Patterns of male sterility within and among populations of the distylous shrub Erythroxylum havanense (erythroxylaceae)

Distyly, a reproductive system characterized by the presence of long-styled (thrum). and short-styled (pin) individuals within a population, has been repeatedly used as a model for the study of the evolution of the reproductive systems in plants. Erythroxylum havanense is a distylous species in which most thrum plants fail to develop a fertile androecium, thus behaving as male-sterile or partially male-sterile plants. Short-styled (thrum) individuals have an increased performance as female parents, thereby compensating for their loss of male fitness. Previous studies of populations within close proximity to each other suggest that E. havanense may be involved in a process of gender specialization in which, unlike other heterostylous species, thrum plants are specializing as females and pins (long-styled) as males. In this paper we describe more general patterns of male sterility, one of the first steps in the evolution of gender specialization, among populations of the distylous shrub Erythroxylum havanense. Pollen germination differed among populations (range 0.52 ± 0.03 to 0.06 ± 0.04), and between morphs. Pollen from pin plants was almost two times (1.89) as fertile as that from thrums (0.36 ± 0.03 and 0.19 ± 0.03, pin and thrums respectively). Thrums were significantly more male sterile in four out of five populations. The population where differences between the floral morphs were not apparent showed the lowest levels of pollen fertility. Accordingly, our results indicate that populations of E. havanense show marked differences in pollen fertility and higher male sterility associated with the thrum morph. We hypothesize that differences between morphs could be explained if restorers of male sterility are linked to the distyly haplotype, while differences in genes associated with male sterility could explain the variation among populations. Overall, the prevalence of thrum-biased male sterility across populations suggests that E. havanense is subject to a process of gender specialization.     

Environmentally induced variation in fecundity compensation in the morph-biased male-sterile distylous shrub Erythroxylum havanense (Erythroxylaceae)

The evolution of male-sterile individuals in hermaphroditic species represents the first step in the evolution of sex specialization. For male-sterile individuals to persist they must have some fitness advantage that compensates for their loss of the male function. Female fecundity also depends on environmental factors as those determining the likelihood of pollination and fertilization. Here we assessed the effects of both male sterility and reproductive synchrony (an environmentally affected trait) on the magnitude of female compensation of Erythroxylum havanense, a distylous shrub with morph-biased male sterility. In vitro measurements of pollen germination showed that thrums were more male sterile than pins. The compensatory advantage of thrums changed by a factor of five depending on flowering synchrony. Flowering in synchrony with the population increased fruit production in both morphs. However, because pins that flowered out of synchrony produced almost no fruits, the reproductive compensation of thrums was higher in these circumstances. Because the magnitude of compensation is frequently considered as a key factor in the evolution of sex specialization, the environmentally induced variation in the magnitude of the reproductive compensation of thrum plants may have profound effects on the evolutionary dynamics of the reproductive system of E. havanense.     

Morph-biased male sterility in the tropical distylous shrub Erythroxylum havanense (Erythroxylaceae)

This study explores whether ecological factors, such as pollinators and pollen flow, or variation in pollen and ovule development account for the observed differences (approximately twofold) in the reproductive output of pin and thrum individuals of Erythroxylum havanense. The importance of ecological factors was assessed by means of comparison of the identity of pollinators and the rates of flower visitation, and by performing controlled hand pollinations and measurements of fruit set. In addition, we described the pollen and ovule development of thrum and pin individuals. Our results indicate that pollinators of E. havanense do not distinguish between floral morphs. The differences in fruit set between pin and thrum plants held even after hand pollination and, therefore, the observed differences in reproductive output between floral morphs of E. havanense cannot be explained in terms of asymmetrical pollen flow. There were no differences in the pattern of gynoecium development between the pin and thrum morphs, however androecium development showed marked differences between the morphs, and there was a resemblance between the developmental pathways leading to male sterility of the thrum morph of E. havanense with that of species with cytoplasmic male sterility (CMS).     

Male sterility, fitness gain curves and the evolution of gender specialization from distyly in Erythroxylum havanense

The evolution of dioecy from a monomorphic hermaphroditic condition requires two mutations, one producing females and one producing males. Conversely, a single mutation sterilizing one sexual function in one morph of distylous species would result in functional dioecy because such a mutation also affects the complementary function in the other morph. In this study, we tested these ideas with Erythroxylum havanense, a distylous species with morph-biased male sterility. Based on sex allocation theory we evaluated whether the invasion of thrum females is favoured over the maintenance of this morph cosexuals. Completely male sterile thrum plants obtained higher fitness returns than hermaphrodites or partial male sterile individuals of the same morph, thus favouring the invasion of female thrum plants. We concluded that because fruit production of pin individuals depends on the pollen produced by thrum plants, the invasion of thrum females would result on the evolution of functional dioecy.     

Sequential variation in the components of reproductive success in the distylousJasminum fruticans (Oleaceae)

A 2-year study of three natural populations of the distylousJasminum fruticans showed that mean fruit and seed production were significantly greater in shortstyled plants (thrums) than in long-styled plants (pins). In this study, we investigated the role of four sequential factors which may differentially influence fruit and seed set in the two floral morphs: (1) differences in flowering phenology, (2) a limitation of pollen transfer towards pins, (3) a differential capacity of the two morphs to act through famale and male function and (4) differential fruit abortion in the two morphs. Fruit set was significantly influenced by differences in flowering phenology although there were no differences in flowering time between the two morphs. supplementary pollinations in a natural population significantly increased fruit set and reduced the difference in fruit set between the two morphs in relation to controls, indicating a limitation on pollen transfer which was most severe towards pin stigmas. In reciprocal crosses, seed set was significantly dependent on the paternal and maternal identity of the pin parent. There was no significant variation among thrums for their performance as male or female parent. Furthermore, individual pin plants with relatively high percentage seed set as female parents gave poor seed set as male parents and vice-versa. Whereas fruit removal had no effect on seed number in thrum plants, a greater proportion of viable seeds were produced on pin plants which were left to naturally mature their fruits than on pins which had fruits artificially removed, suggesting the occurrence of selective fruit abortion in pins but not in thrums. The initially greater maternal fitness of thrums due to their greater success as pollen recipients may thus be opposed by increased viable seed set in the pins due to factors acting after the pollination stage. The relative reproductive success of floral morphs in the distylousJ. fruticans is thus differentially influenced by ecological factors occurring at different stages of the reproductive process.     

Asymmetry in pollen flow promotes gender specialization in morphs of the distylous neotropical herb <Emphasis Type="Italic">Arcytophyllum lavarum</Emphasis> (Rubiaceae)

Distyly is a floral polymorphism, characterized by a reciprocal positioning between stigmas and anthers in different flowers, where two floral morphs, long-styled (pin) and short-styled (thrum) occur within the population. Distyly is suggested as one of the routes leading to the evolution of separate sexes in plants. In this evolutionary pathway, pollinators may disrupt the complementarity of pollen transfer between morphs. Consequently, the floral morphs gradually specialize as either male or female. A key process required for gender specialization in distylous plants is a deviation of the realized functional gender (i.e. the proportion of genes transmitted to the next generation via pollen donation and seed production) from the potential functional gender (i.e. the expected contribution of male and female function to reproductive success from the number of ovules or pollen grains produced by each morph). I selected the distylous herb Arcytophyllum lavarum (Rubiaceae) to determine if asymmetry in pollen flow promotes differences in seed production, pollen donation and a discrepancy between the potential and the realized functional genders in pin and thrum floral morphs. Pollen flow in A. lavarum is asymmetric and the pin morph is more efficient at performing cross-pollination than the thrum morph. Conversely, the thrum morph produced two times more seeds than the pin morph. Male and female contributions to the potential functional gender were equivalent in both morphs. However, the pin morph transmitted more genes through pollen donation and the thrum morph more through seed production than expected from their potential functional genders. These results support the hypothesis that if pollinators consistently promote asymmetric pollen flow between morphs over generations, it is possible that gender specialization may evolve to the extreme of dioecism from an original distylous condition.     

DEMONSTRATION OF CRYPTIC INCOMPATIBILITY IN DISTYLOUS AMSINCKIA DOUGLASIANA

Hand pollinations experiments with distylous Amsinckia douglasiana A. DC. (Boraginaceae) revealed that legitimate (intermorph) crosses produce more seed than self-pollinations and that self-pollinations yield more seed than illegitimate (intramorph) crosses. We tested the occurrence of cryptic incompatibility in both style-length morphs by pollinating them with a mixture of legitimate and illegitimate pollen, each homozygous for a different electrophoretically distinguishable allele of phosphoglucose isomerase. The success of intramorph pollen was greatly reduced in mixture; only about 4% of the offspring of both pins and thrums were sired by illegitimate pollen. One pin and one thrum had sufficiently high seed set that the results cannot be attributed to selective abortion of embryos. In those individuals, discrimination against illegitimate pollen must have occurred on the stigma or in the style. Based on our findings, it appears that illegitimate matings are rare in natural populations. The progeny of legitimate crosses was biased toward pins, which suggests some regulation of morph ratios independent of the style-length locus. Prezygotic discrimination against the thrum genotype or selective abortion of embryos with the thrum genotype are both possible, but selective mortality of seedlings with the thrum genotype cannot be ruled out for some families.     

Pollen donation patterns in a tropical distylous shrub (PSYCHOTRIA SUERRENSIS; RUBIACEAE)

Selection on male function has been invoked to explain various floral features, including number of flowers, flower size, and flower color. Here I describe two experiments designed to examine the efficiency of distyly in promoting male floral function, as measured by successful pollen dispersal to stigmas. In both experiments, I performed emasculations to control the type of pollen locally available in a natural population of Psychotria suerrensis, a tropical shrub. In the “natural-recipients” experiment, I allowed each floral morph to donate pollen on alternate days to emasculated flowers of each morph. In the “paired-recipients” experiment, I attached paired cuttings of each morph to individual donor plants. The results of both methods were consistent. Pollen borne on low anthers (from pin plants) was transferred most efficiently to low stigmas (on thrum plants). Pollen borne on high anthers (from thrum plants) was dispersed in equal amounts to flowers of both morphs. The results suggest that distyly is only partially effective in achieving efficient pollen donation. Male function of pins is enhanced by the polymorphism, but male function of thrums is not. A supplemental pollination experiment illustrates that seed set in this species is predominantly pollen limited, reducing the importance of male function, in comparison with species where seed set is primarily resource limited.     

PARENTAL EFFECTS ON SEED DEVELOPMENT AND SEED YIELD IN RAPHANUS RAPHANISTRUM: IMPLICATIONS FOR NATURAL AND SEXUAL SELECTION

The possibility that sexual selection operates in angiosperms to effect evolutionary change in polygenic traits affecting male reproductive success requires that there is additive genetic variance for these traits. I applied a half-sib breeding design to individuals of the annual, hermaphroditic angiosperm, wild radish (Raphanus raphanistrum: Brassicaceae), to estimate paternal genetic effects on, or, when possible, the narrow-sense heritability of several quantitative traits influencing male reproductive success. In spite of significant differences among pollen donors with respect to in vitro pollen tube growth rates, I detected no significant additive genetic variance in male performance with respect to the proportion of ovules fertilized, early ovule growth, the number of seeds per fruit, or mean individual seed weight per fruit. In all cases, differences among maternal plants in these traits far exceeded differences among pollen donors. Abortion rates of pollinated flowers and fertilized ovules also differed more among individuals as maternal plants than as pollen donors, suggesting strong maternal control over these processes. Significant maternal phenotypic effects in the absence of paternal genetic or phenotypic effects on reproductive traits may be due to maternal environmental effects, to non-nuclear or non-additive maternal genetic effects, or to additive genetic variance in maternal control over offspring development, independent of offspring genotype. While I could not distinguish among these alternatives, it is clear that, in wild radish, the opportunity for natural or sexual selection to effect change in seed weight or seed number per fruit appears to be greater through differences in female performance than through differences in male performance.     

ADVANTAGES OF BEING A SPECIALIST FEMALE IN NODIOECIOUS SILENE VULGARIS S.L. (CARYOPHYLLACEAE)

According to sex allocation theory, to maintain a mutant male-sterile plant in a population of hermaphrodites such a plant must compensate its loss of fitness caused by inhibition of pollen production with a higher reproductive success through its female function. In the present study of a gynodioecious population of Silene vulgaris (Caryophyllaceae) I show that hermaphrodites not only benefit from outcrossing, in that progeny from outcrossed flowers are more vigorous than those from selfed flowers within an individual plant, but they also suffer heavily from self-pollination between different flowers of the same individuals, which could be demonstrated in experimentally made male-sterile (emasculated) individuals. Seeds from the emasculation period were heavier and germinated better than when the same individual was an intact hermaphrodite. Naturally male-sterile (female) individuals produced more fruits due to flowers staying open longer for pollen to arrive via some vector. However, the higher seed number alone could not provide the fitness advantage needed for females to be maintained in the population, but females also produced heavier seeds as compared to the hermaphrodites. Differences in seed survival and seedling establishment in the field are expected to add the advantages necessary for female plants to be selectively plausible.     

EFFECTS OF POLLEN DONOR IDENTITY ON OFFSPRING QUALITY IN WILD RADISH,RAPHANUS SATIVUS

If pollen donor performance during mating correlates with differences in offspring growth and fitness, processes that sort among potential mates may directly improve offspring fitness. Here seeds sired by three pollen donors on ten maternal plants were grown for eight weeks in the greenhouse. The performance of the pollen donors during pollination and fertilization was known from a previous experiment. There were significant effects of paternity on two measures of early growth: leaf number and plant height. Paternal effects on three measures more closely related to fitness; final plant weight, day of first flower production, and total flower number were also significant. Under the conditions of this experiment, final plant weight was probably the best predictor of fitness. The pollen donor that sired the largest seeds in the previous experiment sired offspring that were largest after 8 weeks of growth. Half of the plants were grown under low-water conditions. Paternal effects on growth were not masked by the environmental effects. In fact, some paternal effects became stronger under stress. This suggests that paternal effects could also be important in the field. Plants sired by donor A bolted very early when water was limited and would probably have an advantage in a season that was very short due to an early and severe drought. During fertilization and seed filling, seeds sired by this donor were more frequent on water-stressed maternal plants than on control maternal plants (Marshall, 1988). The data from this experiment indicate a connection between pollen donor performance during mating and offspring growth. These results suggest that the processes that sort among potential fathers during pollination, fertilization, and seed filling, may improve offspring quality.     

A comparison of male and female responses to inbreeding in Cucurbita pepo subsp. texana (Cucurbitaceae)

Accurate estimates of inbreeding depression are necessary in order to predict the evolutionary dynamics of a population, but many studies estimate inbreeding depression based solely on components of female function such as fruit set, seed set, and seed quality. Because total fitness is achieved through both male and female functions in hermaphroditic plants, estimates of both male and female fitness are needed to estimate accurately the magnitude of inbreeding depression. Seedlings of a wild gourd, Cucurbita pepo subsp. texana, with coefficients of inbreeding of 0 and 0.75 were planted in an experimental garden, and several components of male and female fitness were measured over the course of the growing season. Fitness in inbred plants was confounded by both maternal and genetic inbreeding effects. Inbred individuals produced significantly fewer fruits than outcrossed individuals, and percentage germination of seeds from inbred individuals was significantly lower than seeds from outcrossed individuals. Inbred plants also produced significantly fewer staminate flowers and marginally fewer and smaller pollen grains per flower. Pollen from inbred plants also grew significantly more slowly in vitro than pollen from outcrossed plants. Multiplicative estimates of inbreeding depression revealed inbreeding depression for both male and female functions in wild gourd, but inbreeding depression through female function was stronger than inbreeding depression through male function.     

Pollination biology and the impact of floral display, pollen donors, and distyly on seed production in Arcytophyllum lavarum (Rubiaceae)

In animal-pollinated plants, two factors affecting pollen flow and seed production are changes in floral display and the availability of compatible mates. Changes in floral display may affect the number of pollinator visits and the availability of compatible mates will affect the probability of legitimate pollination and seed production. Distyly is a floral polymorphism where long-styled (pin) and short-styled (thrum) floral morphs occur among different individuals. Distylous plants frequently exhibit self and intra-morph incompatibility. Therefore changes in morph abundance directly affect the arrival of compatible pollen to the stigmas. Floral morph by itself may also affect female reproductive success because floral morphs may display differences in seed production. We explored the effects of floral display, availability of neighboring compatible mates, and floral morph on seed production in the distylous herb ARCYTOPHYLLUM LAVARUM. We found that floral display does not affect the mean number of seeds produced per flower. There is also no effect of the proportion of neighboring legitimate pollen donors on seed production in pin or thrum flowers. However, floral morphs differed in their female reproductive success and the thrum morph produced more seeds. Hand pollination experiments suggest that differences in seed production between morphs are the result of pollen limitation. Future research will elucidate if the higher seed production in thrum flowers is a consequence of higher availability of pollen donors in the population, or higher efficiency of the pin morph as pollen donor.     

Paternal transmission of a seed size reduction gene varies with age of a primary transformant and seed set is also influenced by gene expression in maternal tissues

We have constructed chimaeric genes that are expressed in embryo and endosperm compartments of the seed, induce dominant seed lethality and have potential to reduce seed size in 75% of seeds within a fruit such as Citrus [7]. The genes are not entirely seed-specific as a proportion of primary test tobacco transformants containing their gene were fully male-sterile [7]. Here we investigated why a proportion of apparently male-fertile transgenic plants showed segregation distortion from the 75% seed lethality expected for a single dominant gene. Reciprocal crosses were conducted between pollen fertile, primary tobacco transformants containing various copies of the CG1-400-RNase gene [7] and wild-type tobacco plants to examine the transmission of the gene through maternal and paternal gametes and also the effects of gene dosage in embryo and endosperm compartments on seed viability and phenotype. Pollen viability, seed set and seed phenotype were examined over a 16 month period to assess stability of gene expression in primary transformants because woody, fruit crops containing these genes will be vegetatively propagated from primary transformants. In male-fertile transformants, the gene was observed to be expressed to varying degrees post-meiotically in pollen over the time period examined resulting in lethality of transgenic pollen and reduced paternal transmission. A variable, low-level maternal expression component was also detected that resulted in seed lethality and influenced morphological variation in the seed lethal phenotype. The maternal and paternal expression components caused seed lethality to range from 50 to 75%. This study indicates the need to select for transformants with stable pollen transmission and high seed expression and raises questions in relation to possible environmental and epistatic effects on gene expression in primary, hemizygous transformants over long growth periods.     

REPRODUCTIVE BIOLOGY OF DISTYLOUS PARTRIDGEBERRY,MITCHELLA REPENS

Long-styled (pin) and short-styled (thrum) plants in populations of Mitchella repens from North Carolina and New York displayed strongly clumped spatial dispersion. This distribution pattern results from the tendency of these prostrate perennials to spread by production of trailing stems with adventitious roots. Both morphs, in both populations, exhibited natural fruit set exceeding 86%. This percentage was not increased by hand-pollination. Although the number of ovules per pair of ovaries was fixed at eight in each morph, numbers of seeds per fruit in naturally pollinated flowers differed significantly between pins (4.3) and thrums (6.4) in the North Carolina population. In the New York population, no significant differences were found (pins had 5.0 seeds per fruit; thrums, 4.4). Artificial pollinations in both populations showed low seed set in intramorph cross-pollinations as compared with intermorph crosses. The flowers of M. repens were visited by native species of Bombus, which visited many flowers in each patch of plants. These insects moved frequently from patch to patch, effecting intermorph cross-pollination and maintaining high levels of seed set. Movement among patches may be promoted by the dispersion of nectar rewards, with flowers about to open or just opened providing maximum amounts of nectar. Calculations of functional gender suggested that in the North Carolina population, thrums contributed more than 75% of the genes transmitted by ovule production. This contrasts strongly with previous studies of unequal sexual contributions in distylous taxa, which have demonstrated greater female contributions by pins. In the New York population, however, nearly equal contributions by pins and thrums through pollen and ovule production occurred, a situation similar to that found in most distylous species. This appears to be the first demonstration of variation in functional gender between populations of a single species.     

Seed sex ratio in dioecious plants depends on relative dispersal of pollen and seeds: an example using a chessboard simulation model

Two principles are important for the optimal sex ratio strategy of plants. (1) Sib mating. Because seed dispersal is restricted, sib mating may occur which selects for a female bias in the seed sex ratio. (2) Local resource competition (LRC). If a plant produces pollen its nuclear genes are dispersed in two steps: first through the pollen and then, if the pollen is successful in fertilizing an ovule on another plant, through the seed. If the plant produces an ovule, its genes are dispersed only through the seed. By making pollen instead of ovules the offspring of a single plant is then spread out over a wider area. This reduces the chance that genetically related individuals are close together and need to compete for the same resource. The effect is the strongest if pollen is dispersed over a much wider area than seeds. Less LRC for paternally vs. maternally derived offspring selects for a male bias in sex allocation. We study the above‐mentioned opposite effects in dioecious plants (with separate male and female individuals), with maternal control over the sex ratio (fraction males) in the seeds. In a two‐dimensional spatial model female‐biased sex ratios are found when both pollen and seed dispersal are severely restricted. If pollen disperses over a wider area than seeds, which is probably the common situation in plants, the seed sex ratio becomes male‐biased. If pollen and seeds are both dispersed over a wide area, the sex ratio approaches 0.5. Our results do not change if the offspring of brother–sister matings are less fit because of inbreeding depression.     

Pollen Flow,Fecundity, and the Adaptive Significance of Heterostyly in Palicourea padifolia (Rubiaceae)1

Palicourea padifolia is a distlous shrub visited primarily by hummingbirds and bees in mid-elevation rain forests in Costa Rica. At Las Cruces Biological Station, the population of P. padifolia is composed of equal numbers of pin and thrum plants and morphs are randomly distributed. Like the majority of distylous species, P. padifolia exhibits a self and intramorph-incompatible mating system. The analysis of stigmatic pollen loads obtained from emasculated flowers shows that pollen flow between floral morphs is disassortative, which supports the Darwinian hypothesis concerning the adaptive significance of heterostyly. Pins experience greater disassortative pollination than thrums. Pins also set more seed than thrums, suggesting that the higher frequency of compatible pollen deposited on pin stigmas results in greater fecundity; alternatively, greater seed set in pins could be due to the differential allocation of resources to male and female function between morphs. Overall, seed set for the Las Cruces popularion of P. padifolia is much lower than the potential maximum. Factors which could contribute to seed set limitation include the frequency and/or spatial pattern of pollinator visits, pollen availability, and resource availability.     

The transition to gender dimorphism on an evolutionary background of self-incompatibility: an example from Lycium (Solanaceae)

Populations of three North American species of Lycium (Solanaceae) are morphologically gynodioecious and consist of male-sterile (i.e., female) and hermaphroditic plants. Marked individuals were consistent in sexual expression across years and male sterility was present throughout much of the species' ranges. Controlled pollinations reveal that L. californicum, L. exsertum, and L. fremontii are functionally dioecious. Fruit production in females ranged from 36 to 63%, whereas hermaphrodites functioned essentially as males. Though hermaphrodites were mostly male, investigation of pollen tube growth reveals that hermaphrodites of all dimorphic species were self-compatible. Self-fertilization and consequent inbreeding depression are commonly invoked as important selective forces promoting the invasion of male-sterile mutants into cosexual populations. A corollary prediction of these models is that gender dimorphism evolves from self-compatible ancestors. However, fruit production, seed production, and pollen tube number following outcross pollination were significantly higher than following self-pollination for three diploid, cosexual species that are closely related to the dimorphic species. The data presented here on incompatibility systems are consistent with the hypothesis that polyploidy disrupted the self-incompatibility system in the gynodioecious species leading to the evolution of gender dimorphism.     

Do differences in plant and flower age change mating patterns and alter offspring fitness in Raphanus sativus (Brassicaceae)?

When more pollen is present on stigmas than needed to fertilize all ovules, selection among pollen grains may occur due to effects of both pollen donors and maternal plants. We asked whether increasing plant age and flower age, two changes in maternal condition, altered the pattern of seed paternity after mixed pollination. We also asked whether changes in seed paternity affected offspring success in an experimental garden. While flower age did not affect seed paternity, there was a dramatic shift in pollen donor performance as plants aged. These differences were seen in the offspring as well, where the offspring of one pollen donor, which sired more seeds on young plants, flowered earlier in the season, and the offspring of another pollen donor, which sired more seeds on old plants, flowered later in the season. Thus, change in maternal condition resulted in altered seed paternity, perhaps because the environment for pollen tube growth was different. The pattern of seed paternity and offspring performance suggests that pollen donors may show temporal specialization.