Factors Affecting Intrafruit Pattern of Ovule Abortion and Seed Production in Hormathophylla Spinosa

Plant Syst. Evol. 239: 215–229 (2003) DOI 10.1007/s00606-003-0009-y

Factors aﬀecting intrafruit pattern of ovule abortion and seed production in Hormathophylla spinosa (Cruciferae)

J. M. Go´mez and R. Zamora

Departamento de Biologı´a Animal y Ecologı´a, Facultad de Ciencias, Universidad de Granada, Granada, Spain

Received April 29, 2002; accepted January 30, 2003 Published online: July 31, 2003 Springer-Verlag 2003

Abstract. We investigated the pattern of intrafruit Key words: Cruciferae, Hormathophylla spinosa, seed production in Hormathophylla spinosa (Cru- intrafruit seed position, ovule abortion, seed- ciferae) over a 7-yr period. H. spinosa fruit have ovule ratio, sibling rivalry. two chambers, each chamber containing two ovules, and usually situate perpendicular to the infructescence axis with one chamber above the other. The percentage of ovules ripening to seeds Introduction is usually lower than 50%. In addition, we found Only a fraction of the ovules per fruit become a consistent position-dependence in seed ripening seeds in the most plant species (Casper and probability, since more than 90% of the ripe Wiens 1981; Casper 1984, 1988; Lee 1988; seeds are arranged in the lower chamber of the Stephenson 1992). Several main non-exclusive fruit. This pattern of seed production is not produced by the direct or indirect effect of seed predispersal factors account for most of this predators, by pollen limitation or by nonrandom loss in reproductive potential, outstanding the fertilizations. By contrast, fruit removal experi- effect of pollen, resource, seed predators and ments showed that sibling rivalry can partially fruit architecture (Willson and Burley 1983; explain the seed production pattern, be the cause Haig and Westoby 1988; Lee 1988; Charles- of the reduction in the seed number from the worth 1989; Diggle 1995, 1997). uppermost threshold of 50%. Moreover, experi- Pollen availability (‘‘pollen limitation’’) or mental manipulations of fruit orientation suggest incompatibility (‘‘self-incompatibility system’’) that the position-dependence in seed production cause many ovules remain unfertilized in is the cause but not the effect of seed ripening. several plant species, decreasing seed number We presume that some architectural effect is per fruit (Haig and Westoby 1988, Barrett producing a significant increase in the ripening 1988a, Vander Kloet 1991, Stephenson 1992, probability of the ovules arranged in the lower chamber, causing thus the observed pattern in the Burd 1994). Furthermore, fertilized ovules can intrafruit seed production in H. spinosa and abort during development because of inbreed- severely constraining the maximum number of ing depression (Wiens et al. 1989, Husband and seeds per fruit to two. Schemske 1996), which is specially harmful in 216 J. M. Go´mez and R. Zamora: Intrafruit pattern of seed production in Hormathophylla spinosa selfing species with a mixed mating system, due This plurality of causes governing seed to the random joining of genetically similar number per fruit dramatically difficults the gametes after endogamous pollination which analysis of the exact mechanisms shaping seed causes the expression of deleterious recessive production in many plant species, and claims alleles, which has been described as ‘‘muta- for integrative studies which consider the tional load’’ (Charlesworth and Charlesworth combination of several factors working at the 1987; Charlesworth 1989; Wiens et al. 1987, same time (Marshall and Folsom 1992, Camp- 1989; Fuss and Sedgley 1991; Vander Kloet bell and Halama 1993). Despite of this, most 1991; Byers 1995; Rigney 1995). Inbreeding studies have assumed that each limiting factor depression, however, can also occur in out- usually acts independently on seed production crossing species, if there is a high probability of (Casper and Niesenbaum 1993). In the present fertilization by mates genetically more similar study, we investigate the potential causes of the than expected by random (Barrett 1998b). observed intrafruit patterns of seed produc- Resource limitation, whether nutrients or tion in Hormathophylla spinosa (L.) Ku¨ pfer water, contributes to the loss of some ovules (Cruciferae), a Mediterranean high-mountain per fruit both directly by decreasing the ability stunted shrub. H. spinosa bears many (up to of fertilized ovules to ripe to seed (Bierzych- 8000) small (2–3 mm diameter), white to pink, udek 1981, McDade 1983, Willson and Burley actinomorphic flowers consistently displaying 1983, Gorchov and Estabrook 1987, Lee 1988, four ovules (Go´mez and Zamora 1992). These Stephenson 1992), as well as indirectly by flowers are visited by more than 70 species of increasing the competition between the embry- insects belonging to 16 families and five orders, os arranged in the same ovary, which has been though the ant Proformica longiseta is the described as ‘‘sibling rivalry’’ (Kress 1981; predominant pollinator (Go´mez and Zamora Uma Shaanker et al. 1988; Ganeshaiah and 1992, 1999). In addition, this plant species is Uma Shaanker 1988; Arathi et al. 1996, 1999; also pollinated by wind in high-altitude pop- Cheplick 1996). Moreover, not only number ulations (Go´mez and Zamora 1996). H. spin- but also pattern of intrafruit seed production osa appears to be self-incompatible when are usually affected by resources, since severe pollinated with pollen coming from the same stress can encourage seeds to ripen in positions flower, and does not produce seeds by spon- offering greater access to resources (Lee 1988, taneous autogamy, in spite of more than 10% Ehrleń 1992, Ho 1992). Similarly, seed preda- of the flowers have pollen grains on the stigma tors reduced seed number per fruit in many before opening (Go´mez and Zamora 1996). plant species directly by consuming ripe seeds, Nevertheless, this crucifer can produce some and indirectly by increasing the probability of seeds by geitonogamy, although experimental abortion of the seeds arranged in the same geitonogamous hand-pollinations produce a fruit than the attacked ones (Ellison and significantly lower amount of seeds per fruit Thompson 1987, Anderssen 1988). Finally, than that of xenogamous pollinations (Go´mez according to the ‘‘architectural effects hypoth- and Zamora 1996). Although its flowers can esis’’, the pattern of seed production can also produce up to 4 seeds, they usually set 2 ripe be provoked in some plants by intrinsic seeds (Go´mez and Zamora 1992). This occurs factors limiting the ripening of ovules located despite the seed production per fruit is not in some positions (Diggle 1995). The proxi- pollen limited in this crucifer (Go´mez and mate causes of these architectural effects are Zamora 1996), and it is not affected by selective still unknown (Diggle 1997), although cum- exclusions of main pollinators (Go´mez and mulating evidence is showing that it can have Zamora 1992). The specific objectives of this important effect on the observed pattern of study are to 1) describe the intrafruit pattern of seed production (Medrano et al. 2000 and seed production, comparing the level of ovule references therein). fertilization with the level of seed maturation J. M. Go´mez and R. Zamora: Intrafruit pattern of seed production in Hormathophylla spinosa 217 and quantifying the level of seed abortion per chamber, as well as a left and a right placenta fruit, 2) quantify the temporal (3 years) and (Fig. 1). spatial (3 populations, 80 plants) variability in After pollination, the ovules can be fertilized this reproductive component, and 3) experi- setting ripe seeds, fertilized but aborting during mentally investigate the potential causes pro- development or remain unfertilized. A ripe seed is voking this observed pattern in seed uniformily brown and the embryos and full cotyledons are totally developed. On the contrary, an production. aborted seed is invariably dark brown, with shrivelled cotyledons and embryo. The size of Materials and methods aborted seeds was highly variable, depending on H. spinosa fruit structure. The H. spinosa fruit has the moment of the development occurring the two carpels and two placentas, but is secondarily abortion. Ovules aborted immediately after fertil- divided into two chambers by means of a thin ization (Type I Aborted Seed) are much smaller membrane called the septum (see Hill and Lord than ovules aborted later in the development (Type 1987), each chamber containing two ovules. Thus, II Aborted Seed). Finally, virgin ovules are consis- in each chamber, there is one ovule belonging to tently the same creamy white color, lanceolate in one of the two placentas. The four ovules are shape and of a far smaller size than aborted or ripe symmetrically arranged at the same distance from seeds. the style and the stigma, as well as from the Ripe seeds are depredated within the fruit by a vascular tissue that feeds the fruit. However, when weevil seed predator, Ceutorhynchus sp. nov. (Cur- beginning to ripe, fruit orientation shifts to situate culionidae), which completes its development with- perpendicular to the infructescence axis and with in the fruit (Go´mez and Zamora 1994). one chamber above the other, maintaining both Study area and plant populations. The study placentas at the same distance from the ground. was carried out in the Sierra Nevada (Granada Thus, in a ripe fruit, there is an upper and a lower province, SE Spain). We selected three populations

Fig. 1. Schematic representation of a H. spinosa fruit. A Longitudinal view showing a chamber with two seeds and the screem dividing the two chambers (septum). B Transversal view showing the two chambers, the septum dividing them, and one aborted seed in the upper chamber 218 J. M. Go´mez and R. Zamora: Intrafruit pattern of seed production in Hormathophylla spinosa of H. spinosa above the timberline, situated at 2160 number of ovules per fruit is invariably four in m (Population A), 2550 m (Population B) and 3130 H. spinosa, the number of fertilized but aborted m (Population C). These populations were separate ovules per fruit, if the mutational load hypothesis from each other by more than 5 km, and each is true, must fit a binomial distribution of k ¼ 4 population comprised more than 100 reproductive with parameter p ¼ probability that a fertilized plants. At the beginning of the study, we tagged 40 ovule does not ripen. We have found this proba- reproductive plants chosen arbitrarily in popula- bility using a method similar to that of Gorchov tion A, and 20 plants in the remaining plant and Estabrook (1987) and Rocha and Stephenson populations. (1990), according to which this probability is the Natural pattern of within-fruit seed produc- ratio of the total number of ripened ovules (that is, tion. The proportion of ovules developing into number of seeds) to the number of total fertilized seeds in each mature fruit (‘‘SO ratio’’ hereafter), ovules. Thus, using as a denominator of that ratio and the position of the seeds within the fruit, were the number of fertilized ovules instead of the total examined in 1754 fruits during 1989, in 1989 fruits number of ovules per fruit, we avoided the noise during 1990 and in 1906 fruits during 1991. Fruits caused by other factors acting during other phases were collected from the 80 tagged plants. Each fruit of the reproductive cycle of the plant which could was opened in the laboratory to count, under a interact with mutational load. Because every plant binocular microscope (16x), the number of fully is genetically unique, we have used a different p for developed intact seeds, the number of seeds depre- each of the 80 plants (Herrera 1990). Finally, dated by Ceutorhynchus, the number of fertilized we fitted the observed distribution of aborted but aborted ovules and the remaining number of seeds/fruit for each plant with a simulated theo- virgin ovules. The SO ratio was calculated pooling retical distribution (n ¼ 100 fruit), using as p those intact plus depredated seeds, to eliminate the effect found for that particular plant. Plants with less of the weevil seed predators. than 30 fruit examined were omitted from the The position of 2131 seeds belonging to 1906 analyses. fruits collected from the 80 tagged plants was Effect of weevil predation. To experimentally analysed in 1991, noting whether they were in the test whether the seed predation by Ceuthorhynchus upper or lower chamber and in the right or left affected the probability of an ovule to abort in the placenta. The affinity of a seed to ripen within a same fruit, weevils were excluded in 1991 from 10 chamber already containing another seed was inflorescences in each of 7 plants, by covering the studied in 1990 (533 fruits) and 1991 (621 fruits) flowers with a screen and applying glue to the stem by noting, in fruits with two seeds, whether these (Tanglefoot ). In addition, another 10 inflores- seeds were together or not. We have used exclu- cences in each of 44 plants were left as control. At sively two-seeded fruits to enable all possible the end of ripening, we collected the exclusion (130 spatial combinations of the seeds (Andersson fruits) and control fruits selecting in the latter those 1990). Fruits with more than two seeds, due to depredated by weevils (254 fruits). We quantified their structure (Fig. 1), always had seeds in both the number of fertilized ovules per fruit, the chambers. In addition, we contrasted the size of the proportion of ovules which aborted or ripened, seeds growing alone in a chamber or growing with the proportion of ripe seeds attacked by curculio- a sibling seed. nids and the number of intact seeds per fruit in each Effect of mutational load. In 1991, we tested treatment. whether ovule abortion and the resulting SO ratio Effect of water limitation. To determine the in the 80 permanently labelled H. spinosa individ- impact of drought on SO ratio, we experimentally uals is produced by mutational load. According to watered in 1991 six plants living at 2550 m a.s.l. this hypothesis, because the seeds are determined every two days with two liters each (Experimental exclusively by the genotype of the zygote, the group). The watering begun before plant flowering number of seeds aborted per fruit should be and ended after every plant had fully ripened its random, the failure of a fertilized ovules being fruits. Additionally, we tagged five plants located independent of the failure of other fertilized ovule in the same population and microhabitat as within the same fruit (Gorchov and Estabrook experimental plants, but not watered (Control 1987, Charlesworth 1989, Herrera 1990). Since the group). The SO ratio of fruits belonging to both J. M. Go´mez and R. Zamora: Intrafruit pattern of seed production in Hormathophylla spinosa 219 experimental (140 fruits) and control (394 fruits) unmanipulated as control. After rotation, both plants was analyzed in the laboratory. chambers were at the same distance from the Effect of flower and fruit removal. To study the ground and the placentas were one above the other. effect of competition between embryo arranged in Rotation was managed when flowers were already the same ovary on SO ratio, in 1995 we experi- pollinated, and was attained by bending the mentally removed sinks from inflorescences, and inflorescence stems by attaching them to the main quantified the SO ratio of remaining fruits (Med- branches of the shrubs. Fruit did not re-orientate rano et al. 2000). If resources are limiting for seed after rotation. In the second experiment (180 production and competition between embryo there- Rotation) carried on in 1993, we selected 10 plants fore influence SO ratio, we would expect the from population B and in each tagged 50 fruits, 25 remaining flowers to have a lower level of embryo artificially rotated 180 and the rest without abortion and to produce more seeds per flowers manipulation as control. After each rotation, we after removing competing sinks (Uma Shaanker changed the original position of the chambers, et al. 1988, Klinkhamer et al. 1994, Diggle 1997). moving the lower chamber to the upper part and In addition, to distinguish between sibling rivalry vice-versa. Placentas were left at the same distance and other potential hypothesis also explaining the from the ground. theoretical increases in SO ratio after sink removal Data analysis. SO ratio was contrasted be- (i.e. maternal control), we studied the effect of both tween populations and years by a repeated-mea- the time and intensity of reproductive structures sures ANOVA (Proc GLM, SAS 1997), since we removal on SO ratio and seed position. For this, we used the same 80 plants for the three years. In this tagged 12 inflorescences in each of 10 plants at 2550 model, we used the population as between-subject m a.s.l. We divided the inflorescences in two groups factor, year as within-subject factor and plants as of six each one. In one group, we removed flowers subjects. Differences in the distributions of ripe before fertilization (Flower Removal treatment), seeds and fertilized ovules per fruit were yearly whereas in the other group we removed fertilized analyzed by Kolmogorov-Smirnov tests, pooling fruits when they were still green and tiny (Fruit data from all plants and using a normal approx- Removal treatment). In each group, to analyse the imation (Z statistic). effect of removal intensity, we removed 20% of the The effect of the position in the probability of flowers or fruits in each of two inflorescences (Low maturation was analyzed using several techniques. Intensity treatment), 50% in other two inflores- We contrasted the observed distribution of the cences per group (Medium Intensity treatment), seeds sampled in 1991 with an expected distribution and 70% in the remaining two (High Intensity assuming no selection by chamber (whether upper treatment). In addition, we tagged another four or lower) or placenta (whether left or right), and inflorescences per plant as control. In total, we thus built considering that the probability of a seed analysed 68 fruits belonging to High intensity to be arranged in either of the two chambers or treatment, 111 belonging to Medium Intensity, placentas is 0.5. The observed and expected distri- 137 belonging to Low Intensity and 202 belonging butions for chambers and placentas were contrasted to control. by a multivariate analysis of contingency with Experimental manipulation of fruit orienta- interaction term (Proc CATMOD, SAS 1997), tion. To further study any putative architectural testing each factor by means of a Wald Chi square effect of fruit on the SO ratio and intra-fruit seed (Rocha and Stephenson 1990). position, we experimentally manipulated the orien- The affinity of a seed to ripen within a chamber tation of H. spinosa fruits. The experiments were already containing another seed was studied by carried out at the beginning of the fruiting period, contrasting the observed distributions of seeds in when fertilized ovules were still undeveloped, but the same chamber with a theoretical, Bernoulli when the fertilization period had finished and the distribution with p ¼ 0.33. According to the null green fruit started to rotate. hypothesis of no site selection, the probability of In the first experiment (90 Rotation) carried finding two seeds in the same chamber is 2/6. To on 1992, we selected 7 plants in population B and calculate the significance, we used the chi-square tagged, in each plant, 150 fruits, 75 of which had test, with correction of continuity when sample size been artificially rotated 90 and the rest left was greater than 400 (Proc CATMOD). In 1991 we 220 J. M. Go´mez and R. Zamora: Intrafruit pattern of seed production in Hormathophylla spinosa also studied the effect of the placenta, obtaining the fertilized ovules. However, only 238 (2.63%) frequency of fruits with both seeds in the same or fruit had more than two ripe seeds (whether different placenta. Using the same procedure, we intact or depredated), and thus a consistent contrasted, by a chi-square the observed distribu- significant difference appeared every year be- tion with an expected distribution with p ¼ 0.33. To tween the frequency distribution of the number differentiate the effect of the placenta from the of fertilized ovules and the number of seeds per effect of the chamber, we selected in 1991 all the fruit (1989: Z 12.31, n 1082 fruits; 1990: fruits with the two seeds in different chambers ¼ ¼ (n ¼ 88 fruits), thereby fixing the position of the Z ¼ 15.61, n ¼ 1444 fruits; 1991: Z ¼ 14.51, seed with respect to the chamber, and using the n ¼ 1906 fruits; P<0.0001 all years, Kolmogo- placenta as the only factor of change. In this rov-Smirnov test). This difference was exclu- particular case, with no placenta selection, the sively due to the abortion of the remaining percentage of fruits with the seeds in the same or a fertilized ovules, since the number of seeds different placenta was 50% in both cases. Thus, by preyed upon by weevils have been included a chi-square, we contrasted the observed distribu- within the category of ripe seeds. Consequently, tion with an expected one with p ¼ 0.5 (Proc SO ratio was low, 32.3% ± 2.0, averaging the CATMOD). Analogously, we selected all fruits three populations and years. with the seeds arranged in different placentas There was significant inter-annual differ- (n 573 fruits) and followed the same procedure ¼ ence in SO ratio (F ¼ 5.26, P ¼ 0.02, rm- as before, contrasting the observed frequency of 1,70 ANOVA), being 30.4±0.1% (n 1754 fruits) fruits with the seeds in the same or a different ¼ chamber with an expected distribution with p ¼ 0.5. in 1989, 36.3±0.1% (n ¼ 1989 fruits) in 1990 The effects of water addition and weevil and 30.3±0.1% (n ¼ 1906 fruits) in 1991. exclusion on SO ratio were tested by nested There was also significant between-population ANOVAs (Proc GLM), since different plants were differences in SO ratio (F2,70 ¼ 3.33, P ¼ 0.04). used per treatment. In these analyses plants were Plants from populations B (SO ra- nested within treatment. The effect of fruit rotation tio ¼ 30.5±0.1%) and A (29.6±0.1%) during and flower/fruit removal on seed number, was the three years had an SO ratio significantly tested by two-way mixed model ANOVAs, with lower than plants from the population C a-posteriori pair-wise comparison (Tukey-Kramer’s (40.3±0.1%; P<0.05 all years, Tukey-Kramer t-test). t-test). No plant during the three years had a Due to the unbalanced data, to generate the SO ratio greater than 70%. F-ratio values of the fixed factors we used the More than 88% of the 2131 seeds analyzed denominator MS synthesis according to the model II provided by SAS. The effect of the factors on the in 1991 ripened in the lower chamber of the position of the seed within the fruit, since the fruits, reveling a strong significant difference dependent variable is in this case categorical, has between the observed frequency of the seeds been analysed by a logit model (Proc. CATMOD), arranged in the lower chamber and the expected using a Wald Chi-square (Rocha and Stephenson frequency (Multivariate Wald v2 ¼ 11.65, P ¼ 1990). 0.0006). On the contrary, 53.7% of the seeds When more than one comparison was made were arranged in the left placenta and 46.35% using the same analytical model, the sequential in the right placenta of the studied fruits, Bonferroni technique was used to select the critical with no effect of the placenta (Wald v2 ¼ probability level (Rice 1989). All means are 1 SE. 0.83, P ¼ 0.36) or the interaction between placenta and chamber (Wald v2 ¼ 0.74, P ¼ 0.39). Results 77.2% of the two-selected fruits analyzed Pattern of within-fruit seed production. 99.62% in 1990 and 85.8% in 1991 had both seeds of the fruits analysed during the three years located in the same chamber. Thus, there was a (n ¼ 5649 fruits) had at least one fertilized significant difference between observed and ovule, and 55% of the fruits had 3 or 4 expected distributions of fruits with two seeds J. M. Go´mez and R. Zamora: Intrafruit pattern of seed production in Hormathophylla spinosa 221 in the same chamber (1990: v2 ¼ 207.84, percentage of aborting ovules per fruit was P<0.0001; 1991: v2 ¼ 355.32, P<0.0001). Up similar in fruits excluded from weevil seed to 98% of the fruits with the seeds in the same predators (34.87 ± 6.85%) than in control chamber had the seeds located in the lower fruits (39.16 ± 2.61%; F1,240=1.69, P ¼ 0.20). chamber. There were also significant differences The correlations between seed aborted per fruit between the observed and expected distribu- and weevil abundance per fruit in permanently tions of the seeds with respect to the placenta labelled plants were in all cases non-significant (v2 ¼ 138.95, P<0.0001), since only 6.4% of (P>0.1 for all year x population combinations, the seeds were arranged in the same placenta. Spearman rank correlations). However, after adjusting each factor, only the Water limitation. Although the whole chamber significantly affected to the position model of the experiment was significant 2 of the seeds (v ¼ 259.65, P<0.0001), since (F11,68 ¼ 3.56, P ¼ 0.0001), water did not in- 91.6% of the fruits with two seeds in different crease the SO ratio (F1,68 ¼ 0.08, P ¼ 0.78), placentas (581 fruits) had the seeds in the same being similar in watered (28.9±7.9) and con- chamber. On the contrary, of the 88 fruits with trol plants (30.0±9.1). two seeds in different chambers, 55.7% had the Flower and fruit removal. Removal date seeds in different placentas, with no difference (F1,501 ¼ 19.82, P<0.0001), but not removal between the observed and expected distribu- intensity (F2,501 ¼ 0.01, P ¼ 0.47), affected SO tions (v2 ¼ 0.57, P>0.1). Seeds growing alone ratio. Fruits belonging to fruit-removal treat- (2.73±0.04 mm in length, n ¼ 69) were similar ment (40.94±0.02) had a significantly higher in size as seeds sharing a chamber (2.75±0.02 SO ratio than fruits belonging to either control mm, n ¼ 390; F1,457 ¼ 0.22, P ¼ 0.64, one-way (34.39±0.03) and flower-removal treatment ANOVA). (29.72±0.02, all comparisons P<0.001 ac- Mutational load. The observed distribu- cording to the Bonferroni-Dunn post-hoc test). tions of the number of aborted seed per fruit in However, the interaction term was also signif- each plant were not consistent with the muta- icant (F2,501 ¼ 7.28, P ¼ 0.001), since as ob- tional load in most plants, since the observed served in Fig 2, SO ratio was significantly distributions significantly departed from the higher in fruits from medium- and high- expected distributions (Table 1). There was a intensity in fruit-removal treatment than in pattern in this difference, with the probability control. of abortion of two or more fertilized ovules per The proportion of seeds filled either in the fruit being much lower than expected accord- lower or in the upper chamber did not vary ing to theoretical binomials. That is, the depending on removal intensity (v2 ¼ 1.55, probability that a fertilized ovule would abort P ¼ 0.67), but did vary depending on removal was not independent of the abortion of other date (v2 ¼ 11.56, P ¼ 0.003). Thus, the percent- fertilized ovules in the same fruit, and age of seeds ripening in the lower chamber was decreased with the number of ovules already 87.5% in control fruits, 78.6% in fruits aborted. Nevertheless, there were between- belonging to flower-removal treatment and population differences in the potential effect 93.0% in fruits belonging to fruit-removal of the mutational load on the abortion of treatment. Lastly, there were no differences fertilized ovules, since 67% of the plants in depending on either removal intensity population C (from C1 to C20 in Table 1) fit (v2 ¼ 3.91, P ¼ 0.27) or date (v2 ¼ 4.16, the expected binomials whereas 10% of the P ¼ 0.12) in the seed position when there were plants fitted the expected distributions in the only two seeds per fruit. In every case, more remaining two populations (Table 1). than 70% of two-seeded fruits had both seeds Effect of weevil predation. The whole model ripening together in the same chamber. of weevil exclusion experiment was significant Experimental manipulation of fruit orienta- (F5,240 ¼ 2.30, P ¼ 0.0001). However, the tion. The number of seeds did not vary 222 J. M. Go´mez and R. Zamora: Intrafruit pattern of seed production in Hormathophylla spinosa

Table 1. Frequency distributions of the number of aborted seeds per fruit in each tagged plant with more than 30 analyzed fruits. Probability refers to the probability of a fertilized ovule does not ripe in that plant (see Methods) Plant Number Probability v2 Signiﬁcance Departure from expected n of aborted ovules of fruits per fruit

01234 ) A1 43 0.65 9.467 0.0500 + + + ) A2 52 0.88 24.808 0.0001 + + + + ) A3 41 0.73 14.987 0.0047 + + + )) A4 33 0.74 24.946 0.0001 + + ))) A5 35 0.44 20.593 0.0004 + + ))) A6 37 0.70 8.103 0.0879 A7 46 0.69 26.896 0.0001 + + + )) A8 53 0.62 38.522 0.0001 + + ))) A9 48 0.61 48.052 0.0001 + + ))) A10 39 0.68 14.391 0.0061 + + ))) A12 49 0.57 16.523 0.0024 + + ))) A13 49 0.55 9.294 0.0500 + + + )) A14 34 0.73 15.727 0.0034 + + + )) A15 46 0.66 7.088 0.1313 A16 45 0.57 14.881 0.0066 + + + )) A17 52 0.55 32.253 0.0001 + + ))) A18 40 0.67 10.820 0.0287 ) ++ )) A19 46 0.60 8.169 0.0856 A21 45 0.44 26.288 0.0001 + + ))) A22 44 0.62 29.257 0.0001 + + ))) A23 35 0.47 9.392 0.0500 + + + )) A24 41 0.67 15.734 0.0034 + + ))) A25 45 0.60 15.573 0.0036 + + + )) A26 41 0.58 8.097 0.0800 A27 48 0.42 17.421 0.0010 + )))) A28 37 0.54 10.048 0.0396 + + + ) + A29 39 0.52 8.955 0.0500 + + ))+ A30 45 0.55 12.329 0.0151 + + + )) A31 43 0.66 29.584 0.0001 + + ))) A32 37 0.52 10.787 0.0291 + + ))) A33 45 0.54 5.084 0.2788 A34 54 0.52 18.449 0.0010 + ) + )) A35 37 0.48 12.988 0.0113 + + ))) A36 42 0.73 23.331 0.0001 + + + )) A38 41 0.53 9.999 0.0404 + + ))) A39 39 0.49 19.223 0.0007 + + ))) A40 47 0.41 11.172 0.0247 + + ))) B1 35 0.50 6.756 0.1493 B3 53 0.49 18.846 0.0008 + + ))) B4 52 0.58 29.822 0.0001 + + ))) B5 44 0.56 19.005 0.0008 ) ++ )) B6 35 0.44 14.790 0.0052 + + ))) B7 39 0.58 25.035 0.0001 + + ))) J. M. Go´mez and R. Zamora: Intrafruit pattern of seed production in Hormathophylla spinosa 223

Table 1 (continued) Plant Number Probability v2 Signiﬁcance Departure from expected n of aborted ovules of fruits per fruit

01234 B8 39 0.35 10.793 0.029 + + ))) B10 45 0.97 99.155 0.0001 + + + )) B11 45 0.60 11.499 0.0215 + + + )) B12 46 0.53 11.219 0.0242 + + + )) B13 53 0.49 12.533 0.0138 + + ))) B14 46 0.39 11.777 0.0191 + + ))) B15 46 0.29 7.432 0.1147 B18 36 0.46 14.679 0.0054 + + ))) B19 50 0.48 16.609 0.0023 + + ))) B20 39 0.51 9.761 0.0446 + + + )) C1 34 0.45 4.617 0.3289 C2 51 0.29 7.961 0.0930 C5 31 0.65 9.599 0.0478 + + ))) C7 43 0.29 4.889 0.2989 C8 33 0.27 15.347 0.0040 + )))) C9 41 0.69 36.824 0.0001 + + + )) C10 39 0.36 9.288 0.0500 + )))) C11 40 0.23 3.054 0.3834 C12 41 0.44 6.925 0.1399 C13 53 0.36 5.622 0.2292 C14 49 0.30 9.095 0.0500 + )))) C15 47 0.24 4.024 0.2589 C16 43 0.41 6.082 0.1931 C17 49 0.51 17.102 0.0018 + + ))) C18 57 0.51 7.002 0.1358 C19 44 0.39 2.412 0.6604 C20 37 0.36 6.313 0.1769

between treatments in the 90 rotation exper- percentage of type I aborted seeds did not iment, the SO ratio being highly similar in differ between treatments (67.41±3.11 vs. rotated (34.7±0.7) and control fruits 63.56±2.37), the percentage of type II aborted (34.5±0.7, Table 2). This experiment did not seeds was higher in rotated fruits (8.30±2.42) affect, in two-seeded fruits, the position of the than in control fruits (2.43±0.65, Table 3). seeds with respect to the chamber (v2 ¼ 0.50, Most of the type II aborted seeds in both P ¼ 0.78), since 85.5% of the rotated fruits and treatments were arranged in the upper cham- 88.4% of the control fruits had both seeds ber. arranged in the same chamber. The 180 The 180 rotation experiment did alter the rotation experiment, however, did show be- proportion of seeds located in the upper or tween-treatment differences in the number lower seeds (v2 ¼ 9.3, P ¼ 0.002). Only 83.7% of seeds per fruit, with a significantly lower of the ovules which were fertilized in the lower SO ratio in rotated fruits (23.3±0.01) than chamber continued to ripe to seed in the upper in control ones (33.0±0.1; F19,373 ¼ 7.49, P< chamber after fruit rotation, whereas 94.5% of 0.001, Table 3). However, although the the fertilized seeds in the lower chamber set 224 J. M. Go´mez and R. Zamora: Intrafruit pattern of seed production in Hormathophylla spinosa

tation does not increase SO ratio in this plant species (Go´mez and Zamora 1996). It is also unlikely that late, postzygotic self-incompatibility be the cause of SO ratio reduction in H. spinosa, since, contrary to main predictions of this hypothesis (Seavey and Bawa 1986, Montalvo 1992, Stace 1995), ovules aborted at a variety of stage and some seeds are consistently produced per fruit. Moreover, our results suggest that ovule abortion was not provoked by inbreeding depression due to mutational load, since most plants did not fit distribution of aborted seeds expected by this hypothesis (see Table 1). In fact, xenogamous Fig. 2. Effect of fruit and flower removal in SO ratio hand-pollinated fruits, which have highly con- (means ± 1 SE) in Hormathophylla spinosa. Means with different letters are differents at a <0.05after trolled pollen quality, continued to produce pair-wise ANOVA comparisons and Bonferroni around two seeds (Go´mez and Zamora 1996), sequential corrections both generally arranged in the same chamber. Nonrandom fertilization can produce position-dependent maturation of seeds (Ste- seeds in control fruits ripened. Moreover, there phenson and Bertin 1983; Wiens et al. 1987; was a significant decrease in the percentage of Marshall and Ellstrand 1986, 1988; Marshall fruit containing two seeds in the same chamber 1991; Snow and Spira 1991a). However, (v2 ¼ 19.72, P ¼ 0.0001), since 89.4% of the H. spinosa fruits have the four ovules arranged control fruits but only 70.0% of the rotated in a square, at the same distance from the fruits had both seeds in the same chamber. stigma. This probably results in a random distribution of both male and female gameto- phytes, which highly preclude the probability Discussion of nonrandom fertilization (Andersson 1990). Our long-term study has shown that in all As a consequence, the position-dependent populations and years, the number of seeds per maturation observed in H. spinosa is not fruit does not overpass 50% of the available produced by selective abortion due to either ovules. In addition, there was an intense maternal, paternal or offspring control. In fact, position-dependent maturation of the seeds. most studies showing position-dependent mat- Around 90% of the seeds were arranged in the uration have been done with plant species lower chamber of the fruit. Similarly, more bearing elongated fruits, such as legumes or than 85% of the seeds of the two-seeded fruits siliques (e.g. Horovitz et al. 1976, Bawa and grew in the same chamber (the lower one). Webb 1984, Lee and Bazzaz 1986, Hill and Thus, although we performed the observations Lord 1987, Marshall and Ellstrand 1988, and experiments in different years, our results Rocha and Stephenson 1990, Marshall 1991, clearly showed a spatio-temporally consistent Snow and Spira 1991b, Marshall and Folsom reproductive pattern. 1992, Namai and Ohsawa 1992). Pollen limitation and fertilization ef- Effects of seed predators. Seed predators fects. SO ratio decreased in the H. spinosa by can sometimes have additional impacts on the ovule abortion, indicating that it was not SO ratio of some plant species by indirectly produced by pre-fertilization factors, as self- provoking the abortion of additional seeds incompatibility or pollen limitation. In fact, we within the same fruit (e.g. Andersen 1988). have previously shown that pollen supplemen- However, our results have experimentally J. M. Go´mez and R. Zamora: Intrafruit pattern of seed production in Hormathophylla spinosa 225

Table 2. Summaries of the mixed model ANOVAs on SO ratio (Proportion of ovules ripening to seed per fruit), and type I and II Aborted Seeds after rotating fruits 90 and 180. Plants (¼block) is a random factor. Df = degree of freedom, SS = sum of squares Experiment Source of variation df SS F P 90 rotation SO Treatment 1 0.00 0.07 0.799 Plant 6 1.96 52.42 0.0001 Plant · Treatment 6 0.04 0.34 0.916 Residuals 993 18.22 180 rotation SO ratio Treatment 1 0.21 6.33 0.029 Plant 9 0.03 6.62 0.0001 Plant · Treatment 9 0.06 4.76 0.0001 Residuals 373 4.14 Type I Aborted Seed Treatment 1 0.21 4.39 0.06 Plant 9 0.31 8.15 0.0001 Plant · Treatment 9 0.55 5.53 0.0001 Residuals 372 8.51 Type II Aborted Seed Treatment 1 0.09 3.28 0.09 Plant 9 1.67 3.15 0.0001 Plant · Treatment 9 0.26 1.28 0.246 Residuals 373 9.18 demonstrated that Ceuthorhynchus decreased Shaanker 1988, Cheplick 1996, Medrano et al. H. spinosa SO ratio only by direct consump- 2000, Go´mez and Fuentes 2001), and may be a tion of seeds, but not by affecting the abortion consequence of relaxation in the competition rate of the remaining ovules in the fruit between ovaries for maternal resources, sup- (Go´mez and Zamora 1994). Moreover, be- porting sibling rivalry as a mechanism con- cause of the lack of correlation between ovule trolling SO ratio in H. spinosa. However, this abortion per plant and decrease in seed attack increase in seed number per fruit could also be by weevils, ovule abortion cannot be consid- a mechanism actively used by the mother to ered as a maternal defense against seed preda- maintain the original seed production after tors. This is also suggested by the fact that high manipulations (Ehrleń 1992, Vaughton 1993). rates of ovule abortion occur yearly in popu- Three main factors, nevertheless, suggest that lation C, a site where no weevils are present. it was not due to maternal regulation. Firstly, Sibling rivalry. Our experiments suggest the effect of the experimental manipulation that sibling rivalry can partially explain SO was manifested only after fruit removal, when ratio in H. spinosa. Thus, when we removed seeds were developing and could compete with green fruits, the SO ratio increased in H. spin- their sibs, but not after flower removal. osa, and plants that we left only 50% and 20% Secondly, mother regulating after sink removal of the initial fruits significantly increased the should be affected by intensity of removal, number of seeds per fruit. An increase in seed since it would distribute the resources among production after sink removal has been the different number of flowers left per inflo- observed in other plant species (e.g. Uma rescences (the Integrated Physiological Units). Shaanker et al. 1988, Ganeshaiah and Uma Thirdly, the unability of the plants of taking 226 J. M. Go´mez and R. Zamora: Intrafruit pattern of seed production in Hormathophylla spinosa advantage of additional water by increasing now lower chamber. Secondly, most type II SO ratio suggests a poor capacity of short- aborted ovules in both control and experimen- term regulation of this reproductive compo- tal treatments in the rotation experiment were nent (Herrera 1990). in the upper chamber. Thirdly, a lower pro- Nevertheless, sibling rivalry cannot fully portion of the ovules arranged in the lower explain why, after 180 rotation of fruits, the chamber that were fertilized successfully set seeds started to abort in the now upper seed after rotating the fruits (83.7%) than in chamber and to ripen in the now lower control fruits (94.5%) in which these ovules chamber; in fact, according to this hypothesis, maintained in the original lower chamber. the spatial distribution of the chamber would That is, abortion rate quickly increases when be a consequence of the zygote development ovules are in the upper chamber, irrespective and not the contrary, as this active change in to that these ovules were originally arranged in maturation/abortion suggests. Nor this hy- the lower chamber. Fourthly, additional sup- pothesis can explain why after missing the port is provided by the fact that a significantly chamber effect in the observational analysis of higher proportion of two-seeded fruit (30%) the seed position, the probability of two seeds had both seeds arranged in different chambers maturing in the same or different placenta is after fruit rotation. This may be because seeds the same. In fact, according to the hypothesis, in the lower chamber after rotation (originally the ovules, regardless of being in the same or a in the upper chamber and consequently con- different chamber, should ripen in different demned to abortion) started to ripen; but placentas to avoid mutual inhibition, since, as because we rotated fruit after the seeds had pointed out by Wiens et al. (1987), the carpel is started to develop, only some seeds were presumably the unit of interovular competi- rescued. Unfortunately, the exact causal mech- tion. But the main problem for accepting the anisms producing this strong chamber-depen- hypothesis of sibling rivalry as the only factor dence in seed ripening probability, which can affecting H. spinosa SO ratio is that it is unable successfully explain both the SO ratio and the to explain why neither the experimental flower intrafruit pattern of seed production in and fruit removal increased the number of H. spinosa, are still unknown. We think that seeds per fruit to higher than 2 (SO ratio this strong chamber-dependent seed ripening >50%), and why the zygotes consistently probability might be considered an architec- developed in the lower chamber. It seems that tural effect (sensu Diggle 1995, 1997). the uppermost number of seeds produced per fruit appears to be firmly fixed at about two, JoseÁntonio Ho´dar and Daniel Garcıá helped and when sibling rivalry occurs, it could only us during the field work. 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