Inventario de la infraestructura que rodea la Hacienda "El

Summary

We present data on late-acting inbreeding depression in pollen performance, siring success and seed production in Echium vulgare. Pollen viability and rate of pollen tube

growth were both lower for pollen from plants derived from selfing than for pollen from plants derived from outcrossing. Pollen tube numbers within the styles did not differ for pollen from plants derived from selfing or outcrossing. A pollination experiment with two mixtures of pollen from plants derived from selfing or outcrossing, revealed a significant decline of 55% in siring success for pollen from plants derived from selfing. A second experiment with a complete diallel design revealed inbreeding depression for both siring success of the offspring (32.8%) and a decline in seed production of the offspring (34.8% - 40.6%). In addition, results indicated a heritable component for seed number per flower. Offspring fitness, measured as seed production and siring ability, can be severely affected by late-acting inbreeding depression. Inbreeding depression values for male and female functions were not correlated. Both functions must therefore be considered when calculating inbreeding depression.

Introduction

Inbreeding depression is considered a major force in the evolution of plant reproductive systems (Darwin 1876, 1877, Maynard Smith 1978, Lloyd 1980, Charlesworth & Charles- worth 1987). Inbreeding depression is the reduction in fitness of offspring produced by polli- nation with closely related pollen donors, compared to the fitness of offspring after pollination with unrelated pollen donors. Ideally, the entire life cycle of the progeny, from seed to seed production, is measured to determine offspring fitness, and thus the magnitude of the inbreeding depression. Yet most published estimates of inbreeding depression are predominantly based on early life stages, i.e. seed production of the maternal plant, germination of the seeds, survival of the seedlings and size of the offspring (e.g. Waser & Price 1993, Trame et al. 1995, survey in Husband & Schemske 1996, Fischer & Matthies

1997, Byers 1998, del Castillo 1998, Hardner 1998). Estimates of early inbreeding depression may underestimate the cumulative lifetime inbreeding depression. Although offspring have been screened for quality at reproductive stages several times, data are mostly restricted to measurements of e.g. days to flowering, number of flowers or number of seeds of the offspring (Waser & Price 1993, survey of 25 species in Husband & Schemske 1996). The reduction in the female fertility as a result of inbreeding depression ranges from -0.09 in

Eichhornia paniculata to 0.74 in Clarkia tembloriensis with a selfing rate of 0.49 (reviewed

in Husband & Schemske 1996). The number of seeds measures only the female function of a hermaphrodite plant and on average thus only half of the reproductive success. The effect of inbreeding depression on the male function of plants needs to be studied to get a more reliable estimation of the magnitude of inbreeding depression in plant populations.

Late-acting inbreeding depression at the stage of pollen production has been described for Mimulus guttatus (Willis 1993, Carr & Dudash 1997). Offspring derived from selfing pro-

duced approximately 60% fewer pollen grains than offspring derived from outcross polli- nations after one generation of selfing (Fig. 1 in Carr & Dudash 1997). In addition, stainability of the pollen after one generation of selfing was 15% to 40% lower (Fig. 1d in Willis 1993), declining to 60% lower after four generations of selfing (Fig. 1 in Carr & Dudash 1997). Collinsia heterophylla also showed a decline in the stainability of pollen from

plants derived from selfing (Mayer et al. 1996). However, this effect was less than 7% and

occurred only in two populations out of four (Mayer et al. 1996). In Phacelia dubia the

progeny derived from outcrossing tended on average to have a significantly higher frequency of normal pollen grains than those derived from plants which were produced by selfing, with a difference of 9% (del Castillo 1998). Selecting for high and moreso for low ovule number in Malva moschata by means of selfing also produced severe inbreeding depression in via-

bility of the pollen (T.J.Crawford, pers. comm.). These studies of differences in pollen quality were not continued up to the siring of seeds. As far as we know, only the study by Jóhannsson

et al. (1998) has measured effects of inbreeding on siring success directly as functional siring

ability. Pollen from plants of Cucurbita texana derived from selfing had significantly slower

growing pollen tubes in vitro. This effect was on average approximately 8% (Fig. 1 in

Jóhannsson et al. 1998). The pollen from plants derived from selfing also sired fewer seeds

under conditions of pollen competition with a tester line on Cucurbita pepo. Their

identify separate effects of slower pollen tube growth or post-fertilization causes of differential siring success.

Here we present a study on late-acting inbreeding depression in pollen performance, siring success and seed production in Echium vulgare. In an earlier study of E. vulgare on

average no differences in seed production were found between self- and outcross pollinations (Chapter 4). Thus, there are no indications of early inbreeding depression during seed production (Chapter 4) of this mainly outcrossing species (Rademaker 1998). However, late- acting inbreeding depression could influence its reproductive dynamics. By comparing the total reproductive success of selfed and outcrossed progeny we quantify the magnitude of late-acting inbreeding depression during reproductive stages of the progeny for both male and female function.

Materials and Methods

Species

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

stem, cymes diverge on which flowers develop sequentially (Nicholls 1987). Each day new flowers open on 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, then after about one day the style elongates and the two stigmatic lobes 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 the same plant simultaneously. The selfing rate of male-fertile plants in the field in 1996 was estimated with molecular paternity analyses and ranged between 0 and 30% in six individuals (Rademaker 1998). Echium vulgare is a

gynodioecious species. In Meyendel, near The Hague, where plants were collected, about 12% of all individuals are male-sterile (Klinkhamer et al. 1991). This male sterility is

heritable (Klinkhamer & de Jong, unpubl. data). A clear distinction can usually be made between (self-compatible) hermaphrodite individuals with perfect flowers that produce fertile blue pollen, and male-steriles with female flowers that produce infertile yellow pollen. The yellow pollen appears to be collapsed and unstainable with methylene blue when viewed under the microscope. Apparent male-steriles with yellow pollen did not occur in our experiments.

Selection and cultivation of the plants

In earlier experiments about a third of the individuals produced more seeds after selfing than after outcrossing (Chapter 4 and 7), but over all parental combinations used, there was no difference on average between the number of seeds produced after selfing compared to out- crossing (Chapter 4 and 7). For our experiments we selected the parental individuals (P gene- ration) and their number of descendants (F1 plants) to conform to these proportions of seed

production: in earlier hand-pollinations, two parental individuals (i and j) produced relatively many seeds after selfing compared to outcrossing, whereas two parental individuals (a and b)

produced relatively few seeds after selfing. The different parental combinations of i, j, a and b were included in the experiment with both selfing and outcrossing. The comparison between selfed and outcrossed plants is thus a comparison within relatives and partially eliminates the contribution of genotypic maternal effects (Lynch 1988). The labelling of the plants is as in Chapter 4.

Seeds were germinated on filter paper; seedlings were potted in 3-L pots filled with 50% sand and 50% potting soil and randomly placed in a growth chamber under controlled conditions. Day and night temperatures were, respectively, 20˚C and 15˚C, and relative humi- dity ranged between 60% and 85%. To induce flowering, after 7 weeks the plants received a cold treatment at 5˚C. After six weeks of cold, the temperature was raised to 22˚C and 18˚C during, respectively, day and night. Additionally the plants received four droplets of gibberellin (21.75% in H₂O) in the middle of the rosette two times a week to ensure the production of a flowering stalk (Wesselingh et al. 1994). The final height of the plants ranged

between 70 and 90 cm and did not differ between selfed and outcrossed plants. The plants used in the experiments were all at the same developmental stage.

We studied pollen viability, number of pollen grains on the stigma, number of pollen tubes and pollen tube growth and performed two experiments on siring success. Unless other- wise stated, two-sided levels of significance are given.

Pollen viability

To obtain a rough estimate (Heslop-Harrison et al. 1984) of the level of late-acting inbreeding

depression in the viability of the pollen in the anthers, we stained the pollen and determined the percentages viable and non viable pollen.

Two individuals of each parental combination derived from selfing were used, based on the availability of individuals of different genotypes: progeny of the crosses (j x j) (=maternal individual x paternal individual), (a x a) and (b x b). One individual of the parental combinations derived from outcrossing was used from the crosses (j x a), (a x j), (j x b) and (b x j). Pollen was collected from several ripe anthers of each plant, diluted in methylene blue (Dafni 1992) and analysed under a light microscope. Percentages of stainable pollen were scored for each plant for 330 to 760 pollen grains. Differences between plants in percentage stainable pollen were tested with Wilcoxon tests.

Pollination method

Flowers were emasculated with forceps before the style elongated and before anthers dehis- ced. Flowers were pollinated by rubbing the pollen firmly on 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 five visible pollen grains that adhere permanently to the stigma (unpubl. data, cited in Chapter 4). The treated flowers were marked with a small drop of paint to identify the pollen donor. All plants were pollinated within one period of 50 days. Approximately three weeks after the last pollinations, the number of developed seeds per flower was counted.

Number of pollen grains on the stigma, number of pollen tubes and pollen tube growth Based on availability of individual plants, the parental combinations derived from selfing, (i x i) and (j x j), and the parental combinations derived from outcrossing, (j x a) and (j x b), were used, with two individuals of each parental combination. For each parental combination of individual plants, in the F1 generation five flowers were pollinated. After 5 h. the styles were

collected, fixed in ethanol with acetic acid (4:1) and stored in 70% ethanol. The tissue was softened overnight in 8N KOH, stained in 0.006% aniline blue in 0.15 M K₂HPO₄ and viewed with a fluorescence microscope (Martin 1959). The number of pollen grains still adhering to the visible side of the stigma was counted and analysed with Kruskal-Wallis tests. Stigmas without any visible pollen grains were excluded for the analysis of pollen tubes. The number and the length of the pollen tubes was recorded, using an ocular micrometer. The differences between the parental combinations in number and length of the pollen tubes were analysed with Kruskal-Wallis tests.

Siring success and number of seeds per flower Experiment 1

To examine late inbreeding depression in siring success of pollen of F1 plants, a mix of pollen

from plants derived from either selfing or outcrossing was applied on five randomly chosen recipient plants. These recipient plants were collected at least 100 m apart in the nature re- serve of Meyendel, near The Hague.

One pollen mixture was made from six plants derived from selfing (from two sibships from each of the three parental combinations (j x j), (a x a) and (b x b)), and another pollen mixture was made from four plants derived from outcrossing (parental combinations (j x a), (a x j), (j x b) and (b x j)). The mixtures were made with an equal number of anthers of each parental combination. Each mixture was applied to 20 to 30 flowers of the recipient plants, a total of 40 to 60 flowers per maternal individual. All pollinations were carried out within one day. The resulting numbers of seeds per flower were counted. The binomially distributed data of the numbers of seed per flower were analysed with a GLM procedure with a logit link function (McCullagh & Nelder 1989, SAS Institute 1993). The effects of factors entered later in the analysis are adjusted for the effects of the earlier entered factors (SAS PROC GENMOD,

type 1). The numbers of seeds per flower were adjusted for the effect of maternal individual as a main factor.

Experiment 2

To examine late inbreeding depression on seed-set and siring success of F1 plants derived

from selfing and outcrossing, we pollinated flowers with pure pollen of different pollen donors in a greenhouse and counted the resulting number of seeds per flower. The ten plants used were the same individuals as were examined for pollen viability. All plants were polli- nated in a complete diallel design. Each plant received pollinations with pure pollen from each other individual; 25 to 30 flowers were pollinated for each parental combination, which yields a total of 250 to 300 flowers per maternal individual. The resulting numbers of seeds per flower were counted.

Because number of seeds per flower and siring success may have a heritable com- ponent, we first tested for this by regression of the F1 generation on midparent values. For the

seed number per flower, this regression was significant (see results section) and we therefore had to separate the effect of heritability and the effect of late-acting inbreeding depression. To test for late-acting inbreeding depression, we regressed the F1 recipient plants derived from

outcrossing against their midparent values and subsequently tested whether the residuals of this regression line for the F1 recipient plants derived from selfing were significantly smaller

than zero. To quantify the reduction in number of seeds caused by inbreeding depression, the difference between the slopes of the two regression lines for either plants derived from out- crossing or plants derived from selfing was calculated.

For the male function (siring success) there is no significant heritability (Chapter 4 and this experiment). The binomially distributed data of number of seeds per flower were analysed for the effect of late-acting inbreeding depression on the male function (siring success) with a GLM procedure (SAS PROC GENMOD, type 1). The number of seeds per flower

was adjusted for the effect of week of pollination, and the effect of the different pollination types was analysed within individual plants. We distinguished three different pollination types: i) self-pollination within one plant (denoted as self); ii) outcross-pollination with pollen from F1 plants derived from selfing (denoted as S); and iii) outcross-pollination with

pollen from F1 plants derived from outcrossing (denoted as C). All three pollination types

were on F1 recipient plants derived from selfing (denoted as S) and on F1 recipient plants

derived from outcrossing (denoted as C). So, for example, C x S denotes a maternal plant derived from outcrossing, pollinated with pollen from a father derived from selfing, and S self denotes a recipient plant derived from selfing that is self-pollinated. An example of the combination of the pollination types on different types of recipient plants is shown in Fig. 1. Multiple comparisons between the means of the different pollination types on the recipient plants either derived from selfing or from outcrossing were analysed with t-tests. Significance

levels were corrected for multiple comparisons with an improved Bonferroni test (Haccou & Meelis 1992). P generation F₁ generation a x j x b S x S a x a(S) a x j(C)j x a(C) b x j(C)j x b(C) b x b(S) S x C C x C C x S C self S self Figure 1:

Example of the different pollination types on maternal plants. a, j and b denote different individuals of the P generation. They produce the F1 generation by selfing and outcrossing, labeling the F1 gene-

Relation between male and female reproductive success

Pollen viability, siring success and number of seeds per flower were measured on the same individuals. We could therefore calculate the correlation between these two components of male fitness and female fitness.

Results

Pollen viability

The percentages of viable pollen differed between the group of F1 plants derived from selfing

and the group of F1 plants derived from outcrossing (normal approximation Z=3.24;

p=0.0012). Pollen from plants derived from selfing contained on average 89.9% (se=0.011) stainable pollen grains (ranging from 84.0% to 95.1% for parental combinations (j x j) and (b x b), respectively), and pollen from plants derived from outcrossing contained on average 93.2% (se=0.016) stainable pollen grains (ranging from 87.3% to 96.9% for parental combinations (j x a) and (b x j), respectively).

Pollen number on stigma

On average, 8.89 (se=0.331) pollen grains were adhering permanently to the visible part of the stigma after hand-pollination. The back side of the stigma on the slide is not visible under the microscope, so the number of pollen present on the stigma is underestimated. The number of pollen grains adhering to the visible part of the stigmas did not differ significantly between pollen donors (normal approximation Z=−0.418; p=0.675)

Pollen tube number

The number of pollen tubes in the style after approximately 5 h. was on average 1.13 (se=0.109). There was no difference between F1 maternal plants derived from selfing and out-

crossing (Fig. 2A; p=0.9589) nor between F1 pollen donors in the six combinations of pollina-

tions (see Fig. 2A; p=0.1095). Pollen tube growth

The average length of the pollen tubes in the style after 5 h. ranged between 0.92 and 2.28 mm. Within the styles of maternal plants derived from selfing, the pollen tubes grew more slowly than in the styles of plants derived from outcrossing (Fig. 2B; p=0.0071). The length of the pollen tubes depended on the origin of the pollen donor (Fig. 2B; p=0.0087). Excluding the self-pollinations, pollen from plants derived from selfing grew on average 1.12 mm (se=0.146), whereas pollen from plants derived from outcrossing grew on average 2.05 mm (se=0.162). Self-pollinations within the different maternal plants (S self and C self) did

Table 1:

GLM analysis of the number of seeds per flower in Echium vulgare after pollinations on five

randomly collected individuals with a pollen mixture derived from selfing and a pollen mixture derived from outcrossing.

Factor d.f. F-value P-value

Individual plant 4 8.24 <0.0001 Pollen mixture self or outcross 1 19.72 <0.0001

not differ significantly in pollen tube lengths compared to outcross pollinations (compare S self with SxS and C self with CxC). There was thus no immediate effect of selfing on pollen tube growth, but pollen from plants derived from selfing grew slower in the next generation. Number of seeds per flower: Experiment 1

Averaged over all randomly chosen maternal plants, the mean number of seeds per flower was 0.55 (se=0.058). Maternal individual influenced the mean number of seeds (Table 1). The mean number of seeds per flower ranged from 0.36 to 1.20. However, the significance level of the difference between siring success of pollen from plants derived from selfing or outcrossing was even greater (Table 1). The mixture of pollen from plants derived from outcrossing sired on average 0.77 (se=0.096) seeds per flower, whereas that from plants derived from selfing sired on average only 0.34 (se=0.064) seeds per flower, a decrease of 55.8%. 2.00 1.60 1.20 0.80 0.40 0.00 A v er age number of pollen t ubes ( mm) 3.00 2.40 1.80 1.20 0.60 0.00 S self S x S S x C C self C x S C x C