COSTS OF SELF-POLLINATION IN A SELF-INCOMPATIBLE PLANT,POLEMONIUM VISCOSUM

Plants of Polemonium viscosum are strongly self-incompatible, yet have floral features that enforce spatial and temporal separation of pollen and stigma presentation. In this study, we address the widely held view that such barriers to selfing are maintained because they reduce interference by self pollen in performance of outcross grains. In the first year of the study we found that 24 hr precedence of self pollen on the stigma (24S), reduced outcross pollen germination by 32%, and seed set by 40% in flowers of P. viscosum. In the second year significant negative effects of self pollen on compatible pollen germination were seen when self pollen was applied four hours before (4S) or immediately prior (S×) to outcross pollen. Surprisingly, however, in the second year of study, seed set was not reduced with as much as 50% inhibition of compatible pollen germination. Results suggest that by interfering with normal functioning of outcross grains, incompatible self pollen can increase pollen limitation in P. viscosum. Reduction in seed production as a result of this phenomenon should select for displacement of pollen presentation from stigma receptivity.     

A REEXAMINATION OF THE POLLEN-DONATION HYPOTHESIS IN AN EXPERIMENTAL POPULATION OF ASCLEPIAS EXALTATA

The evolution of large floral displays in hermaphroditic flowering plants has been attributed to natural selection acting to enhance male, rather than female, reproductive success. Proponents of the “pollen-donation hypothesis” have assumed that maternal resources, rather than levels of effective pollination, limit fruit set. We investigated the pollen-donation hypothesis in an experimental population of poke milkweed, Asclepias exaltata, where effective pollination did not limit fruit set. Specifically, we examined the effects of flower number per plant, and flower number per umbel on male reproductive success (number of fruits sired) and female reproductive success (number of fruits matured). In 1990, a paternity analysis was performed on fruits collected from 53 plants whose inflorescences were not manipulated. Flower number per plant was significantly correlated with male success, but not with plant gender. Flower number per plant was also significantly correlated with female success, but umbel number and stem number per plant together explained more than half (58%) the variation in female success. The percentage of fruit set was not significantly correlated with flower number per plant. Plants with large floral displays did not disproportionately increase in male reproductive success, relative to female success, as predicted by the pollen-donation hypothesis. In 1991, the effect of flower number per umbel on male and female reproductive success was investigated. Flower number per umbel was manipulated on four umbels per plant by removing flowers to leave 6, 12, or 18 flowers in each umbel. Plants with the largest umbels effectively pollinated twice as many flowers on other plants, but produced only 1.35 times as many fruits as plants with 6 and 12 flowers per umbel. Relative maleness of plants with large umbels was nearly twice that of small and medium umbels. Although these observations are consistent with the pollen-donation hypothesis at the level of umbels, they are problematic, because much of the variation in flower number per umbel exists within, rather than among, plants in natural populations. Thus, plants consist of both reproductively male (large) and female (small) inflorescences, which act to increase total reproductive success. It is therefore inappropriate to explain the evolution of large floral displays in milkweeds solely in terms of potential male reproductive success.     

REPRODUCTIVE BIOLOGY OF ASCLEPIAS EXALTATA

An analysis of the relationships between plant size and survivorship and reproductive success was carried out by sampling four populations of the herbaceous perennial milkweed Asclepias exaltata in Virginia from 1980 to 1982. The annual survivorship rate (about 65%) is the lowest measured for any species of Asclepias. Survivorship was strongly size-dependent but showed no clear relationship with previous history of fruit production. Non-flowering plants were significantly smaller than flowering plants and showed very strong (r > 0.87) correlations between root dry weight and stem or leaf dry weight. Flowering plants were similar to nonflowering plants in root: shoot ratio (approximately 1:1) but differed in that root dry weight was not strongly correlated with stem or leaf dry weight. Components of inflorescence size were strongly correlated within a given level of comparison (e.g., stems per plant with flowers per plant) but less strongly correlated between levels (e.g., stems per plant with flowers per stem). Number of fruits per plant and percentage fruit-set were positively correlated with every component of inflorescence size. Although overall fruit-set was low (about 2%), fruits that were initiated had a high probability of surviving to maturity. There was no evidence of an early period of high fruit abortion: a relatively constant proportion of fruits aborted between each age class.     

ROLES OF POLLEN AND OVARY AGE IN FOLLICLE PRODUCTION OF THE COMMON MILKWEED ASCLEPIAS SYRIACA

Using cross-pollination experiments, pollinia viability of the common milkweed Asclepias syriaca L. (Asclepiadaceae) did not change significantly over the five-day life of the flowers, but stigma receptivity decreased over threefold over this period. During most of the season pollinators inserted over 90% of their pollinia into flowers during the first two days following anthesis. However, pollinators were scarce during the first few days of flowering, and they inserted over 60% of their pollinia into flowers three or more days old. This behavior potentially decreased outcrossing success and increased the relative male contribution of the flowers. Pollinator visitation patterns may play an important role in determining the functional sexuality of these plants.     

VARIATION IN THE POLLINARIA,ANTHERS, AND ALAR FISSURES OF COMMON MILKWEED (ASCLEPIAS SYRIACA L.)

We measured 10 characters of pollinaria, anthers, and alar fissures from the flowers of seven clones of common milkweed Asclepias syriaca L. to test whether significant differences existed among them. Strong among-clone differences existed for most of these characters. However, these differences were not correlated with each other, except for width of the alar fissure and width of the translator arm. Thus, these floral structures vary mostly in shape, rather than size per se. Few correlations among characters occurred at a within-clone level. Clones growing in the open and shade differed in half of the characters measured, but habitat did not substantially change the among-clone size patterns observed. The alar fissure–translator arm relationship might promote differential uptake of pollinia; however, A. syriaca does not exhibit a clear separation into donor and recipient clones.     

GENETIC CONSEQUENCES OF RARITY IN ASTER FURCATUS (ASTERACEAE), A THREATENED,SELF-INCOMPATIBLE PLANT

Aster furcatus is a rare, self-incompatible plant with fewer than 50 known populations throughout its range. We verified self-incompatibility in A. furcatus by conducting experimental self- and cross-pollinations and by examining seed set in a small population comprised of a single clonal genet. We examined variation at 22 electrophoretic loci in 23 populations of A. furcatus from across its range in Wisconsin, Illinois, Indiana, and Missouri. Except for two rare alleles found in single individuals in three populations, all loci but one of those examined were fixed for single alleles. The only variable locus (triosephosphate isomerase, TPI-1) tended to exhibit genotype frequencies in Hardy-Weinberg equilibrium or with a slight excess of heterozygotes. Although overall gene diversity was extremely low, TPI genotype frequencies were indicative of an outcrossing plant. We examined the subpopulation genetic structure among clonal plants within one Wisconsin population in greater detail. F statistics indicated that much of the genetic variation at the polymorphic TPI locus was due to differentiation among populations. We discuss the implications of self-incompatibility and low levels of genetic variation for the evolution and conservation of Aster furcatus and other rare plants with similar breeding systems.     

ULTRASTRUCTURE OF DEVELOPING AND MATURE NONARTICULATED LATICIFERS IN THE MILKWEED ASCLEPIAS SYRIACA L. (ASCLEPIADACEAE)

The ultrastructure of developing and mature nonarticulated laticifers in Asclepias syriaca L. (the common milkweed) was studied by conventional fixation and staining techniques and by osmium impregnation techniques. The mature laticifer protoplast in A. syriaca possesses a large central vacuole with an intact vacuolar membrane. Formation of this vacuole apparently results from dilation and subsequent enlargement of endoplasmic reticulum and possibly in part by fusion of smaller vacuoles and limited cellular-lytic autophagy. Widespread digestion or autophagy of cytoplasm within vacuoles is not evident. Nuclei, mitochondria, dictyosomes, and small vesicles are the most prominent components distributed in the peripheral cytoplasm. Plastids appear to degenerate as the laticifer matures. The specialized cellular component, latex, which is the vacuolar content of the laticifer, is interpreted to be produced in the cytoplasm and subsequently incorporated into the large central vacuole. Rubber globules, the most prominent latex component, are surrounded by a membrane that does not have a trilaminate structure. Globules are associated with an electron-dense fibrillar component in the vacuole.     

MATERNAL AND PATERNAL EFFECTS ON FOLLICLE PRODUCTION IN THE MILKWEED ASCLEPIAS SYRIACA (ASCLEPIADACEAE)

We measured follicle production from a diallel cross among ten clones of the common milkweed Asclepias syriaca, to assess the relative contributions of maternal and paternal parents. Specific parental combinations differed in the ability to set fruit, indicated by a significant nuclear specific effect accounting for 28% of the observed variance in follicle production. Several mechanisms might contribute to this effect, including shared incompatibility alleles and expression of zygotic genotypes. The nuclear general effect was not significant, however, suggesting a lack of additive genetic variation for offspring control of fruit maturation. Maternal effects also had an important effect on follicle production, as demonstrated by a significant reciprocal general effect (26% of the variance), almost entirely due to a large maternal component. The small reciprocal general variance component attributable to paternal effects, and nonsignificant reciprocal specific effect, indicating little maternal parent-zygote interaction, suggest that female choice through selective follicle maturation was not important in this experiment. The clones varied in proportion of reproductive output through female function, but a significant tradeoff between male and female success was not detected.     

EVIDENCE FOR LONG-DISTANCE POLLEN DISPERSAL IN MILKWEEDS (ASCLEPIAS EXALTATA)

We investigated pollen-mediated gene flow and interspecific matings in natural populations of poke milkweed, Asclepias exaltata. Genetic data from 16 polymorphic isozymes allowed unambiguous identification of the diploid paternal genotype for 225 singly sired fruits collected from six populations in Shenandoah National Park, Virginia. Paternity analysis of progeny arrays revealed that 29%–50% (39.2%, mean) of the seeds produced in these populations were sired by plants located outside each target population. Variation among populations appears to be related to isolation distance (Kendall's τ = ?0.69, N = 6, P > 0.1), although the negative correlation was statistically significant only when the results from a previous study of A. exaltata were also included in the analysis (Kendall's τ = ?0.78, N = 7, P < 0.05). The coupling of standard paternity exclusion analysis on individual seeds (12 seeds/fruit) with estimation of cryptic gene flow from Monte Carlo simulations accounted for only 65% of the gene flow detected using the progeny-array analysis. These results suggest that standard paternity exclusion, incorporating cryptic gene flow, will inherently underestimate actual gene flow rates when the number of paternal parents is smaller than the number of seeds per fruit (i.e., matings are correlated). Indirect estimates of the number of migrants per generation (Nm ≈ 2.3) obtained from Nei's genetic diversity statistic (G_ST) are much smaller than realized gene flow measured in 1990 (Nm = 9.4, progeny-array method). In addition, interspecific hybridization with common milkweed, Asclepias syriaca, via long-distance pollen dispersal was detected in two fruits (0.8%). Thus, pollen-mediated gene flow and infrequent interspecific matings provide mechanisms to lower genetic differentiation among populations to maintain novel, low-frequency alleles within populations of A. exaltata.     

ENVIRONMENTAL INFLUENCES ON NECTAR PRODUCTION IN MILKWEEDS (ASCLEPIAS SYRIACA AND A. EXALTATA)

Cross-comparisons of nectar production data are complicated because different workers use bags made of various materials to exclude animal visitors. Using clonal populations of Asclepias syriaca and A. exaltata in northern Virginia, we carefully measured the effects of four bagging treatments (bridal veil, pellon, paper, plastic) on microenvironment (temperature, relative humidity) and nectar production (volume, concentration, sucrose amount) over the course of a day. In general, bridal veil bags changed the microenvironment least relative to unbagged controls. Plastic bags resulted in higher temperatures and constantly higher relative humidities. Temperature and relative humidity were also elevated, though less dramatically, in paper and pellon bags. Under more humid conditions, flowers contained larger volumes of more dilute nectar. Therefore, researchers who wish to obtain nectar production data that reflect natural field conditions should use bridal veil, or a material with similar properties, to bag inflorescences. We also performed a watering experiment, involving the addition of the equivalent of a 10-cm rain to the A. syriaca plot. After watering, nectar volumes and sucrose amounts were increased approximately twofold.     

PATERNITY ANALYSIS IN A NATURAL POPULATION OF ASCLEPIAS EXALTATA: MULTIPLE PATERNITY,FUNCTIONAL GENDER,AND THE “POLLEN-DONATION HYPOTHESIS”

Recently, some evolutionary biologists have argued that selection on the male component of fitness shapes the evolution of reproductive characters in angiosperms. Floral features, such as inflorescence size, that lead to increased insect visitation without a concomitant increase in seed production are viewed as adaptations to enhance the probability of fathering seeds on other plants. In tests of this “pollen donation hypothesis,” male reproductive success has usually been measured indirectly by flower production, pollinator visitation, or pollen removal. We tested the pollen donation hypothesis directly by quantifying the number of seeds sired by individual genotypes in a natural population of poke milkweed, Asclepias exaltata, in southwestern Virginia. Multiple paternity was low within fruits, a fact which allowed us to use genotypes of progeny arrays to identify a unique pollen parent for 85% of the fruits produced in the population. Seeds sired (male success) and seeds produced (female success) were significantly correlated with flower number per plant (for male success, r = 0.32, P > 0.05; for female success, r = 0.66, P > 0.001). While the number of pollinaria removed, the usual estimator of male success in milkweeds, was highly correlated with numbers of seeds sired (r = 0.47; P > 0.001), it was even more highly correlated with numbers of seeds produced (r = 0.71, P > 0.001). Analysis of functional gender indicated that plants with many flowers did not behave primarily as males. In fact, individuals with the highest total reproductive success contributed equally as males and females. Furthermore, estimates of gender based on numbers of flowers produced or pollinaria removed overestimated the number of functional males in the population. In pollen-limited species, such as many milkweeds, proportional increases in both male and female reproductive success indicate the potential for selection to shape the evolution of large floral displays through both male and female functions.     

BIFURCATION OF THE STEM APEX IN ASCLEPIAS SYRIACA

In Asclepias syriaca the overall inflorescence is an anthoclad in which the peduncles are non-axillary, each occurring about 60° away from the axil of a leaf. Ontogenetically, a peduncle is initiated when the stem apex expands laterally and bifurcates into separate apices, neither of which is subtended by any type of organ. One of the two apices continues as the functional apex of the stem (bifurcating again at each subsequent node), and the other functions as the apex of the peduncle. The peduncle first produces a bract and, then, a pedicel in the axil of the bract. Subsequent pedicels are each axillary to separate bracts. The pedicels, therefore, can be interpreted as ordinary lateral branches. However, because the bifurcations of the stem apex are not associated with subtending organs, the branching of the stem does not conform to expected monopodial or sympodial systems in the angiosperms. This suggests the possibility that each bifurcation of the stem apex is a true dichotomy. The anthoclad axis, thus, is a series of dichotomies. Although such a series may have been phylogenetically derived from a monopodial or sympodial ancestor, it is also possible that it may have been retained from a primitive, dichotomizing inflorescence.     

PHYSICOCHEMICAL PROPERTIES OF THE PROTEOLYTIC ENZYME FROM THE LATEX OF THE MILKWEED,ASCLEPIAS SPECIOSA TORR. SOME COMPARISONS WITH OTHER PROTEASES : III. KINETICS OF THE HEAT INACTIVATION OF PAPAIN,BROMELIN, AND ASCLEPAIN

1. The rates of heat inactivation of papain, bromelin, and asclepain were determined at several different temperatures. Papain was by far the most resistant to heat. 2. The destruction of papain at 75–83° and bromelin at 55–70° followed the course of a first order reaction, except that for longer times of heating, bromelin (at 60–70°) was inactivated more rapidly than the first order equation required. 3. The rate of inactivation of asclepain at 55–70° followed the second order equation. 4. The critical thermal increments of inactivation of papain and bromelin, calculated with the van''t Hoff-Arrhenius equation, were of the same high order that has been found for protein denaturation. The increment for asclepain was somewhat lower.     

PHYSICOCHEMICAL PROPERTIES OF THE PROTEOLYTIC ENZYME FROM THE LATEX OF THE MILKWEED,ASCLEPIAS SPECIOSA TORR. SOME COMPARISONS WITH OTHER PROTEASES : I. CHEMICAL PROPERTIES,ACTIVATION-INHIBITION,pH-ACTIVITY,AND TEMPERATURE-ACTIVITY CURVES

1. A study has been made of the properties of a hitherto unreported proteolytic enzyme from the latex of the milkweed, Asclepias speciosa. The new protease has been named asclepain by the authors. 2. The results of chemical, diffusion, and denaturation tests indicate that asclepain is a protein. 3. Like papain, asclepain dots milk and digests most proteins, particularly if they are dissolved in concentrated urea solution. Unlike papain, asclepain did not clot blood. 4. The activation and inhibition phenomena of asclepain resemble those of papain, and seem best explained on the assumption that free sulfhydryl in the enzyme is necessary for proteolytic activity. The sulfhydryl of asclepain appears more labile than that of papain. 5. The measurement of pH-activity curves of asclepain on casein, ovalbumin, hemoglobin, edestin, and ovovitellin showed no definite digestion maxima for most of the undenatured proteins, while in urea solution there were well defined maxima near pH 7.0. Native hemoglobin and ovovitellin were especially undigestible, while native casein was rapidly attacked. 6. Temperature-activity curves were determined for asclepain on hemoglobin, casein, and milk solutions. The optimum temperature was shown to increase with decreasing time of digestion.     

PHYSICOCHEMICAL PROPERTIES OF THE PROTEOLYTIC ENZYME FROM THE LATEX OF THE MILKWEED,ASCLEPIAS SPECIOSA TORR. SOME COMPARISONS WITH OTHER PROTEASES : II. KINETICS OF PROTEIN DIGESTION BY ASCLEPAIN

1. The kinetics of milk clotting by asclepain, the protease of Asclepias speciosa, were investigated. At higher concentrations of enzyme, the clotting time was inversely proportional to the enzyme concentration. 2. The digestion of casein and hemoglobin in 6.6 M urea by asclepain follows the second order reaction rate. The rate was roughly second order for casein in water. 3. Evaluation of the nature of the enzyme-substrate intermediate indicates that one molecule of asclepain combines with one molecule of casein or hemoglobin in urea solution. 4. Inhibition by the reaction products was deduced from the fact that the digestion velocity of hemoglobin in urea solution varied with the asclepain concentration in agreement with the Schütz-Borissov rule.     

THE GENETIC CONSEQUENCES OF WORKER ANT POLLINATION IN A SELF-COMPATIBLE,CLONAL ORCHID

The self-compatible orchid Microtis parviflora is pollinated by the flightless worker caste of the ant Iridomyrmex gracilis. The orchid is clonal and forms small patches, usually less than 1 m², of disconnected individual ramets. Ant pollinators visited and revisited a limited proportion of available inflorescences, and 40% of all flower visits occurred within plants promoting self-pollination. Pollen labels indicated that self-pollination accounted for 51% of the pollen transfers, although pollen carryover extended beyond 16 flowers on 2 or 3 inflorescences. The distribution of ant movements between plants was leptokurtic with a mean of 12.4 ± 14.9 cm and a maximum of 89 cm, but a high proportion of movements were within clones accentuating the level of self-pollination. However, some pollen transfers between inflorescences of unlike genotypes contributed to a low incidence (max = 8%) of outcrossing. In 12 patches examined by electrophoresis, the density varied from 11 to 61 inflorescences per m² and a maximum of only 4 genotypes were detected. Electrophoretic analysis revealed populations were highly inbred: only 23% (N = 17) of the loci were polymorphic and the mean gene diversity h, was 2.7%. Heterozygotes were observed in only one population given a mean fixation index F, of 0.982. These results reflect the combined effects of restricted ant foraging and clonality. Nevertheless, while ant foraging was restricted, some outcrossing occurred and in the absence of clonality it is likely that ant foraging would have yielded a mixed mating system similar to those reported for a wide array of insect pollinators. Given the ability of ants to generate pollen flow, the reasons for the rarity of ant pollination appear to lie elsewhere.