Late-acting inbreeding depression in both male and female function of Echium vulgare (Boraginaceae)

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1 Heredity 83 (1999) 162±170 Received 15 December 1998, accepted 27 April 1999 Late-acting inbreeding depression in both male and female function of Echium vulgare (Boraginaceae) CHANTAL MELSER*, ARJAN BIJLEVELD & PETER G. L. KLINKHAMER Institute of Evolutionary and Ecological Sciences, Leiden University, PO Box 9516, 2300 RA Leiden, The Netherlands 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 sel ng than for pollen from plants derived from outcrossing. Pollen tube numbers within the styles did not di er for pollen from plants derived from sel ng or outcrossing. A pollination experiment with two mixtures of pollen from plants derived from sel ng or outcrossing, revealed a signi cant decline of 55% in siring success for pollen from plants derived from sel ng. A second experiment with a complete diallel design revealed inbreeding depression for both siring success of the o spring (32.8%) and a decline in seed production of the o spring (34.8%± 40.6%). In addition, results indicated a heritable component for seed number per ower. O spring tness, measured as seed production and siring ability, can be severely a ected 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. Keywords: inbreeding depression, o spring quality, pollen quality, pollen tube, seed production. Introduction *Correspondence. melser@rulsfb.leidenuniv.nl Inbreeding depression is considered a major force in the evolution of plant reproductive systems (Darwin, 1876, 1877; Maynard Smith, 1978; Lloyd, 1980; Charlesworth & Charlesworth, 1987). Inbreeding depression is the reduction in tness of o spring produced by pollination with closely related pollen donors, compared to the tness of o spring after pollination with unrelated pollen donors. Ideally, the entire life cycle of the progeny, from seed to seed production, is measured to determine o spring tness, 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 o spring (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 o spring have been screened for quality at reproductive stages several times, data are mostly restricted to measurements of, e.g. days to owering, number of owers or number of seeds of the o spring (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 sel ng 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 e ect of inbreeding depression on the male function of plants needs to be studied to obtain 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). O spring derived from sel ng produced»60% fewer pollen grains than o spring derived from outcross pollinations after one generation of sel ng ( gure 1 in Carr & Dudash, 1997). In addition, stainability of the pollen after one generation of sel ng was 15% to 40% lower ( gure 1d in Willis, 1993), declining to 60% lower after four generations of sel ng ( gure 1 in Carr & Dudash, 1997). Collinsia heterophylla also showed a decline in the stainability of pollen from plants derived from sel ng (Mayer et al., 1996). However, this e ect was less than 162 Ó 1999 The Genetical Society of Great Britain.

2 LATE-ACTING INBREEDING DEPRESSION 163 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 signi cantly higher frequency of normal pollen grains than those derived from plants which were produced by sel ng, with a di erence of 9% (del Castillo, 1998). Selecting for high and especially for low ovule number in Malva moschata by means of sel ng also produced severe inbreeding depression in viability of the pollen (T. J. Crawford, pers. comm.). These studies of di erences in pollen quality were not continued up to the siring of seeds. As far as we know, only the study by Jo hannsson et al. (1998) has measured e ects of inbreeding on siring success directly as functional siring ability. Pollen from plants of Cucurbita texana derived from sel ng had signi cantly slower-growing pollen tubes in vitro. This e ect was on average» 8% ( gure 1 in Jo hannsson et al., 1998). The pollen from plants derived from sel ng also sired fewer seeds under conditions of pollen competition with a tester line on Cucurbita pepo. Their experimental design with pollination against a tester line under pollen competition does not identify separate e ects of slower pollen-tube growth or postfertilization causes of di erential 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 di erences in seed production were found between self- and outcross pollinations (Melser et al., 1997). Thus, there are no indications of early inbreeding depression during seed production (Melser et al., 1997) of this mainly outcrossing species (Rademaker, 1998). However, late-acting inbreeding depression could in uence 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 owering stem, cymes diverge on which owers develop sequentially (Nicholls, 1987). Each day new owers open on each cyme. Flowers are hermaphrodite with ve anthers and four ovules. The four ovules are arranged in a square. Flowers are protandrous: rst 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 ower, sel ng by geitonogamy can still occur because owers in the male and female phase are present on the same plant simultaneously. The sel ng rate of malefertile plants in the eld 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 (P. G. L. Klinkhamer & T. J. de Jong, unpubl. obs.). A clear distinction can usually be made between (self-compatible) hermaphrodite individuals with perfect owers that produce fertile blue pollen, and male-steriles with female owers that produce infertile yellow pollen. The yellow pollen appears to be collapsed and unstainable with methylene blue when viewed under the microscope. Apparent malesteriles 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 sel ng than after outcrossing (Melser et al., 1997 and unpubl. data), but over all parental combinations used, there was no di erence on average between the number of seeds produced after sel ng compared to outcrossing (Melser et al., 1997 and unpubl. data). For our experiments we selected the parental individuals (P generation) and their number of descendants (F 1 plants) to conform to these proportions of seed production: in earlier hand-pollinations, two parental individuals (i and j) produced relatively many seeds after sel ng compared to outcrossing, whereas two parental individuals (a and b) produced relatively few seeds after sel ng. The di erent parental combinations of i, j, a and b were included in the experiment with both sel ng and outcrossing. The comparison between selfed and outcrossed plants is thus a comparison within relatives and partially eliminates the contribution of genotypic maternal e ects (Lynch, 1988). The labelling of the plants is as in Melser et al. (1997). Seeds were germinated on lter paper; seedlings were potted in 3-L pots lled with 50% sand and 50% potting soil and randomly placed in a growth chamber under controlled conditions. Day and night temperatures were 20 C and 15 C, respectively, and relative humidity ranged between 60% and 85%. To induce owering, after 7 weeks the plants received a cold treatment at 5 C. After 6 weeks of cold, the temperature was raised to 22 C and 18 C during day and night, respectively. Additionally the plants received four droplets of gibberellin (21.75% in H 2 O) in the middle of the rosette

3 164 C. MELSER ET AL. two times a week to ensure the production of a owering stalk (Wesselingh et al., 1994). The nal height of the plants ranged between 70 and 90 cm and did not di er 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 otherwise stated, two-sided levels of signi cance 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 of viable and nonviable pollen. Two individuals of each parental combination derived from sel ng were used, based on the availability of individuals of di erent genotypes: progeny of the crosses (j j) ( ˆ maternal individual paternal individual) (a a) and (b b). One individual of the parental combinations derived from outcrossing was used from the crosses (j a), (a j), (j b) and (b 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±760 pollen grains. Di erences 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 dehisced. Flowers were pollinated by rubbing the pollen rmly on the lobes of the stigma with the end of a toothpick, which was covered with para lm. With this method of handpollination, 90% of the owers received at least ve visible pollen grains that adhered permanently to the stigma (unpubl. data, cited in Melser et al., 1997). The treated owers were marked with a small drop of paint to identify the pollen donor. All plants were pollinated within one period of 50 days. Approximately 3 weeks after the last pollinations, the number of developed seeds per ower 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 sel ng, (i i) and (j j), and the parental combinations derived from outcrossing, (j a) and (j b), were used, with two individuals of each parental combination. For each parental combination of individual plants, in the F 1 generation ve owers were pollinated. After 5 h the styles were collected, xed 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 K 2 HPO 4 and viewed with a uorescence 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 length of the pollen tubes was recorded, using an ocular micrometer. The di erences 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 ower Experiment 1 To examine late inbreeding depression in siring success of pollen of F 1 plants, a mixture of pollen from plants derived from either sel ng or outcrossing was applied on ve randomly chosen recipient plants. These recipient plants were collected at least 100 m apart in the nature reserve of Meyendel, near The Hague. One pollen mixture was made from six plants derived from sel ng (from two sibships from each of the three parental combinations (j j), (a a) and (b b)), and another pollen mixture was made from four plants derived from outcrossing (parental combinations (j a), (a j), (j b) and (b j)). The mixtures were made with an equal number of anthers of each parental combination. Each mixture was applied to 20±30 owers of the recipient plants, a total of 40±60 owers per maternal individual. All pollinations were carried out within 1 day. The resulting numbers of seeds per ower were counted. The binomially distributed data of the numbers of seed per ower were analysed with a GLM procedure with a logit link function (McCullagh & Nelder, 1989; SAS Institute, 1993). The e ects of factors entered later in the analysis are adjusted for the e ects of the earlier entered factors (SAS PROC GENMOD, type 1). The numbers of seeds per ower were adjusted for the e ect of maternal individual as a main factor. Experiment 2 To examine late inbreeding depression on seed-set and siring success of F 1 plants derived from sel ng and outcrossing, we pollinated owers with pure pollen of di erent pollen donors in a greenhouse and counted the resulting number of seeds per ower. The 10 plants used were the same individuals as were examined

4 LATE-ACTING INBREEDING DEPRESSION 165 for pollen viability. All plants were pollinated in a complete diallel design. Each plant received pollinations with pure pollen from each other individual; 25±30 owers were pollinated for each parental combination, which yields a total of 250±300 owers per maternal individual. The resulting numbers of seeds per ower were counted. Because number of seeds per ower and siring success may have a heritable component, we rst tested for this by regression of the F 1 generation on mid-parent values. For the seed number per ower, this regression was signi cant (see Results section) and we therefore had to separate the e ect of heritability and the e ect of late-acting inbreeding depression. To test for late-acting inbreeding depression, we regressed the F 1 recipient plants derived from outcrossing against their mid-parent values and subsequently tested whether the residuals of this regression line for the F 1 recipient plants derived from sel ng were signi cantly smaller than zero. To quantify the reduction in number of seeds caused by inbreeding depression, the di erence between the slopes of the two regression lines for either plants derived from outcrossing or plants derived from sel ng was calculated. For the male function (siring success) there is no signi cant heritability (Melser et al., 1997 and this experiment). The binomially distributed data of number of seeds per ower were analysed for the e ect of lateacting inbreeding depression on the male function (siring success) with a GLM procedure (SAS PROC GENMOD,type1). The number of seeds per ower was adjusted for the e ect of week of pollination, and the e ect of the di erent pollination types was analysed within individual plants. We distinguished three di erent pollination types: (i) selfpollination within one plant (denoted as self); (ii) outcross-pollination with pollen from F 1 plants derived from sel ng (denoted as S); and (iii) outcross-pollination with pollen from F 1 plants derived from outcrossing (denoted as C). All three pollination types were on F 1 recipient plants derived from sel ng (denoted as S) and on F 1 recipient plants derived from outcrossing (denoted as C). So, for example, C S denotes a maternal plant derived from outcrossing, pollinated with pollen from a father derived from sel ng, and S self denotes a recipient plant derived from sel ng that is self-pollinated. An example of the combination of the pollination types on di erent types of recipient plants is shown in Fig. 1. Multiple comparisons between the means of the di erent pollination types on the recipient plants either derived from sel ng or from outcrossing were analysed with t- tests. Signi cance levels were corrected for multiple comparisons with an improved Bonferroni test (Haccou & Meelis, 1992). Relation between male and female reproductive success Pollen viability, siring success and number of seeds per ower were measured on the same individuals. We could therefore calculate the correlation between these two components of male tness and female tness. Results Pollen viability The percentages of viable pollen di ered between the group of F 1 plants derived from sel ng and the group of F 1 plants derived from outcrossing (normal approximation Z ˆ 3.24; P ˆ ). Pollen from plants derived from sel ng contained on average 89.9% (SE ˆ 0.011) stainable pollen grains (ranging from 84.0% to 95.1% for parental combinations (j j) and (b b), respectively), and pollen from plants derived from outcrossing contained on average 93.2% (SE ˆ 0.016) stainable pollen grains (ranging from 87.3% to 96.9% for parental combinations (j a) and (b j), respectively). Pollen number on stigma On average, 8.89 (SE ˆ 0.331) pollen grains were adhering permanently to the visible part of the stigma P generation a x j x b Fig. 1 Example of the di erent pollination types on maternal plants. a, j and b denote di erent individuals of the P generation. They produce the F 1 generation by sel ng and outcrossing, labelled in the F 1 generation by S and C, respectively. F 1 generation S x S axa (S) axj (C) jxa (C) bxj (C) jxb (C) bxb (S) S x C C x C C x S C self S self

5 166 C. MELSER ET AL. Average number of pollen tubes (mm) Average length of pollen tubes (mm) (a) (b) s s c c s c S self S x S S x C C self C x S C x C Parental combination Fig. 2 (a) Average number and (b) length of pollen tubes (SE) in the style for Echium vulgare in a complete diallel pollination experiment, classi ed by the origin of the recipient plant (either derived from sel ng S or outcrossing C), pollinationtype of sel ng within one plant (S self and C self), outcrosspollination with pollen from plants derived from sel ng (S S and C S) and outcross pollination with pollen from plants derived from outcrossing (S C and C C). 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 di er signi cantly between pollen donors (normal approximation Z ˆ )0.418; P ˆ 0.675). Pollen-tube number The number of pollen tubes in the style after» 5 h was on average 1.13 (SE ˆ 0.109). There was no di erence between F 1 maternal plants derived from sel ng and outcrossing (Fig. 2a; P ˆ ) nor between F 1 pollen donors in the six combinations of pollinations (see Fig. 2a; P ˆ ). c Table 1 GLM analysis of the number of seeds per ower in Echium vulgare after pollinations on ve randomly collected individuals with a pollen mixture derived from sel ng and a pollen mixture derived from outcrossing Factor d.f. F-value P-value Individual plant < Pollen mixture self or outcross < 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 sel ng, the pollen tubes grew more slowly than in the styles of plants derived from outcrossing (Fig. 2b; P ˆ ). The length of the pollen tubes depended on the origin of the pollen donor (Fig. 2b; P ˆ ). Excluding the self-pollinations, pollen from plants derived from sel ng grew on average 1.12 mm (SE ˆ 0.146), whereas pollen from plants derived from outcrossing grew on average 2.05 mm (SE ˆ 0.162). Self-pollinations within the di erent maternal plants (S self and C self) did not di er signi cantly in pollen-tube lengths compared to outcross pollinations (compare S self with S S and C self with C C). There was thus no immediate e ect of sel ng on pollen-tube growth, but pollen from plants derived from sel ng grew slower in the next generation. Number of seeds per ower: expt 1 Averaged over all randomly chosen maternal plants, the mean number of seeds per ower was 0.55 (SE ˆ 0.058). Maternal individual in uenced the mean number of seeds (Table 1). The mean number of seeds per ower ranged from 0.36 to However, the signi cance level of the di erence between siring success of pollen from plants derived from sel ng or outcrossing was even greater (Table 1). The mixture of pollen from plants derived from outcrossing sired on average 0.77 (SE ˆ 0.096) seeds per ower, whereas that from plants derived from sel ng sired on average only 0.34 (SE ˆ 0.064) seeds per ower, a decrease of 55.8%. Number of seeds per ower: expt 2 Female reproduction Averaged over all parental combinations in the diallel pollination design, the mean number of seeds per ower was 0.42 (SE ˆ 0.016). The number of seeds produced by the F 1 generation plants was correlated at the margin of signi cance

6 LATE-ACTING INBREEDING DEPRESSION Fig. 3 Regression of the number of seeds per ower of the F 1 generation in expt 2 on the mid-parent value of the P generation for each parental combination of Echium vulgare. Closed dots are plants derived from sel ng; open dots are plants derived from outcrossing. Regression lines are based on all data (continuous line; y ˆ 0.764x), on plants derived from sel ng (dashed line; y ˆ 0.592x) and on plants derived from outcrossing (dotted line; y ˆ 0.996x). No. of seeds per flower in F 1 generation Mid-parent value for no. of seeds per flower (R 2 ˆ 0.383; n ˆ 10; P ˆ 0.056) with the mid-parent value of the number of seeds produced by the P generation (Fig. 3), which indicates a slight heritable component for the number of seeds produced. The regression line for the outcrossed F 1 recipient plants with an intercept xed at 0 has a slope of (SE ˆ 0.179). For this regression line, the residuals of the maternal plants derived from sel ng (X) were signi cantly smaller than zero (X (avg) ˆ )0.203; t 5 ˆ 5.027; P ˆ 0.002). This represents 34.8% of the mean number of seeds per ower, compared with the plants derived from outcrossing (X ˆ 0.584). Thus plants derived from sel ng produce fewer seeds than expected on the basis of their mid-parent values, indicating late-acting inbreeding depression. The di erence between the slopes of the regression lines for the number of seeds produced derived from sel ng vs. outcrossing gives an alternative estimate (40.6%) of the decrease in number of seeds produced in the F 1 generation as a result of late-acting inbreeding depression. Male reproduction There was no signi cant correlation between the siring success of the F 1 generation plants and their mid-parent values, indicating that the siring success is not signi cantly heritable (P ˆ ). The e ect of paternal inbreeding depression was signi cant (Table 2; Fig. 4). Over all parental combinations, pollen from plants derived from sel ng (S self, S S and C S) sired on average 0.34 (SE ˆ 0.020) seeds and pollen from plants derived from outcrossing (C self, S C and C C) sired on average 0.52 (SE ˆ 0.027) Table 2 GLM analysis of the number of seeds per ower in Echium vulgare after single-donor pollinations in expt 2 with three pollination types: (i) self-pollination within one plant; (ii) outcross-pollination with pollen from F 1 plants derived from sel ng; and (iii) outcross-pollination with pollen from F 1 plants derived from outcrossing Factor d.f. F-value P-value Week < Maternal plant derived from < sel ng or outcrossing Individual plant < Pollination type < seeds. For the outcross pollinations, plants derived from sel ng (S S and C S) sired 32.8% (0.39 vs. 0.58) fewer seeds than plants derived from outcrossing (S C and C C). For the four plants derived from outcrossing, early acting inbreeding depression is detectable in the zygotes: fewer seeds per ower were produced after self-pollination (C self) compared to pollinations with pollen from other individuals that were derived from outcrossing (C C; Fig. 4). For the six plants derived from sel ng, no e ect of early acting inbreeding depression was found. Over all 10 individual plants in the experiment, on three individuals more seeds per ower were produced after sel ng compared to outcrossing (Table 3). The interaction between pollination type and maternal

7 168 C. MELSER ET AL. Average number of seeds per flower a b a s s c c s c b S self S x S S x C C self C x S C x C Parental combination Fig. 4 Average number of seeds per ower (SE) for Echium vulgare in expt 2, classi ed by the origin of the recipient plant and pollen donor (either derived from sel ng S or outcrossing C), pollination-types of sel ng within one plant (S self and C self), outcross pollination with pollen from plants derived from sel ng (S S and C S), and outcross pollination with pollen from plants derived from outcrossing (S C and C C). Capital and small letters denote di erent Tukey tests. plant was not signi cant and was excluded from the nal analysis. Relation between pollen viability and seed number per ower With a one-sided test, there was a signi cant correlation between pollen viability and siring success (r ˆ 0.462; n ˆ 10; P ˆ 0.045). A c A B Relation between maternal and paternal seed production There was no signi cant correlation between the average seed number per ower produced as a maternal plant and the average seed number per ower sired as a pollen donor (r ˆ 0.178; n ˆ 10; P ˆ ). Discussion The results of our study unequivocally show that lateacting inbreeding depression can severely reduce the fertility of o spring derived from sel ng. Not only was the female function of the hermaphroditic owers of the o spring derived from sel ng reduced by 34.8±40.6%, but also a decline in siring success of 32.8% to 55.8% was detected. Di erent stages of pollen performance were a ected. Pollen stainability was lower in o spring derived from sel ng, but the di erence was small. However, pollen viability is only one component of male tness. Moreover, low pollen stainability was not re ected in a lower number of pollen on the stigma, nor in fewer pollen tubes. The length of the pollen tubes shows a greater e ect of late-acting inbreeding depression. Pollen tubes from plants derived from sel ng grew slower than tubes from plants derived from outcrossing, in accordance with the ndings of Jo hannsson et al. (1998). Pollen competition in the style can cause a shift from the percentages of paternal genotypes present on the stigma to the resulting percentages of the paternity of the seeds (Mulcahy, 1979; Snow & Mazer, 1988; Snow & Spira, 1991, 1996). In each mixed pollination the pollen from plants derived from outcrossing would then outcompete the pollen from plants derived from Table 3 Average number of seeds per ower in Echium vulgare for the di erent parental combinations in expt 2 Maternal individual Mean in outcrossj j (1) j j (2) b b (1) b b (2) a a (1) a a (2) j b b j j a a j pollinations Paternal individual j j (1) j j (2) b b (1) b b (2) a a (1) a a (2) j b b j j a a j Mean in outcross pollinations

8 LATE-ACTING INBREEDING DEPRESSION 169 sel ng. Our results probably underestimate the e ects of sel ng on siring ability, because we used pure, singledonor pollinations, giving all pollen donors equal access to ovules. In Melser et al. (1997), however, no di erences in the success of pollen donors at siring seeds were found between single- and mixed-donor pollinations. Early inbreeding depression in seed-set and survival of the o spring also has to be included in calculations of the life-time inbreeding depression. Among the parental generation that was tested for selective abortion in Melser et al. (1997), inbreeding e ects at the stage of seed production were on average zero. In contrast, the data in the present experiment suggest that sel ng results in fewer seeds than outcrossing (at least in plants that were derived from outcross pollination). An extensive study of early inbreeding depression in seed germination, plant growth and survival under eld conditions is in progress. Preliminary results indicate a decline of»10% in germination and early seedling survival (Melser et al. unpubl. obs.). In the literature, a life-time inbreeding depression of 50% has been mentioned as the threshold limit for sel ng to be selectively favoured (Lloyd, 1980; Lande & Schemske, 1985). The threshold of 50% will be even lower if pollen discounting a ects the availability of pollen for outcross pollinations (de Jong et al., 1993; Holsinger & Thomson, 1994). With an overall level of late-acting inbreeding depression of 34±48%, the life-time inbreeding depression of E. vulgare is close to or even exceeds 50%, and this preliminary estimate suggests that outcrossing should be favoured. As the sel ng rate of E. vulgare under natural conditions is between 0 and 30% (Rademaker, 1998), the species would be considered by Husband & Schemske (1996) as an outcrossing species (sel ng rate <45%). The considerable inbreeding depression of over 30% in the reproductive stage in E. vulgare supports the hypothesis that, with a low sel ng rate, considerable inbreeding depression remains. Nearly half of the 40 outcrossing species analysed by Husband & Schemske (1996) showed inbreeding depression during growth and female reproduction (i.e. number of owers and number of seeds) of the o spring. Although the number of di erent individual parents in this experiment was limited to ve, the seed material was collected from those which showed the most extreme variation in number of seeds from sel ng vs. outcrossing in earlier pollination experiments (Melser et al., 1997), reducing parent sampling error (Lynch, 1988). Our experiment conforms to the fraction of onethird of the plants producing more seeds after sel ng compared to outcrossing in the parental generation. Also, in our experiment the average number of seed per ower after self-pollination in the parental generation is equal to that after cross-pollination. Strikingly, in this experiment, the parental combination (j j) produced relatively more seeds per ower after self-pollinations than after cross-pollinations, and parental combinations (a a) and (b b) produced relatively fewer seeds per ower after self-pollinations. The parental generation of j, a and b in Melser et al. (1997) has similar di erences in seed production between self- and cross-pollination, revealing a heritable component for the production of seeds per ower. The inbreeding depression in both male and female tness observed in this study suggests that this phenomenon occurs in both genders, and is not correlated between genders. Estimates of late-acting inbreeding depression, accounting only for the female function, cannot therefore legitimately be extrapolated to overall late-acting inbreeding depression. 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