Selective seed abortion
and offspring quality
Melser, Chantal
Selective seed abortion and offspring survival Proefschrift Universiteit Leiden
Cover illustration: Frank van der Meer Graphics: Martin Brittijn
Printing: Haveka, Alblasserdam
Selective seed abortion and offspring quality
PROEFSCHRIFT
ter verkrijging van
de graad van Doctor aan de Universiteit te Leiden, op gezag van de Rector Magnificus Dr. D.D. Breimer,
hoogleraar in de faculteit der Wiskunde en Natuurwetenschappen en die der Geneeskunde, volgens besluit van het College voor Promoties
te verdedigen op dinsdag 4 december 2001 te klokke 14.15 uur.
door
Chantal Melser
geboren te Eindhoven in 1968
Promotiecommissie
Promotor: Prof. dr. E. van der Meijden Copromotor: dr. P.G.L. Klinkhamer
Referent: dr. P.H. van Tienderen (Universiteit Utrecht) Overige leden: Dr. R. Bijlsma (Universiteit Groningen)
Prof. dr. P.M. Brakefield
Mw. Prof. D. Charlesworth (University of Edinburgh) Prof. dr. R. Hoekstra (Universiteit Wageningen)
Chapter 1 General Introduction. ... 9
Chapter 2 Embryo abortion in a natural population of Cynoglossum officinale (Boraginaceae). ... 23
Chapter 3 Selective seed abortion increases offspring survival in Cynoglossum officinale (Boraginaceae). ... 41
2001 American Journal of Botany: in press Kuenenprijs 2001. Chapter 4 Selection on pollen donors by Echium vulgare (Boraginaceae) ... 57
1997 Sexual Plant Reproduction 10: 305-312 Chapter 5 Late-acting inbreeding depression in both male and female function of Echium vulgare (Boraginaceae)... 73
1999 Heredity 83: 162-170 Chapter 6 Embryo selection, abortion and inbreeding depression. I. Cynoglossum officinale (Boraginaceae). ... 87
Chapter 7 Embryo selection, abortion and inbreeding depression. II. Echium vulgare (Boraginaceae). ... 107
Chapter 8 Predicting the optimal seed number per flower for two species with effects of sex allocation and selective seed abortion. ... 131
Chapter 9 General summary and discussion... 151
References ... 163
Nederlandse samenvatting ... 171
Nawoord... 179
Curriculum vitae ... 181
Publicatielijst ... 183
1
General Introduction
Selection among offspring
Mate choice
Already long ago, Darwin (1871) distinguished two different aspects of sexual selection:
1) sexual competition among the members of one sex, usually male, to mate with individuals of the other sex, usually female, and 2) preferences of one sex, usually female, for particular mates. Bateman (1948) noted that sexual selection might also occur in plants and thereby generalized the issue. Mate choice can be effective before and after fertilization. Mate choice after fertilization is commonly known as selective embryo abortion. Willson and Burley (1983) hypothesized that mate choice by abortion both in animals and plants can cause evolutionary changes when a primary effect of post-mating destruction of potential offspring by a female initiates adaptations by males to avoid such abortion.
Animals
Selection among mates in animals is well known. In animals, the most obvious mate choice occurs before fertilization. Mate choice leads to a selection for preferred characteristics and the evolutionary outcome of this selection is visible by differences in morphology or behavior of the male animals. For pre-copulatory selection of potential mates, the organisms should perceive a signal about the quality of the candidates before copulation. One of the most appa- rent morphological examples is the excessive ornamentation in birds. The choosing sex might take the trait under selection as an honest signal for the genetic quality of the potential mate.
However, as Fisher pointed out (1958), in a "runaway process" the trait might be exaggerated by evolution of selection on the trait itself, and might not be an honest signal of genetic quality anymore. Although, one can also argue that exaggerating a costly trait, still gives a reliable estimation of the potential mate to cope with extra difficult conditions (=conditional handicap). Mate choice in animals before fertilization is not always based on morphology, but might also occur on the basis of behavior of the competing potential mates. An example of behavior that was studied recently is the drumming wolf spider, in which females prefer males with a high drumming rate (Mappes et al. 1996). Males vary in their ability to invest in drumming, which suggests that drumming is an honest signal of male quality.
Selection among mates in animals may also be delayed and can occur after copulation.
For several species it is suggested that sperm from different mates competes for fertilization.
This sperm competition seems be enhanced by the behavior of the female animals. Adders (Vipera berus) copulate frequently, while this high frequency does not increase litter size (Madsen et al. 1992). This suggests that the female adder benefits from multiple copulations through the genetic quality of her offspring. In Swedish sand lizards (Lacerta agilis), usually females mate with more than one male (Olsson et al. 1996a). The reproductive output of the female lizards is not enhanced by additional matings, and more closely related males sire fewer offspring per copulation than do more distantly related males (Olsson et al. 1996a). In these sand lizards, brother-sister matings have reduced offspring viability (Olsson et al.
1996b). These data suggest that female lizards are capable of subtle discrimination among potential mates after copulation and increase their fitness in this way. Baker and Bellis (1993) found in humans that in periods of infidelity, females change their orgasm patterns and favor
the sperm of the extra-pair male in this way, presumably raising his chances of success in sperm competition with the female's partner. Selection among offspring may also occur post- zygotically, i.e. after fertilization. With recent techniques, genetic deficiencies in the human fetus are detected post-zygotically and in many countries parents can choose to abort a seriously injured fetus. Moreover, recent evidence from East Asia suggest that human parents use prenatal sex testing to abort fetuses selectively because they are of the female sex (Goodkind 1996). In those regions, daughters are more costly than sons are and the benefits for the parents are lower. The traditionally postnatal discrimination against young daughters is likely to lower the quality of female offspring compared to male offspring.
Plants
Hypothesis of selective seed abortion
At first sight, plants are unable to choose among potential mates because of their sessile life form and limited possibilities of behavior. Pollination is established by external agents as wind or animals and is hardly under control of the plants involved. Although, with the protan- drous development of flowers (the female and male function of the flower is separated in time and the stigma becomes receptive only after the ripening of the pollen) self-pollination within one flower is avoided as occurs e.g. in Plantago (Bos et al. 1995). A more subtle discrimi- nation among potential mates in pollination is not feasible by morphology or development of the flowers. Selection among potential mates can thus only occur after pollination, either pre- or post-zygotically. Females are likely to evaluate certain aspects of quality of male gametes from various sources. Pre-zygotic selection among mates occurs when there are differences in speed or success of pollen germination, or in competition among the pollen tubes when they grow down the stigma to reach the ovary in order to establish the fertilization. Post-zygotic selection occurs by abortion of a part of the embryos after fertilization. In contrast to pre- zygotic selection, abortion of embryos lowers the average number of seeds per flower. The costs of abortion and the necessary additional flower production can be balanced by a higher average quality of the offspring and increased male fitness through the extra flowers.
The hypothesis of selective seed abortion was firstly mentioned explicitly by Darwin (1883) and received a considerable amount of attention in the last quarter of the 20th century (e.g. Janzen 1977, Willson 1979, Stephenson 1981, Stephenson & Bertin 1983, Willson &
Burley 1983, Lee 1988, Marshall & Ellstrand 1988, Marshall & Folsom 1991). Selective abortion of embryos of relatively low quality later in life can free resources for higher quality offspring and thereby increase the fitness of the maternal plant. This effect is expected to be most distinct if resources for producing seeds are limited. This hypothesis assumes a negative relationship between the probability for offspring of being aborted and their potential fitness later in life. The genes that are deleterious to the embryo are assumed to be related to a low quality of the offspring later in life as well (Mulcahy 1979). However, there should be a distinction between the abortion of offspring that carries lethal alleles and is unviable under any circumstance, and the selective abortion of viable embryos. With aborting offspring with lethal alleles, there is no female control on the process. Only if embryos are potentially viable, the mate choice of the maternal plant can affect her fitness. Such offspring selection may act between self- and cross-pollination, among different outcross pollen donors, or within off-
spring of a single pollen donor. At the beginning of the nineties, the stream of publications about this subject ceased. Recently, the subject of mate choice in plants received renewed attention because of the applications of molecular techniques for paternity analyses. Pollen can be applied in a mixture on the stigma, allowing to trace the most successful pollen donor by analyzing the paternity of the resulting offspring. Moreover, selection among offspring from one single pollen donor can be detected. In this thesis we will describe a study on selection between different pollen donors and the effect of selective seed abortion on the fitness of the maternal plant.
Possible regulatory mechanisms of seed abortion
Different mechanisms may lead to selective seed abortion (Lee 1988). These mechanisms will not be investigated in this thesis. However, in this introduction I want to provide background information of the possible mechanisms. There are two ways in which control of abortion can be established, either developmental or hormonal.
Developmental regulation
One of the possible mechanisms of embryo abortion deals with the morphology and develop- ment of the growing embryo with its nourishing veins of the maternal tissue. A fast growing embryo attracts many resources, while a slow growing embryo attracts fewer resources (Lee 1988). The veins that supply the nourishment to a fast growing embryo will transport large amounts of resources. This might extend the diameter of the veins, which makes the transport of the nourishment easier and fast (pers. comm. P. Wolswinkel). The veins that supply a slow growing embryo with resources transport less and will not extend their diameter. Through smaller veins fewer resources can flow. For both fast and slow growing embryos this is a self- reinforcing process. At a certain moment the slow growing embryos will starve.
The hypothesis that abortion is physiologically caused by starvation of the less compe- titive embryos, assumes that embryos are competing for a resource. However, the integrated physiological unit of a plant, that acts relatively autonomous with respect to the assimilation, distribution and utilization of e.g. carbon, varies greatly among species (Westoby & Rice 1981, Watson & Casper 1984). Every flower might receive carbon from its neighboring axillary leaf and be relatively independent of other carbon sources within the plant. If flowers are independent of each other for their supplies, this will limit the competition among em- bryos on different flowers. Still, even if carbon is distributed locally rather than over the whole plant, nutrients have to be taken up by the roots and transported to all growing organs of a plant. Embryos may then compete strongly for a sufficient supply of nutrients.
Hormonal regulation
Another possible physiological cause of abortion is the increase or decrease of different hormone levels in parts of the plants (Lee 1988). Plant hormones play an important role in fruit maturation (Swain et al. 1993) and leaf senescence (Sagee et al. 1980, Osborne 1989, Engvild 1989). It has been suggested that these processes are related to the abortion of seeds (Swain et al. 1993). The study of Mohan Raju et al. (1996) on Dalbergia sissoo and the study of Krishnamurthy et al. (1997) on Syzygium cuminii suggest that maturing fruits/seeds excrete a substance that promotes abortion of younger fruits/seeds. When analyzed with a HPLC, this
substance seems in both species to be closely related to the plant hormone indoleacetic acid.
Indoleacetic acid is known to be among the major growth metabolites produced during early seed and fruit development (Engvild 1987, 1989, Katayama et al. 1988) and to induce abor- tion of tissues to which it was applied (Nooden & Leopold 1978). Under the assumption that later initiated seeds are aborted by hormones that are excreted by earlier initiated seeds, there should be an advantage of earlier initiated seeds over late initiated seeds. This is a possible explanation for decreasing seed numbers along the season. If hormones function to establish abortion of seeds of lesser quality, then seeds with a higher potential total fitness should excrete more abortive hormones relative to the seeds of a lower quality or should be less affected by the general hormone level.
Maternal regulation versus embryonal regulation
Although a difficult subject for research because there are many confounding effects in- volved, it remains an interesting question whether selective seed abortion is under maternal or embryonal control or both.
If selective seed abortion is under maternal regulation, the maternal plant should have indications about the potential fitness of the different embryos. The contact between the ma- ternal tissue and the growing pollen tube before fertilization offers the first possible screening of the quality of the pollen donor (Westoby & Rice 1982, Law & Cannings 1984, Haig &
Westoby 1989). After fertilization, the maternal tissue is surrounding the developing embryo.
Moreover, the 3n endosperm consists of two chromosome sets of the maternal plant, and one paternal chromosome set. The endosperm might act as a compromising tissue between the evolutionary interests of the maternal parent and the offspring (Westoby & Rice 1982, Uma Shaanker et al. 1996). A highly functional and persistent endosperm (produced for one third by the pollen donor) might facilitate an equitable resource allocation to developing offspring and quench competition among developing embryos for resources. In addition, the maternal plant has the majority of the genetic composition of the endosperm and might control the nourishment of the embryo. If nutrient availability is limiting seed maturation, the maternal plant might direct the resources to the offspring with the highest potential quality.
The embryos themselves might also induce selective abortion. Stearns (1992) sug- gested that embryos might be in a selective arena competing for resources. Quality differences among embryos might occur because of their genetic composition, possibly caused by e.g.
inbreeding depression. The strongest competitors acquire most resources, grow the fastest, and will outcompete the less competitive embryos, which might die from starvation. If the embryo's ability to acquire resources reflects the potential quality later in life, the surviving embryos will be the ones of the highest fitness. In this case, the maternal plant may have control over the level of abortion, but not over the outcome of the selection. With a higher abortion level, the selection will be stronger than with a lower abortion level, but the selection will remain in the same direction.
A new way of exploring this old question is with QTL (quantitative trait analysis). A genetic marker might be associated with seed abortion. The genome of the mother plant might be screened for this marker, and an association with the abortion marker would indicate maternal control over the level of abortion. Also the part of the genome of the mother plant that will be part of the offspring might be screened for an associated marker that is linked
with abortion. With such an abortion marker in this part of the genome, embryonal dependent abortion would be suggested.
In this thesis I focus primarily on the possible fitness advantage of selective seed abortion and I largely ignore the underlying mechanism of selective seed abortion. Intuitively the most appealing assumption and the most widely used in life history studies, is the hypothesis that selection and abortion of seeds can be a passive process for the maternal plant. Embryos inter- act in a selective arena (Bookman 1983, Stearns 1992) and compete for resources for growth and development. I do not exclude a more active role for the maternal plant in terms of screening the quality of embryos actively and directing abortive hormones to the embryos of presumably lower quality. However, an active maternal role is not required to establish selec- tive seed abortion that could increase the mean quality of the offspring.
Empirical evidence of selective seed abortion and remaining problems
Abortion of embryos lowers the average number of seeds per flower. This will result in an overproduction of flowers compared to seed set. Flowers may be produced that do not pro- duce seeds. This "overproduction" of flowers seems to be the rule rather than the exception for many species. Another obvious possible explanation for low seed set is that ovules were not pollinated. In a review by Burd (1994), pollen limitation has been found at some times or in some sites for a majority of species (159 of 258 species). However, very often pollen is not the limiting factor for seed production while the number of female flowers exceeds fruit set (e.g. 59.3% seed to ovule ratio with additional outcross-pollination of populations in the field, extracted from Burd's survey (1994) by Korbecka et al. (in prep). This offers a potential for selective seed abortion, which should increase the fitness of the maternal plant.
To estimate the relative importance of selective seed abortion for the fitness of the maternal plant, we want to know i) the level of seed abortion in natural populations, ii) whether the aborted seeds are viable, iii) whether there is selection among different pollen donors, and iv) whether higher abortion rates lead to higher quality offspring compared to offspring from lower abortion rates. With various experimental designs, these questions have been investigated with different species. Although the amount of data available is small, the results of those experiments suggest that selective seed abortion might indeed be a relevant factor to explain the overproduction of flowers. However, yet no consistent picture arises, because the experimental designs used present some major problems or because experiments investigate only a part of the total picture.
Abortion rate in natural populations
Very few studies estimate the level of seed abortion of unmanipulated plants, and as far as I know, no estimates have been made under fully natural conditions. Only if all ovules are fertilized, the number of seeds per flower can give a reliable estimate of the actual abortion rate. To confirm pollination success, embryos should be detected in the ovules. In an experi- mental hand-pollinated population of Oxalis magnifica, Guth and Weller (1986) cleared the ovules to detect if embryos were present. They found an average fertilization rate between 48- 92%, and an abortion rate of 3-48%. In two natural populations with extra hand pollination of Epilobium angustifolium, an ovule clearing technique has been used too (Wiens et al. 1987).
A fertilization rate of 97% and an abortion rate near 30% were found. However, those populations were all additionally hand-pollinated and might not represent abortion rates under natural pollination. The level of seed abortion under natural conditions determines the poten- tial for embryo selection by seed abortion.
Are aborted embryos viable?
Previous experiments to show abortion of viable embryos dealt with manipulated plants from which flowers or ovules were removed to increase the seed set in the remaining reproductive structures. This has been successfully done for a number of species (Cassia fasciculata (Lee
& Bazzaz 1986), Agave mckelveyana (Sutherland 1987), Lotus corniculatus (Stephenson &
Winsor 1986), Crypthanta flava (Casper 1983, 1984 and 1988) and Phaseolis coccineus (Rocha & Stephenson (1991)). On the contrary, in Anchusa officinalis (Andersson 1990) and Achillea ptarmica (Andersson, 1993), the remaining reproductive structures did hardly in- crease their seed set. However, these hand-thinning experiments have two major drawbacks and may not give a reliable estimation of the percentage of viable embryos that normally would have aborted. Firstly, the different treatments were applied within one plant. An effect on seed set in the remaining ovules might then be masked if resources can be reallocated between different flowers of the same plant. If a part of the inflorescences is removed, the effect on seed set in the remaining part might be masked if resources can be reallocated between different inflorescences of the same plant. Secondly, viable embryos can still be aborted after hand thinning if species store their resources for another flowering season rather than reallocating them to embryos of lower quality in the actual flowering season. This does not necessarily exclude an effect, but may account for the fact that no effect is found. To prevent reallocation between different flowers or inflorescences of the same plant, treatments should be applied preferably to all inflorescences or flowers of a plant. To prevent storage for another flowering season, monocarpic species can be used.
Selection among pollen donors
Selection of pollen sources will lead to paternity composition of seeds that deviate from the paternity distribution that was originally applied as pollen. Such deviations have been establi- shed for e.g. Campsis radicans (Bertin 1982, 1985 and 1988), Raphanus raphanistrum (Mazer et al. 1986, Ellstrand & Marshall 1986, Marshall & Ellstrand 1988, Marshall 1991, Marshall & Folsom 1992), and Chameacrista fasciculata (Fenster 1991). In Asclepias spe- ciosa (Bookman 1984) and Raphanus sativus (Marshall 1988), pollen donors sired different numbers of seeds. Moreover, the pollen donor that sired most seeds in these species also sired the largest ones, from which the largest offspring grew (Bookman 1984, Marshall & Whit- taker 1989). Apparently, selection among different pollen donors in these species is related to offspring quality. In contrast, in Discaria americana (Medan & Vasellati 1996), the number of seeds sired by a particular pollen donor was not related to their germination success. To assess selection between different pollen donors in more species, the paternity composition of applied pollen has to be compared to the final siring success of the pollen donors.
Higher abortion rates increase average offspring quality
Stephenson and Winsor (1986) showed that with a decreased abortion level, the quality of the offspring was lower. With hand-thinning inflorescences of Lotus corniculatus, the seed set per remaining flower or ovule in the hand thinned inflorescences increased. The offspring from the hand-thinned inflorescences, that also included the offspring that normally would have been aborted, grew more slowly and produced fewer flowers, inflorescences, and fruits compared to the control group (Stephenson & Winsor 1986). Similar results were found with Crypthanta flava (Casper 1983, 1984 and 1988). Seeds produced in an ovule removal treat- ment germinated less (Casper 1988). In Phaseolis coccineus too, the destruction of some ovu- les in the ovary increased the probability that the remaining ovules would produce a mature seed. Compared to seeds from control fruits, the progeny from the experimental fruits were less vigorous also (Rocha & Stephenson 1991). These experiments show that the aborted embryos are viable, but of inferior quality. Apparently, some selection took place in the con- trol groups. However, the paternity of those embryos is not taken into consideration and there is no direct connection with data of selection on pollen donors. Moreover, the removal of reproductive structures can by itself also affect the outcomes of the studies. Casper (1988) gave three reasons why the results of such experiments should be taken with caution. Firstly, the treatment itself might damage the remaining ovule or seed, and therefore result in off- spring of lower quality. Secondly, with removing prematurely reproductive structures, one might upset initial source-sink relationships and affect nutrient flows. Thirdly, forcing a flower to distribute resources to an ovule that it normally would not have matured, might itself result in an inferior seed. In order to study selective embryo abortion, one should pre- ferably use a design with undamaged plants.
Inbreeding depression and selection against selfing.
If there are recessive, deleterious alleles present in the population, the average quality of selfed offspring will be lower than the quality of outcrossed offspring. This decrease in quality is commonly referred to as inbreeding depression (δ) and is calculated as the performance of selfed offspring (Ws) compared to the performance of outcrossed offspring (Wc): δ=1-Ws/Wc (e.g. Charlesworth & Charlesworth 1987). With a high level of inbreeding depression, selection against selfing would increase the average quality of the offspring.
Inbreeding depression can be high through accumulation of recessive deleterious alleles if selfing rates are low (Husband & Schemske 1996). In the case of recessive deleterious alleles, we thus expect that in species with a high inbreeding depression, selection against selfed offspring is strong.
If the deleterious alleles that are present in the population are not recessive, but have an additive effect, then the average quality of selfed offspring is not necessarily lower than the average quality of offspring from outcrossing. In that case, selective seed abortion should not be directed against selfing, but should be directed against individuals with a relatively high amount of deleterious alleles. In the case of additive deleterious alleles, we expect that selection is not consequently directed against selfing.
There is not yet a general statement about the level of recessivity or additivity of deleterious alleles in populations. Johnston & Schoen (1995) estimated in Amsinckia specta- bilis and Amsinckia gloriosa the relative amount of recessive and additive alleles. In A. spec-
tabilis, deleterious alleles were presumed to be recessive. In A. gloriosa, deleterious alleles showed partial dominance (incomplete recessivity) and they form a mixture of recessive and additive deleterious alleles.
Evolutionary consequences of selective seed abortion
If seeds are selectively aborted, this might lead to a fitness advantage of the maternal plant.
Selective seed abortion has consequences for the resource allocation to seeds and flowers and will lead to an overproduction of ovules compared to seed set (Stephenson 1981). Selective seed abortion is, however, not the only hypothesis that explains an overproduction of ovules.
Besides selective seed abortion, the non-exclusive hypotheses of bet hedging and sex allocation have been proposed (Stephenson 1981) to explain an overproduction of ovules.
Bet hedging
Stephenson (1980) has first mentioned the hypothesis of bet hedging. He states that an overproduction of flower compensates for variations in the amount of resources available for fruit maturation or variations in pollination success. If flower losses occur in an unpredictable way, a surplus of flowers can also act as a reproductive insurance (Stephenson 1981, Sutherland 1986). Kozlowski and Stearns (1989) explain the term of bet hedging as follows:
the organisms are "betting" in the sense that they do not know at the start of the reproductive attempt what is the best number of zygotes to produce and they take a risk (make a bet) in producing some definite number. They are "hedging" in the sense that, if they err in any direction, it is in the direction of overproduction. Under this hypothesis, the optimal number of offspring changes unpredictably from breeding attempt to breeding attempt. If bet hedging would be the most important selective force for the overproduction of flowers, seed set should come close to the maximum under optimal conditions.
The model of Kozlowski and Stearns (1989) shows that the hypothesis of bet hedging might explain an overproduction of flowers, under the conditions that abortion of young embryos is not too costly. However, Kozlowski and Stearns also argue that under this hypo- thesis, it pays for the plant to wait as long as possible with the abortion of seeds, to make the evaluation of the quality of the environment as reliable as possible. This hypothesis would thus not explain abortion of early-stage embryos. Moreover, with a prolonged waiting time before abortion, costs of an embryo are likely to increase. They conclude that also selective seed abortion might be the driving force for the overproduction of flowers. This statement is, however, not verified with experimental data.
Sex allocation
The concept of sex allocation (Charnov 1979, 1982, Lloyd 1980, de Jong & Klinkhamer 1989, de Jong et al. 1999) focuses the attention on the male function of flowers. With a fixed amount of resources for reproduction and a trade-off between the production of flowers and seeds, a high seed production will decrease the total amount of flowers. Increasing the female function of the plant (seed production) will thus decrease the male function of the plant (flower production). If the relationship between fitness gain and investment in female and male function is decelerating (i.e. decreasing fitness returns with increasing investments), flower production might have an intermediate optimum. Depending on the shape of both
fitness curves, the ESS (Evolutionary Stable Strategy) number of flowers might be higher than the number of flowers that corresponds to a maximum seed set per flower (Rademaker &
de Jong 2000).
Modeling the optimal number of seeds per flower
Pollen competition before fertilization does not affect the number of seeds per flower.
Because it is not costly for the female plant, this process presents no inherent reason why the plant should not produce the maximum of seeds per flower. However, selective abortion of seeds is one of the hypotheses that may explain an overproduction of flowers. Besides selective seed abortion, sex allocation is also likely to be a major evolutionary force in the production of empty flowers, which act as pollen donors only. Mathematical models, based on experimentally estimated parameters, can quantify the relative importance of the different hypotheses. A model based on sex allocation that did not include selective embryo abortion (Rademaker & de Jong 2000) indicated that for two hermaphrodite species the predicted ESS seed number per flower was lower than 25% of its potential maximum. If seeds are aborted, there is a shift from seed production to flower production. This shift increases pollen production and geitonogamous selfing and thus affects male fitness of the plant. However, hermaphrodite species are not the only species with an overproduction of flowers. Also in male sterile individuals of a hermaphroditic species, seed set is often considerably below the maximum (Klinkhamer et al. 1991). These individuals have no male function and thus this low seed set cannot be explained in terms of sex allocation theory. This seems to indicate that selective embryo abortion potentially can lead to a low seed set per flower. Also for herma- phrodites, a model that predicts the ESS allocation to seeds and flowers should include both sex allocation and selective seed abortion.
Aim and outline of the thesis
In this thesis, I will determine i) the level of seed abortion under natural conditions, ii) the selection of offspring by the maternal plant and iii) the consequences of selective seed abor- tion for offspring quality. In all experiments, hermaphrodite, monocarpic species were used.
The species
We used two model species of the Boraginaceae, Cynoglossum officinale, Hound's tongue, and Echium vulgare, Viper's bugloss. These two species were selected for the following reasons. All members of the Boraginaceae have a fixed number of four ovules per flower, which are arranged in a square. Average seed set per flower is lower than one, although for both species in our study area, pollination is not limiting seed set (de Jong & Klinkhamer 1989, Klinkhamer et al. 1994). Plants were readily available from natural conditions in Meyendel, the Netherlands. The ecology of these species has been studied extensively (van Breemen 1984, Klinkhamer & de Jong 1987, Nicholls 1987, de Jong & Klinkhamer 1989, Klinkhamer et al. 1994), so that cultivation and reproduction were not likely to cause un- expected surprises during the study. Both species produce normally over a hundred flowers per flowering individual, so that several crosses could be performed and repeated within one maternal plant. E. vulgare can be reproduced clonally, which offered the possibility to repro-
duce crosses between different genotypes. Flowers are large enough to mark them indivi- dually, which allowed us to perform single donor crosses on each individual flower. RAPD's (Random Amplified Polymorphic DNA) could be used successfully for paternity analyses. A disadvantage of both species was the long life span before reproduction. C. officinale required at least one cold period that induces flowering (de Jong et al. 1998). The same appeared to be true for E. vulgare. Individuals of a specific genotype required thus a long cultivation time before they were available for a pollination experiment. The two species differ in selfing rate.
C. officinale has a selfing rate between 0 and 70% (Vrieling et al. 1999). The selfing rate in E.
vulgare ranges between 0 and 30% (Rademaker 1998). Species with a relatively high selfing rate have presumably a low level of inbreeding depression (Husband & Schemske 1996) due to purging of the lethal alleles. E. vulgare was thus likely to show a higher level of inbreeding depression than C. officinale. We aimed at including species with this contrasting difference, because we expected that with a higher level of inbreeding depression and recessive deleterious alleles, selective abortion is most likely to act against selfing.
Main questions
The main questions of this thesis are: 1. Is seed abortion selective? and 2. Does selection among developing seeds improve the fitness of the maternal plant?
Firstly, we wanted to measure the level of abortion under natural conditions (Chapter 2). We applied different treatments with nutrients and hand-pollination on plants in a natural field population. Shortly after wilting of the flowers, we collected the ovules and screened them microscopically for embryo presence. We expected to find a higher number of embryos per ovule than later would be present as seeds per ovule. If pollen would be limited, the number of embryos per ovule should increase with hand-pollination. If nutrients were limiting seed production, then the number of seeds per ovule should increase with adding nutrients. In Chapter 3 we assessed if aborted seeds were otherwise viable. By removing three of the four ovules in each flower of experimental plants, we compared the seed set in the remaining ovule with the seed set of control plants. If viable embryos were aborted, we expected a higher seed set in the remaining ovules of the hand-thinned plants.
An analysis of the selectivity of maternal plants with respect to different pollen donors is found in Chapter 4, 6 and 7. With pollinations of different pollen donors, maternal plants were analyzed for their number of seeds per flower after self- and outcross-pollinations with different pollen donors. With selective seed abortion, we expected that flowers of maternal plants would produce different numbers of seeds per flower when pollinated with different pollen sources. To detect whether selective abortion improved the quality of the offspring we did experiments that are described in Chapter 3, 6 and 7. In Chapter 3 we describe an experiment in which we removed three of the four ovules in each flower. We expected that seed set per ovule would increase in the hand-thinned plants, and offspring that normally would have been aborted, would survive also. Under the hypothesis of selective seed abortion, this offspring would have a lower survival or growth compared to offspring from control plants. In Chapter 6 and 7 we describe an experimental design without damaging the plants but instead used the variation in abortion levels during the flowering season to analyze the effect of selective abortion on mean offspring quality. We expected a better offspring survival for the period in which the seed set was relatively low (presumably because many seeds were
aborted) compared to the period in which many seeds per flower were produced. Inbreeding depression is described in Chapter 4, 5, 6 and 7. We expected that if inbreeding depression lowers the quality of the selfed offspring, abortion should select against selfed seeds. In Chapter 4 we describe the inbreeding depression in seed production. The remaining chapters follow the offspring until and including their reproductive phase. We make the link between inbreeding depression and selection against selfing in Chapter 4, 5, 6 and 7. Finally, a mathematical model to detect whether selective abortion is shifting sex allocation towards an overproduction of ovules will be found in Chapter 8. There we analyze the effect of selective seed abortion on two species and analyze the results for different reproductive systems.
To put the different chapters in an ordered overview: Chapter 2 describes the abortion level of seeds in C. officinale under natural conditions. Chapter 3 assesses whether aborted seeds of C. officinale are viable and if artificially decreased abortion levels reduce offspring quality. Chapter 4 describes the selectivity on pollen donors for seed production of E. vulgare.
Chapter 5 describes late acting inbreeding depression in E. vulgare. Chapter 6 and 7 assess seed selection, abortion and inbreeding in both C. officinale and E. vulgare respectively.
Chapter 8 models the optimal seed production for different reproductive systems, with including the parameters for C. officinale and E. vulgare. Chapter 9 discusses the results and summarizes the main conclusions of this thesis.
2
Embryo abortion in natural populations of Cynoglossum officinale (Boraginaceae).
Summary
In many plant species, more flowers and ovules are produced than seem necessary for the production of seeds. Apart from the causal explanation that pollen quantity and quality can limit seed set, considerable attention has been given to the evolutionary hypothesis of selective embryo abortion. Under this hypothesis, the mother plant can increase her fitness by preferentially maturing the offspring of the highest quality. For only very few species it has been established that indeed many embryos are aborted. In this paper, we tested whether pollen was limiting seed production both at the plant and at the individual flower level and we examined embryo abortion in a natural population of Cynoglossum officinale.
Ovules were viewed microscopically. In 46.2% of the ovules embryos were found. The average number of seeds per ovule was smaller and decreased faster in time than the average number of embryos per ovule. As a result, the minimum estimate of the abortion level in- creased with time from 16.4% to 81.8%. Averaged over all flowers, the abortion level is esti- mated at 39%. Embryo or seed production was not limited by either pollen or nutrients. We discuss that both male function and selective embryo abortion can explain the evolutionary advantage of an overproduction of ovules, while bet hedging is an unlikely hypothesis for our study species.
C. Melser, L. Goosen-de Roo, H. Nell and P.G.L. Klinkhamer.
Introduction
Many plant species produce far more ovules than seems to be necessary for the production of their final number of seeds. Selective embryo abortion has been mentioned as one of the evolutionary explanations of this “surplus of flowers”. The overproduction of embryos gives the opportunity for selective maturation of the embryos with the highest fitness later in life.
The selective maturation of offspring with the highest quality could increase the fitness of the maternal parent. This hypothesis has received considerable attention (e.g. Darwin 1883, Janzen 1977, Lloyd 1980, Stephenson 1981, Casper 1984, 1988, Marshall & Ellstrand 1988).
The hypothesis of selective embryo abortion assumes that otherwise viable embryos are aborted. However, little is known about the level of embryo abortion in natural populations.
For numerous species, a low seed production per ovule is not caused by the lack of compatible pollen (e.g. Stephenson 1981, Willson & Burley 1983). Burd (1994) argued that pollen limitation has been underestimated under natural conditions. If so, we would expect a higher seed production when ample additional compatible pollen is applied and levels of em- bryo abortion should be low. Even if the available resources rather than pollination are the limiting factor for the production of mature seeds, pollen availability might still limit the pro- duction of embryos. Individual flowers can then still be pollen limited, while the plant as a whole is not. Under this condition, adding only nutrients will increase the total seed produc- tion, but not equally over all individual flowers. Reduced pollen availability in one flower may increase the seed production in another flower of the same plant, by reallocation of resources. This has been found in e.g. Polemonium foliosissimum (Zimmerman & Pyke 1988), Sorbus aucuparia (Sperens 1996) and Alstroemeria aurea (Aizen & Searcy 1998). Flowers that received extra pollination produced more seeds than control flowers on the same plants, indicating pollen limitation for individual flowers. However, the total seed production of the experimental plants was equal to control plants. Additional pollination might then increase the number of embryos, while the amount of resources available determines the overall level of abortion. It is therefore difficult to infer the level of embryo abortion from pollination experi- ments. However, other methods to estimate levels of embryo abortion present problems as well.
Levels of embryo abortion have been measured indirectly by removing flowers or ovu- les. With this method, an increase in seed set in the remaining flowers or ovules indicates that otherwise aborted embryos can mature into seed (Casper 1984, 1988, Andersson 1990, 1993, Stephenson & Winsor 1986, Lee & Bazzaz 1986, de Jong & Klinkhamer 1989, Chapter 3).
With this method, the extreme estimates of viable embryos that are aborted range from 4% in Anchusa officinale (Andersson 1990) and Achillea ptarmica (Andersson 1993), to 56.8% in Cynoglossum officinale (Chapter 3). However, this method provides only a minimum estima- tion of embryos and provides no reliable extrapolation to the numbers of embryos originally present. It remains unknown whether in the undeveloped ovules a seed was present and abor- ted, or there was none. Other estimates of embryo abortion are based on counts of empty seed coats (Levri 1998) or expanded but later aborted ovules (Lee & Bazzaz 1986; Stephenson 1984). With these estimates, only abortions that occur relatively late in embryo development are detected (Shuraki & Sedgley 1996). Moreover, it is not known whether swollen ovules always contain an embryo (Ohad et al. 1996, Chaudhury et al. 1997), nor whether ovules that
remain small, are never successfully fertilized. Direct views of initiated embryos under a microscope have been made for Crypthanta flava and C. flavoculata (Casper 1983), but only swollen ovules were selected for the preparates. Such samples are biased, because abortion may start before ovules start to develop and unswollen ovules with an embryo present are not included. In addition, unpollinated flowers might be present in the population but are not included, as swelling of the ovule might follow after pollination. In an experimental hand- pollinated population of Oxalis magnifica, Guth and Weller (1986) cleared the ovules to detect if embryos were present. They found an average fertilization rate between 48-92%, and an abortion rate of 3-48%. In two natural populations with extra hand pollination of Epilobium angustifolium, also an ovule clearing technique has been used (Wiens et al. 1987).
A fertilization rate of 97% and an abortion rate near 30% were found. The more embryos are aborted under natural conditions, the higher is the potential for selection on embryo quality.
To determine both the level of pollen limitation and abortion rate in the field, we exa- mined ovules microscopically from plants with and without additional pollination and nutrients. We compared the number of seeds with the number of embryos present per ovule in a natural population of Cynoglossum officinale. We compared these two numbers for different treatments: with and without additional pollination and with and without additional nutrients along the flowering season, to answer the following questions: 1) Are plants pollen or resource limited for total seed production? 2) Are individual flowers pollen limited for seed production? and 3) what is the potential for selective embryo abortion? The results give an opportunity to discuss the probability of alternative hypotheses for embryo selection.
Material and Methods
Species
Cynoglossum officinale (L.) is a rosette forming monocarpic, self-compatible perennial. From the main flowering stem, cymes diverge at which flowers develop sequentially. The flowering period starts at the beginning of May, and lasts four to five weeks. Each day, one flower can open at each cyme, and each flower remains open for about two days. The dull red-purple corolla fades to blue before abscission. In our study area, the most common visitors of flowers are bumblebees, while honeybees are less common (de Jong & Klinkhamer 1989). Flowers are hermaphroditic with five anthers and four ovules. The four ovules are symmetrically arranged in a square and may all develop into a seed. However, in the sand dunes, plants usually have, on average, fewer than one seed per flower (de Jong & Klinkhamer 1989). In a garden experi- ment, we showed that this low seed to ovule ratio is at least partly due to abortion of viable embryos, because removal of part of the ovules in each flower increased seed set in the re- maining ovules (Chapter 3). In our study area, additional hand pollination did not increase average seed set per flower (de Jong & Klinkhamer 1989).
Experimental design
Plants were selected in a natural population of C. officinale in the dunes of Meyendel, near The Hague, The Netherlands. Sixty individuals were marked and each individual was randomly assigned to one of the four following treatments:
1. watering with nutrients (n+);
2. additional hand pollination of all flowers on a plant (p+);
3. watering with nutrients and additional hand pollination (n+p+);
4. control, no nutrients or additional hand pollination (c).
The four groups of plants did not differ significantly in size (measured with length of longest leaf) at the moment of selection in the middle of April 1997. Treatments were applied to all flowers of a plant. For watering with nutrients (n+ and n+p+), we used per plant 100 ml standard Hoagland solution in addition to 2 l water three times a week. The plants assigned to this treatment got a shield of corrugated plastic (diameter 20 cm) around the rosette. This corrugated plastic was dug in the ground with a depth of approximately 10 cm and rose above ground for approximately 5 cm. While preventing a run off of the solution added, this low wall did not shade the leaves. The additional hand pollinations (p+ and n+p+) were applied with a small brush, always with a mixture of pollen from neighboring plants and self-pollen.
In another experiment with C. officinale, it was found that, averaged over several maternal plants, self-pollination yields an equal number of seeds per flower compared to cross- pollination (Chapter 6). Treatments were applied during the whole flowering period, from the 7th of May until the 9th of July 1997, three times a week. Flowers were marked with a drop of paint to identify the date of opening. Three times a week one flower was sampled of each maternal plant (if available) that had opened 4 or 5 days before: well after wilting but before visible swelling of the ovules. All sampled flowers were stored in 70% ethanol. After seed ripening and the natural death of the plants at the end of July, all plants were harvested and stored in plastic bags. The vast majority of the seeds (99.6%) adhered to the plant in their original position. After cleaning the roots from sand, plants were weighed (shoot, root and seeds separately) to determine total dry mass.
For all flowers on each maternal plant, position, date of flower opening and number of seeds were recorded. Empty seed coats were also registrated. For the analysis of the number of seeds per flower, flowering time was divided in four sequential time periods of equal length over all plants. Number of seeds per flower was averaged per maternal plant for those four periods. The number of seeds per flower were analyzed with an anova for repeated measure- ments for differences among the four treatments in number of seeds per flower (SPSS GLM Repeated Measures Analysis) in time. Two severely damaged plants were excluded from the analysis (one from p+, one from n+). Another four plants (one from treatment n+, three from treatment n+p+) had missing values in the fourth time period and could therefore not be included in the total analysis. These plants did not deviate in overall mean number of seeds per flower from the other plants in the same treatment group. Seed production per flower is positively correlated with plant size (Klinkhamer & de Jong 1987). To correct for differences in seed number per flower due to different plant sizes, we included the total dry mass of the plants as a covariate. Mass of plants is not affected by watering with nutrients (de Jong unpubl. results), so we used dry mass of the plants at the end of the experiment. To detect whether the frequency of flowers without any seed differed between treatments, the
distributions of the seeds per flower of the experimental plants were also compared to the distribution of the control group with a G-test. The total production of flowers and seeds per plant were analyzed with an analysis of variance. To obtain normally distributed residuals, the number of flowers and plant mass were ln transformed. Nutrients and pollination were main factors. For this analysis the four plants with missing values in the fourth time period were included.
Embryo observation
The treatment n+p+ and the control group were analyzed for the presence of embryos in the ovules. Samples for embryo observation were taken over all 15 maternal plants per treatment.
The experiment was divided in two periods of equal length. The time-consuming method to obtain microscopical samples did not allow us to use a more detailed time division. For each time period, one to four ovules were screened for each maternal plant (105 samples). For each plant, data were averaged per period to give an equal representation of each maternal plant.
From the samples of the collected flowers that were stored in 70% ethanol, ovules were isolated and dehydrated in ethanol (96% and 100%, each step for 30 min.) and propylene oxide (Agar Scientific Ltd., Stansted, UK) for 1 h. Thereafter, the ovules were infiltrated with Epon (Serva, Heidelberg, Germany) by successive incubations for at least 1 hour with mixtures of Epon and propylene oxide in the ratios 1:2, 1:1 and 2:1, and incubated in pure Epon, overnight. The specimens were embedded in Epon in BEEM capsules. Epon was allowed to polymerize for 48 h at 60oC. Longitudinal serial sections of 8µ m thickness were cut with glass knives on a LKB pyramitome. The sections were collected onto slides, and after
Figure 1:
Embryo of C. officinale in a pre-globular stage of eight cells. Viewed with a light microscope. Scale bar = P
staining by use of toluidine blue 1% in sodium tetraborate 1% (equal volumes of the two stocks) on a hot plate for at least 5 min. at 60oC, mounted in Epon and polymerized. The presence or absence of an embryo in an ovule was investigated with a light microscope. In this way, the stage of embryo development, from the four-cellular stage and upwards, could be determined (Tokč 1976, see Fig. 1).
Analysis of abortion levels
For the two time periods, we compared the mean number of seeds per ovule with the mean number of embryos per ovule per maternal plant for the treatments n+p+ and control. The difference provided a minimum estimate of the average level of abortion. The average number of seeds per flower were divided by four to achieve the number of seeds per ovule. The data were analyzed with manova (GLM multivariate analysis in SPSS), with dry mass of the plants as covariate, treatments as fixed factor, period as random factor, and average number of seeds per ovule, average number of embryos per ovule and abortion level as variables. In this ana- lysis, the average numbers of seeds and average number of embryos were transformed arcsine square root to obtain normally distributed residuals. The factor period showed a significant de- viation of sphericity, therefore we used a very conservative test for the effect of period, which is the ‘lower-bound’ epsilon for adjusting the degrees of freedom (SPSS 1997). To estimate an average lifetime abortion rate, abortion percentages for the two periods were weighed by the number of flowers produced in the two periods.
Figure 2:
Relation between the number of seeds per flower and the total plant mass (at the end of the experiment). Control: no nutrients or additional handpollination. p+: additional handpollination of all flowers on a plant. n+p+: watering with nutrients and additional handpollination. n+: watering with nutrients.
Results
Number of seeds per flower
The average number of seeds per flower over all plants was 1.13 (n=58; se=0.0424). Plants that have a higher mass at the end of the season, also produced more seeds per flower (Fig. 2;
F-value=8.58; df=1,49; p=0.0051). The number of seeds per flower decreased strongly along the season (Fig. 3; F-value=21.591; df=1,49; p<0.0001). Contrary to our expectations, the four treatments did not differ significantly from each other (F-value=0.893; df=3,49; p=0.4515).
When plant mass was not included as a covariate, this result did not change.
To examine whether the lack of sufficient pollination would increase the number of seedless flowers, the frequency distributions of seeds per flower per plant within the experi- mental treatments, were compared to the control group (Fig. 4). Although each treatment differed significantly from the control, the actual differences were very small. The percentage of e.g. empty flowers in the control group was 46.9%, and the application of extra hand polli- nation decreased this percentage only to 43.1%.
Total number of flowers
The heaviest plants at the end of the season had a greater total number of flowers per plant (F- value 110.0, df=1,53 p<0.001). The treatments appeared to have no significant additional effect on the total number of flowers per maternal plant (F-value =2.28, df=3,53 p=0.090).
Figure 3:
Average number of seeds per flower for the different treatments in time - estimated marginal means at mean plant mass (se). control: no nutrients or additional handpollination. p+: additional handpollination of all flowers on a plant. n+p+: watering with nutrients and additional handpollination. n+: watering with nutrients. Time periods are of equal length in days over the flowering period of all plants.
Without including plant mass as a covariate, the treatment effects remained insignificant, indicating that the effect of plant mass did not mask an eventual effect of treatment.
Total number of seeds
The plants with more mass at the end of the season had a greater total number of seeds per plant (F-value=200.9; df=1,53; p<0.0001). The experimental treatments had no effect on the total number of seeds per maternal plant (F-value=2.19, df=3,53 p=0.101). The average mass per seed was not correlated with plant mass at the end of the season (F-value=0.77, df=1,53;
p=0.385). Neither did the treatments affect average mass of the seeds (F-value=0.49, df=3,53;
p=0.693). Without including plant mass as a covariate, the treatment effects remained insignificant, indicating that also without accounting for plant mass, there were no significant differences between the treatments.
Figure 4:
Frequency distributions of seeds per flowers for all plants of the different treatments. A control plants (no nutrients or additional handpollination); B p+ (additional handpollination of all flowers on a plant; compared with control with the G-test: X2=50,7; df=4; p<0.001); C n+ (watering with nutrients; X2=17.3; df=4; p=0.002); D n+p+ (watering with nutrients and additional handpollination;
X2=56.9; df=4; p<0.001).
Abortion levels
Number of embryos per ovule and corresponding abortion levels were determined in the control and n+p+ treatment. The analysis with average number of seeds per ovule, number of embryos per ovule and abortion level per plant as dependent variables and with plant mass as covariate, and treatment and period as factors, yielded significant results (see Table 1). With analyzing only two treatment groups (n+p+ and control) instead of the full four when analyzing the number of seeds per flower, plant mass was not significant in the analysis.
Neither differed the two treatments significantly. Time period was highly significant, indicating overall changes in the variables (embryos, seeds per ovule and abortion level) along the season. The interaction between time and treatment was not significant, indicating that the two treatments reacted similarly in time.
Embryos were recognized from a four-cellular stage until a globular stage. The majo- rity of embryos found were in a pre-globular stage (Fig. 1). Pollen tubes that pass the ovule without fertilizing it were seen inside the hole of the ovule in 11.8% of the preparates in
Table 1:
Manova table for the embryos, seeds and abortion per ovule. Values for embryos and seeds are square root arc sinus transformed.
Factor F value df p value
Intercept 31,80 3 0,000
Plant mass 2,30 3 0,088
Treatment 0,19 3 0,906
Time period 71,80 3 0,000
Treatment x time 0,86 3 0,469
Factor Variable F value df p value
Corrected Model embryo 1,50 4 0,214
Intercept 16,93 1 0,000
Plant mass 0,00 1 0,969
Time period 5,45 1 0,023
Corrected Model seed 49,84 4 0,000
Intercept 86,00 1 0,000
Plant mass 3,11 1 0,083
Time period 193,57 1 0,000
Corrected Model abortus 0,79 4 0,540
Intercept 4,52 1 0,038
Plant mass 0,53 1 0,471
Time period 2,36 1 0,130
