Planta turística presente en la Hacienda "El Prado”

Summary

Less than only one quarter of the ovules of Echium vulgare in the field develop into viable

seeds, even in the absence of pollen limitation. The loss of ovules could enhance the fitness of the maternal parent, if the less fit embryos are selectively aborted. Two pollination experi- ments were performed to examine the selectivity of maternal parents on self-pollen and different cross-pollen sources.

Pollinated with one pollen genotype per flower, self-pollen was, on average, equally successful in siring seeds as cross-pollen. However, the relative success of self-pollen com- pared to outcross-pollen differed significantly among the maternal parents. These results sug- gest that under certain conditions, selfing can be more advantageous for the number of seeds produced than cross-pollinations. Pollen donors differed significantly in outcrossing success. The plants that were more successful in selfing were also more successful as pollen donor in outcross-pollinations. A significant interaction between maternal parent and paternal genotype was absent.

Pollinations with a pollen mixture produced selfed and outcrossed seeds in the same ratios as in the single-donor experiment. Overall, only slight differences were found between the single- and mixed-donor experiment.

Pollen tube growth does not show a significant correlation with the success of the pa- rental genotypes in the mixed-donor experiment, indicating that pollen tube growth is not the determining factor controlling the paternity of the seeds. These results are discussed with reference to possible mediating mechanisms.

Introduction

Many species produce far more ovules than seems necessary for the production of their seeds. In many cases this low seed to ovule ratio is not caused by a lack of compatible pollen (Willson & Burley 1983), although Burd (1994) has argued that effects of pollen limitation have been underestimated. For Echium vulgare, our model plant, the average seed to ovule

ratio is only 0.23, although seed set is not pollen limited in natural populations (Klinkhamer et al. 1994). These results imply that a large fraction of the fertilized ovules abort in an early

stage. This abortion could serve to increase the female fitness if the fittest offspring are selected to ripen to viable seeds. If this selection is under female control, the problem for the maternal parent is how to determine the offspring quality. Stearns (1992) suggested that em- bryos are in a competitive arena for resources. With such a selective mechanism it is con- ceivable that there is a positive correlation between embryo selection and potential offspring quality later in life. Differences in offspring quality may arise in populations with a relative high and variable inbreeding depression. In such populations, differences are most pronoun- ced among selfed progeny.

Emphasis in literature has been on different mechanisms to prevent self-pollination. Pre-pollination mechanisms include spatial or temporal separation of sexes. Post-pollination mechanisms to avoid selfing include self-incompatibility (de Nettancourt 1977). With these mechanisms, there is no possibility to discriminate among individuals of the selfed progeny. Selection among selfed embryos has the advantage that through offspring with a low genetic load, two copies of the genome are passed to the next generation while large investments of resources in offspring with high genetic load can be avoided. Selection against selfing may also lead to cryptic self-incompatibility (Bateman 1956). With cryptic self-incompatibility the self-pollen is able to sire seeds if no cross-pollen is available, but in the presence of cross- pollen, the self-pollen is consistently outperformed and the majority of the seeds will be sired by the cross-pollen. In this way some advantages of both self- and cross-pollination can be combined.

Selection of pollen sources will lead to a deviation in paternity percentages of the seeds from the paternity percentages of the pollen that is originally applied. Such deviations in seed numbers from different pollen sources have been established for a number of species

(Raphanus raphanistrum in Mazer et al. 1986, Ellstrand & Marshall 1986, Marshall &

Ellstrand 1988, Marshall 1991, Marshall & Folsom 1992, Campsis radicans in Bertin 1982,

1985 and 1988, Chamaecrista fasciculata in Fenster 1991). However, the ovules of these

species are not equally arranged. Discrimination among pollen sources could be influenced by position effects if pollen sources are not randomly distributed among ovule positions. For species with seeds which are not linearly arranged, the impact of the pollen source effect has not yet been estimated. Other studies (Stephenson & Winsor 1986, Casper 1984, 1988) report that selective seed abortion increases offspring quality, without relating this to paternal genotype.

Here we describe a study on the number and the weight of the seeds, selectively sired by different pollen sources of Echium vulgare (L), a monocarpic, self-compatible perennial of

calcareous grasslands and dune vegetations. Specifically, we investigated the impact of self- pollination and different cross-pollinations on seed numbers per flower and seed weight. The

experiments addressed the following questions: is there a difference in the number and the weight of the seeds sired by different pollen sources, (1) between self-pollinations and cross- pollinations or (2) among different cross-pollinations, and (3) is there a relation between the success of the self-pollinations and the success as a pollen donor in cross-pollinations. If there appears to be a difference in the number of seeds sired by different pollen sources, another question can be addressed: (4) Can these differences in number of seeds sired by different pollen donors be explained by differences in number of pollen tubes or early pollen tube growth or are they more likely to result from competition among embryos?

Material and methods

Plant species

Echium vulgare (L) is a rosette-forming monocarpic perennial. From the main flowering

stem, cymes diverge at which flowers develop sequentially in time (Nicholls 1987). Each day new flowers open at each cyme. Flowers are hermaphrodite with five anthers and four ovules. The four ovules are arranged in a square. Flowers are protandrous: first the anthers present the ripe pollen in the male phase. Hereafter the style elongates and the two lobes of the stigma diverge and become receptive to pollination. Although protandry and herkogamy reduce self- pollination within one flower, selfing by geitonogamy can still occur because flowers in the male and female phase are present on one plant simultaneously. In our study area male- steriles, individuals producing yellow pollen, occur at a frequency of 7% (Klinkhamer et al.

1994). Plants (presumably different genotypes) were collected in natural populations of E. vulgare in the dunes of Meyendel, near The Hague, the Netherlands. All individuals were

collected more than 100 m. apart. The different plants were propagated vegetatively in growth chambers and the replicates were used in the experiments. Day and night temperature were respectively 20 and 15˚C ± 1˚C. and relative humidity ranged between 60 and 85%.

The average seed yield per flower per plant of E. vulgare in our study area does not

exceed 1.5 seeds per flower, although four ovules are present. Pollen limitation is not the determining factor for the low seed set per flower because additional hand pollination in the field did not increase the seed set per flower (Klinkhamer et al. 1994). Moreover, more than 4

pollen tubes were present in each style in the field with a modal amount of 9 (n=30, unpubl. data).

Single-donor experiment

To examine the effect of selection on different pollen sources after pollination, we pollinated flowers with pure pollen of different pollen donors in a growth chamber and counted the resulting number of seeds per flower.

Ten different genotypes of E. vulgare were used in a complete diallel design. One

plant of each genotype received different pollinations with pure pollen from each genotype, at 19 to 22 flowers, in total circa 200 treated flowers per maternal genotype. Flowers were emas- culated with forceps before the style elongated. Emasculation eliminates the possible effects of variation in degree of herkogamy between genotypes on the selfing rate. Pollen of at least two replicates of each genotype was used for the pollinations. The pollen was applied within three to four hours after collection. Flowers were pollinated by rubbing the pollen firmly on to

the lobes of the stigma with the end of a toothpick, which was covered with parafilm. With this method of hand pollination, 90% of the flowers received at least 5 pollen grains on the stigma (counted under the light microscope, n=175, unpubl. data). The treated flowers were marked with a small drop of paint to identify the applied pollen donor. All plants were circulated within the growth chamber to avoid environmental effects. All pollinations were carried out within a period of 30 days. After pollination of the last flowers used in the experiment, the later opening flowers were pollinated with a random sample of pollen for at least three days, so that the seeds of the last flowers in the experiment also developed in the presence of younger seeds. Approximately three weeks after the last pollinations, the numbers of developed seeds per flower were counted and the seeds were weighed.

The number of seeds per flower was tested for effects of the position of the cyme along the main flowering stem, the position of the flower along the cyme, the date of polli- nation, the maternal genotype, the pollination type (either self- or cross-pollination), the inter- action between maternal genotype and pollination type, the cross-pollen source and the inter- action between maternal genotype and cross-pollen source. The analysis of the binomial distributed data of the seed numbers used a GLM procedure with a logit link function (McCullagh & Nelder 1989, SAS Institute 1993). The effects of later entered factors in the analysis are adjusted for the effects of the earlier entered factors (SAS PROC GENMOD, type I). For significant factors, genotypes are compared with Miller's multi-stage procedure (Haccou & Meelis 1992). The position of the cyme along the main flowering stem appeared to have no effect and was excluded from the final analysis. The weight of the seeds was normally distributed and tested for effects of the same factors with a GLM procedure.

Maternal success (M_i) was calculated as

10 10 , 1 , , = = j i ij j i i F S M

in which S_i,j is the total number of seeds produced by maternal genotype i and sired by pater- nal genotype j, F_i,j is the number of flowers, pollinated on maternal genotype i with pollen of genotype j. Paternal success (P_j) was calculated as

9 10 1 , , , = = ji i j j i j F S P for i≠j

in which S_i,j is the total number of seeds produced by maternal genotype i and sired by pater- nal genotype j. F_i,j is the number of flowers, pollinated on maternal genotypes i with pollen of genotype j. Inbreeding depression (δ) was calculated as 1-M_i,i/M_i,j, in which M_i,i is the mean number of seeds per flower after selfing produced by maternal genotype i, and M_i,j (for i≠j) is the average number of seeds per flower produced after outcross-pollination by maternal geno- type i, averaged over all 9 outcross paternal genotypes j. A negative value of δ signifies a lar- ger production of seeds after self-pollinations compared to cross-pollinations. Dominance (h), or the degree to which deleterious alleles are expressed in the heterozygote, was calculated as the coefficient from the regression of M_i,j on M_i,i+M_j,j (Johnston & Schoen 1995). A value of 0.5 indicates complete additivity, a value of 0 indicates complete dominance, and values be-

tween 0 and 0.5 represent partial dominance of the alleles with the highest fitness. Irrespective of the underlying mechanisms, each value less than 0.5 signifies inbreeding depression.

Mixed-donor experiment

To mimic the natural field situation, in which at least three different pollen sources are present on the stigma (Rademaker, unpublished data), we did a second pollination experiment. In this experiment we pollinated flowers in a complete diallel design with a mixture of pollen of three different pollen genotypes.

Three different clones out of the ten which were used in the single-donor experiment were selected on the basis of the availability of flowering individuals at the start of both expe- riments. Three different replicates of each genotype were used for the pollinations. The flowers used for the pollinations were all at the middle of a cyme. Each paternal genotype contributed five anthers of one flower to the pollen mixture that was applied. For each polli- nation a new mixture was made. For the different genotypes, 4 to 28 flowers were used for counting the numbers of pollen grains per flower. After staining the nuclei of the pollen with DAPI (de Laat et al. 1987), pollen were counted with a flow cytometer (CA-II, Partec). With

this method, the viable pollen grains are counted. These counts were used to estimate the ratio at which pollen of the three genotypes contributed to the pollen mixture. This ratio is equal to the expected ratio at which these three genotypes sire seeds in the offspring if no selection takes place. The expected number of seeds produced by mother i, sired by father j equals:

i j j i S p p p p S 3 2 1 , = _{+ +} ε

in which S_i is the total number of seeds produced by maternal parent i and p₁, p₂ and p₃ denote the number of pollen grains per anther of the three genotypes. The number of seeds sired per paternal genotype was tested for deviations from the expected number of seeds with a G-test (Sokal and Rohlf 1995).

Approximately 3 weeks after the last pollinations, the ripe seeds were collected and sown in a growth chamber. The paternities of 28 to 30 offspring per maternal genotype were analyzed with the use of Random Amplified Polymorphic DNA (RAPD) (Williams et al.

1990). DNA was isolated of samples of the leaves according to Cheung et al. (1993) with the

addition of 2% PVP to the extraction buffer. In the PCR reaction the primers OPF4, OPF7, OPF9, OPF11, OPF12, OPF13, OPF16 and OPF18 (Operon Technologies) were used. For each possible paternal genotype we scored the presence or absence of at least six unique bands (either homo- or heterozygote). If for both outcross-pollen sources all unique bands were absent, we concluded that a seed was selfed. In this way, the chance of incorrectly classifying a seed as selfed is smaller than 2% (1/2)6.

Comparing the single- and mixed donor experiment

If the single- and the mixed donor experiment give different results, this would indicate that pollen- or early pollen-tube competition affect selection on pollen source or that competition between developing embryos is more intense within than between flowers. The two experi- ments were compared with a G-test. We derived the expected values from the single donor experiment and used the results of the mixed donor experiment as observed values. We multi- plied the number of seeds per flower of the single donor experiment sired by paternal parent j

with the pollen ratio of paternal genotype j in the pollen mixture to obtain the total expected number of seeds per flower in this mixed donor experiment. This expected number of seeds per flower was multiplied by the number of flowers used for each parental combination in the mixed donor experiment to obtain the expected number of seeds produced for each parental combination. The expected number of seeds in the mixed donor experiment produced by maternal parent i, sired by paternal parent j equals:

m j i j s j i s j i m j i F p p p p F S S _{, ,} 3 2 1 , , , , , , = _{+ +} ε

in which the indices s and m refer to respectively the single- and mixed donor experiment, S_i,j,s / F_i,j,s is the number of seeds per flower of the single donor experiment produced by maternal parent i and sired by paternal parent j. p₁, p₂ and p₃ denote the number of pollen grains per anther of the three genotypes, and F_i,j,m is the number of flowers in the mixed donor experiment used for the parental combination of maternal parent i and paternal parent j.

Pollen germination and pollen-tube growth

To examine the effect of pollen germination and pollen-tube competition on the non-random siring of seeds, the number and length of the pollen tubes were recorded. We used all nine pa- rental combinations from the mixed donor experiment, and collected the styles (n=69) five hours after pollination with pollen loads from one single donor.

After a time interval of five hours, the fastest pollen tubes were recorded to have grown one third of the style length. The stigmas were fixed in ethanol with acetic acid (4:1) for one hour and stored in 70% ethanol for later observation. Pollen tubes were stained with aniline blue according to Martin (1959). The ripeness of the stigma, the number of pollen grains in the holes of the stigma and the number and length of the pollen tubes were recorded. Unripe stigmas were discarded from the statistical analysis. Pollen was collected of each genotype and over 100 grains per genotype were viewed under a microscope to record the percentage of collapsed pollen. Collapsed pollen proved to be non-viable when stained (Melser, unpubl. data) according to Alexander (1980).

The number of the pollen tubes in the style were normally distributed and were analyzed statistically with an ANOVA (type III). Pollen tube growth per hour was normally distributed after a square-root transformation and were analyzed statistically with an ANOVA (type III).

Results

Single-donor experiment

Averaged over all mothers, the mean number of seeds per flower was 0.43 (se = 0.017). The number of seeds per flower was strongly influenced by the position of the flower along the cyme and by the date of pollination (Table 1). Generally, late flowers produced fewer seeds than early flowers, although the growth chamber conditions were constant. Maternal parents differed in the number of seeds per flower. There was no main effect of pollination type (self- or cross-pollination). This means that averaged over all maternal parents, the pollination with either self- or cross-pollen did not significantly affect the number of seeds per flower

(average 0.41; se=0.057 and 0.42; se=0.018, respectively). However, the response to pollina- tion type differed significantly among maternal parents, as indicated by the interaction term (Table 1 and Fig. 1). In some maternal parents self-pollen was more successful than outcross- pollen, while in others the reverse was true. The inbreeding depression of the different motherplants ranged from 0.754 to 0.625 with a mean value of 0.125 (sd = 0.424). Paternal effects were also present: one pollen donor (D) sired significantly fewer seeds per flower in outcross-pollinations compared to the other pollen donors (p<0.05; Table 1; Fig. 2). No