MULTIPLE PATERNITY AND SELF-FERTILIZATION IN RELATION TO FLORAL AGE IN MIMULUS GUTTATUS (SCROPHULARIACEAE)

To gain some understanding of the mechanisms responsible for the intermediate levels of both multiple paternity and selfing observed previously in the common monkeyflower, Mimulus guttatus, we performed a field experiment to assess the role of 1) multiple paternity through sequential pollinator visits and 2) selfing through corolla abscission. In M. guttatus, flowers remain open for several days; then, in the process of corolla abscission anthers drag past the stigma. We predicted that multiple visitation by pollinators over this period should increase the degree of multiple paternity of sibling arrays, and that corolla dragging may be the primary cause of the observed selfing. In three northern California populations corollas were removed after being open for 1 day (preventing further mating), and the type of matings resulting was compared to matings from adjacent flowers with undisturbed corollas. On average, seed set was twice as great in flowers with unmanipulated corollas, indicating that about half of all matings in the lifespan of a M. guttatus flower occur after 1 day. Pollen supplementation revealed that pollen was limiting in one population. Electrophoretic assay of progeny showed multiple paternity was greater in unmanipulated flowers in the two populations that were not pollen limited, suggesting that sequential pollination may be an important source of multiple paternity. However, no difference was detected in the selfing rate between the two treatments, suggesting that corolla dragging was not a source of selfing even under pollen-limited conditions.     

ROLE OF COROLLA ABSCISSION IN DELAYED SELF-POLLINATION OF MIMULUS GUTTATUS (SCROPHULARIACEAE)

Corollas of Mimulus guttatus are shed 1–9 days post-anthesis, and autofertility is often high. The possibility that corolla shedding causes self-pollination in the absence of pollinators was examined. In one experimental population 82% of seed produced under conditions of pollinator exclusion were due to corolla abscission. Corolla abscission, in the absence of pollinators, is a mechanism of delayed self-fertilization in this taxon.     

POLLEN MORPHOLOGY IN THE GENUS MIMULUS (SCROPHULARIACEAE) AND ITS TAXONOMIC SIGNIFICANCE

The pollen morphology of 117 species and varieties of Mimulus was examined by light and scanning electron microscopy. Five major and 8 more tentative, minor types were found based on the differential correlation of aperture type, exine morphology, pollen grain diameter and other characters: type 1—synaperturate, usually ±spiraperturate, exine perforate to microreticulate with supratectal processes; type II—trocolporate, exine microreticulate (IIa and IIb, supratectal processes absent; IIa, mean polar axis 16–19 μm; IIb, mean polar axis 25–35 μrn; IIc, supratectal processes present); type III—tricolpate, colpus membrane ±psilate. exine with supratectal processes (IIIa, exine microreticulate and 1.4–2.0 μm thick, polar axis ≥ 30 μm; 111b, exine densely perforate and 2.2–2.8 μm thick, polar axis ≤ 23 μm); type IV—tricolpate, colpus covered with spinulose granules (operculate), exine microreticulate with supratectal processes; type V—5–7 stephanocolpate (Va and Vb, colpus margins ±straight and nongranular; Va, exine microreticulate with supratectal spinules; Vb, exine perforate with supratectal spinules or spinulose verrucae; Vc, colpus margins ragged and granular, exine microreticulate with supratectal processes). The pollen data correlate well with geographical and macromorphological data and, where the latter are ambiguous, often provide important clues toward the resolution of conflicting interpretations of infrageneric classification and generic delimitation.     

REPRODUCTIVE ASSURANCE MECHANISMS IN THREE TAXA OF THE MIMULUS GUTTATUS COMPLEX (SCROPHULARIACEAE)

The evolution of inbreeding in plants has often been attributed to selection for the ability to set seed in the absence of mates or pollinators. Mechanisms of reproductive assurance in five populations of mixed mating Mimulus guttatus, three populations of inbreeding M. platycalyx, and two populations of inbreeding M. nasutus were examined in a pollinator-free greenhouse. Reproductive assurance was manifested in all populations by autofertility, vegetative reproduction, or both. The inbreeding taxa had significantly greater levels of autofertility and less vegetative reproduction. Three modes of autofertility were identified: 1) due to corolla abscission only, occurring in three M. guttatus populations; 2) due to both corolla abscission and direct anther-stigma contact by curling of the lower stigmatic lobe into the anthers, occurring in two M. guttatus populations; and 3) direct stigma-anther contact by stigma curling alone prior to corolla abscission, found in each M. platycalyx and M. nasutus population. Stigma-anther distance and its interaction with stigma curling contributed to differences in autofertility among populations. Significant levels of intrapopulation quantitative genetic variation were found for seven of ten traits examined; average levels were similar between inbreeding and mixed mating populations. Genetic variation within populations for autofertility per se was not detected, but significant levels controlling stigma-anther distance were found in two M. guttatus populations. These results show that evolution of inbreeding by natural selection for reproductive assurance is possible in Mimulus, and illustrate the complex changes in floral dynamics and morphology it may involve.     

THE EFFECT OF SERPENTINE ON THE POPULATION STRUCTURE OF SILENE DIOICA (CARYOPHYLLACEAE)

Serpentine soils are rich in heavy metals and have a distinctive flora. Silene dioica is a member of the Scandinavian serpentine plant community but is also widespread outside serpentine soils. To study the population genetic consequences of serpentine stress and the origin and evolution of serpentine populations we analyzed the isozyme genetic structure of S. dioica. Seventeen populations located in the mountains of Västerbotten and Jämtland, central Sweden, were investigated by starch gel enzyme electrophoresis. About one half of the populations grow in serpentine soils and the rest on adjacent non-serpentine sites. Analyses of allele frequencies show that both serpentine and non-serpentine populations in the northern part of the studied area (Västerbotten) are genetically similar. Evidently serpentine does not exert strong selection acting upon isozyme loci. In the south (Jämtland), however, the serpentine populations exhibit genetic differentiation. This allozyme divergence is probably not due to direct selection but rather represents the effects of isolation and genetic drift. The results suggest that S. dioica has colonized serpentine repeatedly and that the tolerant populations have a multiple origin.     

ULTRASTRUCTURE AND DEVELOPMENT OF THE NEXINE AND INTINE IN THE POLLEN WALL OF SILENE ALBA (CARYOPHYLLACEAE)

Nexine and intine development in Silene alba (Caryophyllaceae) was investigated by electron microscopy and enzyme cytochemistry. Nexine-2 forms by deposition of sporopollenin along unit membrane lamellae closely associated with the microspore plasma membrane in the late tetrad stage. After the callose wall dissolves, electron density increases along the tangentially oriented fibers of the proximal primexine, forming nexine-1. When the exine is essentially complete, the intine begins to develop. In the nearly mature microspore, acid phosphatase activity appears in the peripheral cytoplasm just prior to its extrusion into the intine of the mature pollen grain.     

DELETERIOUS MUTATIONS AND GENETIC VARIATION FOR FLOWER SIZE IN MIMULUS GUTTATUS

Abstract.— Mimulus guttatus is a wildflower that exhibits substantial genetic variation in flower size. Here, we test the hypothesis that this variation is caused by deleterious mutations maintained through mutation‐selection balance. The deleterious‐mutation model predicts that rare, partially recessive alleles will be the primary source of variation. We test this prediction by measuring the change in the mean flower size (ΔM) and the directional dominance of flower size (ΔB) within a selection experiment. If variation is due to rare (partially) recessive alleles, ΔB/ΔM is expected to be positive and exceed one. However, we obtain negative values for ΔB/ΔM from three independent selection lines. This result is statistically inconsistent with the deleterious‐mutation model.     

SEX POLYMORPHISM IN SILENE ACAULIS (CARYOPHYLLACEAE) AND THE POSSIBLE ROLE OF SEXUAL SELECTION IN MAINTAINING FEMALES

Pollen accumulation rates and their relationship to stigma morphology and pollinator visitation behavior were compared between female and hermaphrodite sex morphs of the alpine plant Silene acaulis var. subacaulescens (Caryophyllaceae). Although stigmas of female and perfect flowers collected comparable pollen loads, stigmas of female flowers became receptive earlier in anthesis and therefore recruited a larger number of pollen tubes. Together with early receptivity young female flowers had a larger stigmatic area with longer papillae than did perfect flowers. Pollinator behavior also differed between morphs, with bumble bees spending more time probing female than perfect flowers. Differences in stigma receptivity schedules of female and perfect flowers have consequences for different opportunities for sexual selection in the two sex morphs. Female flowers, by providing a more effective gametophytic screen, have the potential to produce higher quality offspring. This is proposed as a further compensatory advantage maintaining females, with a single fitness function, in populations containing hermaphrodites which have both male and female fitness functions.     

UNUSUAL CYTOLOGICAL PATTERNS IN MICROSPOROGENESIS AND POLLEN DEVELOPMENT OF EVOLUTIONARY SIGNIFICANCE IN THE MIMULUS GLABRATUS COMPLEX (SCROPHULARIACEAE)

Four populations of Mimulus glabratus var. utahensis Pennell from the Great Basin and seven of M. glabratus var. fremontii (Bentham) Grant from the New Mexico–Texas–northeastern Mexico area were intercrossed and their F₁ hybrids grown. Cytology and fertility of both the parental populations and the F₁ hybrids were studied. The following cytological abnormalities were observed in microsporogenesis: cytomixis, the stretching of one or more chromosomes from cell to cell; multipolar divisions, separation of the chromosome complement into two or more parts; unequal disjunction; spontaneous polyploidization; and the production of encapsulated pollen tetrads. Typically, these abnormalities were rare or not observed in the parents, were rare in the intravarietal hybrids, but were more common in the intervarietal hybrids. They were closely associated with, in fact were the probably causes of, barriers to gene exchange between these two diploid (n = 15) varieties. Thus, the apparent causes of barriers to gene exchange in intervarietal hybrids are the likely mechanisms for the evolution of aneuploidy and polyploidy so characteristic of the rest of the Mimulus glabratus complex.     

THE TAXONOMIC IMPLICATIONS OF POLLEN MORPHOLOGY IN SOME SOUTH AMERICAN SPECIES OF MIMULUS (SCROPHULARIACEAE)

The pollen grains of South American hexaploid representatives of the Mimulus glabratus complex, section Simiolus, and M. bridgesii, a South American species sometimes included in this complex and sometimes in section Paradanthus, were examined by light and scanning electron microscopy. Mimulus bridgesii has (6-) 4–5-zoniaperturate grains nearly identical to those observed in a complex of species in section Paradanthus. South American members of the M. glabratus complex (M. bridgesii excluded) have the irregularly synaperturate, ± spiraperturate pollen typical of section Simiolus (sensu Grant). Treatment of M. bridgesii as a palynologically aberrant member of section Simiolus is not supported by what is known about the evolution of pollen types in Mimulus and the evolution and dispersal of the M. glabratus complex.     

VARIATION OF SEX ALLOCATION AMONG EIGHT TAXA OF THE MIMULUS GUTTATUS SPECIES COMPLEX (SCROPHULARIACEAE)

Characters related to sex allocation and the mating system were studied in eight California taxa of the Mimulus guttatus complex: M. guttatus, M. nasutus, M. glaucescens, M. Tilingii, M. nudatus, M. laciniatus, M. platycalyx, and M. micranthus, ranked in approximate decreasing levels of outbreeding. Dry weights and lengths of floral parts, pollen and ovule number, and timing of stigmatic closure were measured on plants in the growth chamber. As percent of total flower weight, allocation to stamens and corollas was lowest in M. micranthus (28%), intermediate in M. platycalyx, M. Tilingii, and M. laciniatus (50%), and high in other taxa (60%). Among M. micranthus, M. platycalyx, and M. laciniatus, pollen–ovule ratios ranged from 3.9 to 12.0; ratios for other taxa were 19.3 to 26.6. Taxa with increased male biomass allocation generally show increased outcrossing and increased P/O ratios. Stigma-anther separation and closure of stigma lobes upon touch were positively correlated with outcrossing ratios, P/O ratios, and male allocation. Isozyme variation indicates the inbreeding taxa have been independently derived; thus, these associations of maleness with outbreeding are significant trends in the Mimulus guttatus complex.     

AMONG- AND WITHIN-FLOWER COMPARISONS OF POLLEN TUBE GROWTH FOLLOWING SELF- AND CROSS-POLLINATIONS IN DIANTHUS CHINENSIS (CARYOPHYLLACEAE)

We compared the rate of pollen tube growth following self- and cross-pollinations both among and within flowers of two clones of Dianthus chinensis L. For among-flower comparisons, both styles of a flower were pollinated with either self- or cross-pollen. Within-flower comparisons were made between the two styles of the same flower, one of which was self-pollinated and the other cross-pollinated. Comparisons between flowers indicated that self-pollen grew slower than cross-pollen in both clones. However, differences in the growth rate of pollen tubes from self- and cross-pollinations were greater when comparisons were made between the two styles of the same flower than when pollinations were made in different flowers. These results suggest the existence of interstyle interactions in pollen tube growth.     

PROGENY SEX RATIOS IN DIOECIOUS SILENE LATIFOLIA (CARYOPHYLLACEAE)

Most sex ratios reported for Silene latifolia are female biased. As a result of experiments performed by Correns in the early 1900s, pollen tube competition has generally been accepted as the primary cause of these skewed ratios. We did four sets of hand pollinations in which we varied the size of pollen loads and placement of pollen along the filamentous stigma. The effect of pollen load size on progeny sex ratios was not statistically significant. Of 32 maternal families, 17 contained more females than males (one ratio deviated statistically from 1:1), and 13 contained more males than females. Paternal families exhibited a greater range of sex ratios, including three with a significant female bias and one with a significant male bias. Within experiments, neither the maternal parent nor where pollen was placed had a statistically significant effect on progeny sex ratios; the paternal effect was significant in one experiment. We suggest that sex ratios in Silene latifolia are not necessarily affected by the level of pollen competition. Other factors, including variation among males and sex-linked mortality, may help explain the skewed sex ratios that characterize populations of this species. Further, Correns' observations of excess females may have resulted from his use of interspecific hybrids.     

POLLEN MORPHOLOGY IN THE GENERA MONTTEA AND MELOSPERMA (SCROPHULARIACEAE)

Pollen of the scrophulariaceous genera Melosperma and Monttea was studied with light and scanning electron microscopy. Both have single, radiosymmetric, isopolar, trizoniaperturate pollen grains with usually ruptured colpus membranes, tectate-perforate to semitectate exines with a columellate interstitium and pitted or perforate nexines. Intergeneric differences in pollen morphology are established based on the differential correlation of sculpturing characters. Comparisons of the pollen of Melosperma and Monttea with that of the tribe Minuleae sensu stricto reveals no difference in the case of Melosperma and the existence of intermediate morphologies and “reticulate” similarities in the case of Monttea; the taxonomic significance of this morphological intergradation is considered. The transfer of Oxycladus aphyllus to Monttea is consistent with the pollen evidence, but proposed affinities between Monttea and tribe Antonieae of the Loganiaceae are not supported.     

RESPONSE TO NATURAL ENVIRONMENTAL HETEROGENEITY: MATERNAL EFFECTS AND SELECTION ON LIFE-HISTORY CHARACTERS AND PLASTICITIES IN MIMULUS GUTTATUS

Recent studies in plant populations have found that environmental heterogeneity and phenotypic selection vary at local spatial scales. In this study, I ask if there is evolutionary change in response to environmental heterogeneity and, if so, whether the response occurs for characters or character plasticities. I used vegetative clones of Mimulus guttatus to create replicate populations of 75 genotypes. These populations were planted into the natural habitat where they differed in mean growth, flowering phenology, and life span. This phenotypic variation was used to define selective environments. There was variation in fitness (flower production) among genotypes across all planting sites and in genotype response to the selective environment. Offspring from each site were grown in the greenhouse in two water treatments. Because each population initially had the same genetic composition, variation in the progeny between selective environments reveals either evolutionary change in response to environmental heterogeneity or environmental maternal effects. Plants from experimental sites that flowered earlier, had shorter life spans and were less productive, produced offspring that had more flowers, on average, and were less plastic in vegetative allocation than offspring of longer-lived plants from high-productivity areas. However, environmental maternal effects masked phenotypic differences in flower production. Therefore, although there was evidence of genetic differentiation in both life-history characters and their plasticities in response to small-scale environmental heterogeneity, environmental maternal effects may slow evolutionary change. Response to local-scale selective regimes suggests that environmental heterogeneity and local variation in phenotypic selection may act to maintain genetic variation.     

EFFECT OF DENSITY ON SECONDARY SEX CHARACTERISTICS AND SEX RATIO IN SILENE ALBA (CARYOPHYLLACEAE)

A field survey of plant and flower sex ratio and secondary sex characteristics was made in Silene alba. Female-biased plant sex ratios were found, as seems typical for the species. Sex ratio distribution correlated with a gradient of soil moisture (with the more moist area having a more female-biased ratio) and with changes in the density of Silene (intermediate and higher density areas having greater female bias). The floral sex ratio was significantly female-biased only at the site that was most female-biased in terms of plant sex ratio. Otherwise the population of flowers was significantly male-biased. Male and female plants harvested from the field differed in secondary sexual characteristics. Males had more flowers and invested proportionately more biomass in leaf, but less in root, stem and reproductive tissue than did females. Although both males and females were larger in terms of total dry weight at the moist site, males produced more flowers at the driest (high density) site. Here the female bias in plant sex ratio was intermediate, but the floral sex ratio was significantly male-biased. A glasshouse experiment was performed in which plants were grown at four densities. Density significantly influenced plant survivorship and the probability of flowering, and increased female bias in the pots, but it did not affect patterns of biomass allocation in flowering plants. Patterns of male and female biomass allocation did not differ in the experiment, except in terms of reproductive allocation (greater in females) and allocation to leaf, greater in males, but only at the lowest density. This work urges caution in interpreting differences between males and females in the field as secondary sex characteristics, since we find such properties to be overlapping under experimental conditions. It supports the idea that males and females of a species may sustain different reproductive output under differing conditions.     

CHLOROPLAST DNA AND ISOZYME DIVERSITY IN TWO MIMULUS SPECIES (SCROPHULARIACEAE) WITH CONTRASTING MATING SYSTEMS

Levels of cpDNA and isozyme diversity were contrasted between the mixed-mating M. guttatus and its highly selfing congener M. micranthus (Scrophulariaceae). Compared to M. micranthus, M. guttatus has two to four times higher diversity for both cpDNA and isozyme variation on a species-wide level. The selfing M. micranthus also has 1.5 to three times lower within-population allozyme variation and a greater proportion of its variation distributed among rather than within populations. Chloroplast DNA is here inferred to be uniparentally inherited. Thus the mating system has no effect on the transmission of the chloroplast genome. Since both cpDNA and isozyme variation are similarly reduced in M. micranthus, factors other than the mating system are hypothesized to be responsible for the observed decrease in species-wide genetic variation in M. micranthus. A recent origin of M. micranthus from a limited number of M. guttatus populations is suggested. Consequently, molecular variation is reduced in M. micranthus due to a bottleneck effect. These data generally demonstrate that levels of cpDNA variation may be high enough in some species to infer evolutionary processes below the species level.     

GYNODIOECY IN SILENE VULGARIS (CARYOPHYLLACEAE): PROGENY SUCCESS,EXPERIMENTAL DESIGN,AND MATERNAL EFFECTS

This study documents the comparative success of seeds and seedlings of the perennial gynodioecious-gynomonoecious weed, Silene vulgaris, in the greenhouse. The importance of experimental design is stressed by comparing two different statistical analyses of the data. Seeds were obtained from artificial pollinations in the field: self-fertilization of hermaphrodites, and cross-fertilizations of both hermaphrodites and females. One-way analysis of variance using progeny type (selfed hermaphrodites, outcrossed hermaphrodites, and outcrossed females) as the treatment effect for each seed and seedling variable showed statistically discernable differences among progeny from different cross types. The significance of this type of ANOVA resulted from a reduced error term and positively-biased F ratios. A factorial design showed no significant differences due to type of progeny in seed mass, days to germination, leaf number, area, or biomass at six weeks of age. There were, however, significant differences among seeds attributable to maternal parent for all seed and seedling variables. A higher proportion of seeds from outcrossed individuals germinated compared to that from self-fertilization. When the data were analyzed to include and partition all sources of variation, differences among offspring appeared during germination, rather than during later development. Seed mass, cross type, and sex of maternal parent all significantly affected the likelihood of germination; however, they had decreasing predictive power, respectively. Inbreeding depression in Silene vulgaris may help maintain gynodioecy; however, the pleiotropic effects of both nuclear and cytoplasmic genes for sex expression also may affect fitness and the maintenance of females.     

THE QUANTITATIVE GENETICS OF SEXUAL DIMORPHISM IN SILENE LATIFOLIA (CARYOPHYLLACEAE). II. RESPONSE TO SEX-SPECIFIC SELECTION

A well-established theoretical relationship exists between genetic correlations between the sexes and the dynamics of response to sex-specific selection. The present study investigates the response to sex-specific selection for two sexually dimorphic traits that have been documented to be genetically variable, calyx diameter and flower number, in Silene latifolia. Following the establishment of a base generation with a known genetic background, selection lines were established and two generations of sex-specific selection were imposed. Calyx diameter responded directly to sex-specific selection, and the positive genetic correlation between the sexes was reflected in correlated responses in the sex that was not the basis for selection within a particular line. Flower number showed a more erratic response to sex-specific selection in that selection in some lines was initially in the wrong direction, that is, selection for a decrease in flower number resulted in an increase. These erratic responses were attributable to genotype-environment interaction as reflected in significant heteroscedasticity in variance among families. Correlated responses to selection in the sex that was not the immediate basis for selection indicated the possible existence of a negative genetic correlation between the sexes for this trait. These results test for the first time the impact of genetic correlations between the sexes on the evolutionary dynamics of sexually dimorphic traits in a plant species.     

LEVER ACTION ANTHERS AND THE FORCIBLE SHEDDING OF POLLEN IN TORENIA (SCROPHULARIACEAE)

The anthers of Torenia fournieri were found to shed pollen forcibly by lever action. Anther structure was modified for this function by a flangelike outgrowth of the lateral pollen sac wall forming a lever. When pressed, this lever causes an infolding of the thinner, subadjacent, pollen sac wall forcing pollen from the stomium. A force of 1–1.5 g pressing against the four levers of an anther pair resulted in the forcible shedding of 2,000–3,000 pollen grains in two parallel rows. During the 2-day anthesis the flowers shift from functioning as males to hermaphroditic outcrossers, and yet only have a pollen:ovule ratio of 98.6, a ratio more indicative of facultative autogamy. The outermost pair of anthers functions on the first day of anthesis, while the second, inner pair functions on both days. In each flower, the 2-day anther pair produces approximately twice as many pollen grains as the 1 -day anther pair, a pollen production highly correlated with the length of their functional lives. This difference in pollen production is apparent in the larger size of the 2-day anthers, a size difference that first appears with the initiation of the anther primordia.