sion in gynodioecious Plantago coronopus. New Phy- purge their genetic load? Effects of population size Herrera J. (1987a) Flower and fruit biology in southern tologist, 132,67–76. and mating history on inbreeding depression. Annual Spanish Mediterranean shrublands. Annals of the Kudo G. (2006) Flowering phenologies of animal-polli- Review of Ecology and Systematics, 30, 479–513. Missouri Botanical Garden, 74,69–78. nated plants: reproductive strategies and agents of Charlesworth D., Charlesworth B. (1987) The effect of Herrera J. (1987b) Biolog ıa reproductiva de algunas selection. In: Harder L. D., Barrett S. C. H. (Eds), investment in attractive structures on allocation to especies del matorral de Donana.~ Anales del Jardın Ecology and evolution of flowers. Oxford University male and female functions in plants. Evolution, 41, Botanico de Madrid, 44,483–497. Press, Oxford, UK, pp 61–80. 948–968. Herrera C.M. (2004) Distribution ecology of pollen Kunin W.E. (1993) Sex and the single mustard: popu- Clemente A.S., Rego F.C., Correia O.A. (2007) Seed tubes: fine-grained, labile spatial mosaics in southern lation density and pollinator behavior effects on bank dynamics of two obligate seeders, Cistus Spanish Lamiaceae. New Phytologist, 161, 473–484. seed-set. Ecology, 74, 2145–2160.
634 Plant Biology 20 (2018) 627–635 © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands Garcia-Fayos, Castellanos & Segarra-Moragues Inbreeding in Rosmarinus officinalis
Madeiras A.M., Boyle T.H., Autio W.R. (2009) Stratifi- Petadinou T., Ellis W.N., Margaris N.S., Vokou D. Snow A.A., Spira T.P., Simpson R., Klips R.A. (1996) cation, gibberellic acid, scarification, and seed lot (1995) Constraints on flowering phenology in a The ecology of geitonogamous pollination. In: Lloyd influence on rosemary seed germination. Seed Tech- phryganic community. American Journal of Botany, D. G., Barret S. C. H. (Eds), Floral biology. Studies on nology, 31,55–65. 82, 607–620. floral evolution in animal-pollinated plants. Springer, Maffei M., Mucciarelli M., Scannerini S. (1993) Envi- R Core Team (2017) R: a language and environment New York, USA, pp 191–216. ronmental factors affecting the lipid metabolism in for statistical computing. (v. 3.3.2) R Foundation for Spigler R.B., Ashman T.L. (2012) Gynodioecy to Rosmarinus officinalis L. Biochemical Systematics and Statistical Computing, Vienna, Austria. Available dioecy: are we there yet? Annals of Botany, 109, 531– Ecology, 21, 765–784. from http://www.R-project.org/. 543. Mateu-Andr es I., Aguilella A., Boisset F., Curr as R., Richards A.J. (1997) Plant Breeding Systems. Chapman Stephenson A.G. (1981) Flower and fruit abortion: Guara M., Laguna E., Marzo A., Puche M.F., Pedrola & Hall, London, UK. proximate causes and ultimate functions. Annual J. (2013) Geographical patterns of genetic variation Rodr ıguez-Riano~ T., Dafni A. (2007) Pollen–stigma Review of Ecology and Systematics, 12, 253–279. in rosemary (Rosmarinus officinalis) in the Mediter- interference in two gynodioecious species of Lami- Thomson J.D., Brunet J. (1990) Hypotheses for the ranean basin. Botanical Journal of the Linnean Soci- aceae with intermediate individuals. Annals of Bot- evolution of dioecy in seed plants. Trends in Ecology ety, 171, 700–712. any, 100, 423–431. & Evolution, 5,11–16. McCauley D.E., Bailey M.F. (2009) Recent advances in Roiz L., Dulberger L. (1988) Male sterility in flowers of Ubera-Jim enez J.L., Hidalgo-Fern andez P.J. (1992) the study of gynodioecy: the interface of theory and Rosmarinus officinalis. In: Cresti M., Gori P., Pacini Temporal gynodioecy in Rosmarinus officinalis. In: empiricism. Annals of Botany, 104, 611–620. E. (Eds), Sexual reproduction in higher plants. Harley R. M., Reynolds T. (Eds), Advances in labi- Mitchell R.J., Karron J.D., Holmquist K.G., Bell J.M. Springer, Berlin, Germany, pp 481. atae science. Royal Botanical Gardens Kew, London, (2004) The influence of Mimulus ringens floral dis- Rossello J.A., Cos ın R., Boscaiu M., Vicente O., UK, pp 281–289. play size on pollinator visitation patterns. Functional Mart ınez I., Soriano P. (2006) Intragenomic diver- Vrieling K., Saumitou-Laprade P., Cuguen J., Van Dijk Ecology, 18, 116–124. sity and phylogenetic systematics of wild rosemary H., de Jong T.J., Klinkhamer P.G.L. (1999) Direct Morales R. (2010) Rosmarinus. In: Morales R., Quintanar (Rosmarinus officinalis L. s.l., Lamiaceae) assessed by and indirect estimates of the selfing rate in small and A., Cabezas F., Pujadas A.J., Cirujano S. (Eds), Verbe- nuclear ribosomal DNA sequences (ITS). Plant Sys- large individuals of the bumblebee pollinated naceae, labiatae, callitrichaceae. Flora Iberica, vol. 12. tematics and Evolution, 262,1–12. Cynoglossum officinale L(Boraginaceae). Ecology Let- Real Jard ın Bot anico CSIC, Madrid, Spain, pp 25–445. Segarra-Moragues J.G., Gleiser G. (2009) Isolation and ters, 2, 331–337. Munn e-Bosch S., Alegre L. (2000) Changes in carote- characterisation of di- and tri-nucleotide microsatel- Wiens D., Calvin C.L., Wilson C.A., Davern C.I., Frank noids, tocopherols and diterpenes during drought lite loci in Rosmarinus officinalis (Lamiaceae), using D., Seavey S.R. (1987) Reproductive success, sponta- and recovery, and the biological significance of enriched genomic libraries. Conservation Genetics, neous embryo abortion and genetic load in flower- chlorophyll loss in Rosmarinus officinalis plants. 10, 571–575. ing plants. Oecologia, 71, 501–509. Planta, 210, 925–931. Segarra-Moragues J.G., Carrion Y., Castellanos Wolfe L.M., Shmida A. (1997) The ecology of sex Nakagawa S., Schielzeth H. (2013) A general and sim- M.C., Molina M.J., Garcia-Fayos P. (2016) His- expression in a gynodioecious Israeli desert shrub ple method for obtaining R2 from generalized linear torical and ecological determinants of population (Ochradenus baccatus). Ecology, 78, 101–110. mixed-effects models. Methods in Ecology and Evolu- genetic diversity and structure in a widespread Zunzunegui M., D ıaz-Barradas M.C., Ain-Lhout F., tion, 4, 133–142. Mediterranean plant Rosmarinus officinalis (Lami- Alvarez-Cansino L., Esquivias M.P., Garc ıa-Novo Owens S.J., Ubera J.L. (1992) Breeding systems in labi- aceae). Botanical Journal of the Linnean Society, F. (2011) Seasonal physiological plasticity and atae. In: Harley R. M., Reynolds T. (Eds), Advances 180,50–63. recovery capacity after summer stress in Mediter- in labiatae science. Royal Botanical Gardens Kew, Sinclair J.P., Maxwell G.D., Freeman D.C. (2013) Con- ranean scrub communities. Plant Ecology, 212, London, UK, pp 257–280. sanguineous mating, specialization, and the environ- 127–142. P erez A.J. (1994) Atlas climatic de la Comunitat Valen- ment: how multiple variable interactions affect the Zuur A., Ieno E.N., Walker N., Saveliev A.A., Smith ciana (1961–1990). Generalitat Valenciana, Valencia, evolution of dioecy. American Journal of Botany, G.M. (2009) Mixed effects models and extensions in Spain. 100, 1038–1049. ecology with R. Springer, New York, USA.
Plant Biology 20 (2018) 627–635 © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands 635 Table S1. Name, location and main characteristics of the studied R. officinalis populations. Climate data are year averages from the nearest meteorological stations for the 1961–1990 period (taken from Pérez 1994). Population Longitude Latitude Altitude Distance to the sea Temperature Precipitation Plant density Plant size (W) (N) (m a.s.l.) (km) (ºC) (mm) (ind.ha-1) (dm3) Llíria 39°38'18.99" 0°36'38.08" 200 28.3 16.5 410 9000 154.7 ± 32.5 Pedralba 39°35'05.27" 0°41'30.76" 210 33.4 16.5 434 11900 368.0 ± 64.7 Porta-Coeli 39°39'43.02" 0°29'35.48" 240 20.7 15.9 409 8933 456.0 ± 79.8 Manzaneruela 39°56'59.41" 1°18'49.57" 1050 66.8 11.1 495 9067 225.2 ± 21.7 Remedio 39°38'04.01" 1°08'08.17" 1240 71.1 11.9 564 1433 252.6 ± 51.2 Sacañet 39°51'27.14" 0°42'45.58" 995 47.2 11.6 485 9400 375.1 ± 48.5 Table S2. Synthesis of GLMM for the effect of population (random term), altitude, season of study and their interaction (fixed terms) on flowering variables of R. officinalis. The first two columns indicate the proportion of variance explained by random and fixed terms. The columns for altitude, season and their interaction indicate their respective coefficients (±SE) in the model and significance (bold numbers indicate significant coefficients). Plant size, number of flowers and maximum number of flowers open were log- transformed (log10). The symbol # denotes terms that were not included in the model.
Variables in the fixed term Random term Fixed term ALTITUDE SEASON ALTITUDE x SEASON Plant size 13% 0% -0.0002 ± 0.0001 # # Plant density # 26% -4.6370 ± 2.7940 # # Days flowering 20% 48% -0.0775 ± 0.0213 -30.4207 ± 8.3807 0.0089 ± 0.0098 Number of flowers 18% 34% 0.0006 ± 0.0003 0.0110 ± 0.1266 0.0008 ± 0.0002 Maximum number of flowers open 13% 42% 0.0008 ± 0.0002 -0.0968 ± 0.1208 -0.0008 ± 0.0001 Flowering synchrony 39% 25% 0.0002 ± 0.0001 0.0965 ± 0.0287 -0.0000 ± 0.0000 Femaleness 6% 28% -0.0003 ± 0.0001 0.0709 ± 0.0557 -0.0000 ± 0.0000
Table S3. Synthesis of GLMM for the effect of plant size, plant density and flowering characteristics (fixed term) on the germination rate of seeds of R. officinalis. Individual identities were included as a random term nested to populations. Significant partial coefficients are in bold.
Variance GERMINATION (%) Random factor 4.85 Fixed factor 17.77
Variables in the fixed factor season Coefficient d.f. t-value p-value Intercept -7.7198 ± 0.7693 122 10.0342 <0.0001 2010-2011 -0.6455 ± 0.2965 43 2.1767 0.0350 Plant size 2011-2012 0.1199 ± 0.2659 43 0.4510 0.6543 2010-2011 0.0000 ± 0.0000 43 0.1669 0.8682 Plant density 2011-2012 0.0002 ± 0.0000 43 4.7374 <0.0001 2010-2011 0.0114 ± 0.0024 43 4.7710 <0.0001 Days flowering 2011-2012 0.0182 ± 0.0020 43 9.3110 <0.0001 2010-2011 0.0000 ± 0.0000 43 0.3777 0.7075 Maximum number of flowers open 2011-2012 0.0002 ± 0.0003 43 0.9189 0.3633 2010-2011 5.5042 ± 0.8213 43 6.7015 <0.0001 Flowering synchrony 2011-2012 2.2227 ± 0.7026 43 3.1637 0.0029 2010-2011 0.5384 ± 0.5080 43 1.0598 0.2951 Femaleness 2011-2012 0.8885 ± 0.4008 43 2.2170 0.0320
Table S4. Synthesis of GLMM for the effect of plant size, plant density and flowering characteristics (fixed term) on the allogamy rate of seedlings of R. officinalis. The model was fitted with data for the breeding season 2011–2012 only (see text for details). Individual identities were included as a random term nested to populations. Significant partial coefficients are in bold.
SEEDLING ALLOGAMY Variance (%) Random factor 12.95 Fixed factor 55.04
Variables in the fixed factor Coefficient d.f. t-value p-value Intercept 2.2194 ± 1.8621 77 1.1919 0.2370 Plant size 0.3974 ± 0.4552 77 0.8729 0.3854 Plant density 0.0001 ± 0.0000 77 1.1205 0.2660 Days flowering -0.0036 ± 0.0035 77 1.0307 0.3059 Maximum number of flowers open -0.0005 ± 0.0004 77 1.1936 0.2363 Flowering synchrony -1.2879 ± 1.6638 77 0.7741 0.4413 Femaleness 4.7881 ± 1.0399 77 4.6043 <0.0001