Steven B. Broyles

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.

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 (Kendalls 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 (Kendalls 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 Neis genetic diversity statistic (GST) 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.

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).

Hybridization in North American Asclepias. III : Isozyme evidence

We sampled 100 adult milkweeds along a 3 km transect through the Dolly Sods Wilderness Area in northeastern West Virginia. These plants were measured for 13 flower and 17 leaf characters. We also used horizontal starch-gel electrophoresis to score variation in 11 enzyme loci. A composite character index was constructed that yielded negative scores for species-specific alleles of A. syriaca and positive scores for species-specific alleles of A. exaltata. The 30 plants from the middle portion of the transect were highly variable, with many plants achieving scores near zero. These plants presumably represent a complex hybrid swarm, consisting of F, interspecific hybrids and backcrosses. Plants with intermediate character-index scores were significantly more likely not to flower than were plants with higher (A. exaltata-like) or, especially, lower (A. syriaca- like) scores. On the basis of a study of reproductive characters alone, one might conclude that there is no evidence of hybridization, as there appear to be no intermediates among the 54 flowering plants. Vegetative characters, however, reveal a more complex pattern of variation that agrees better with the isozyme analysis of all 100 plants. It does appear that hybridization and localized in- trogression are occurring between A. exaltata and A. syriaca in the Dolly Sods region. Several unique features of milkweed reproduction appear to promote interspecific hybridization and to magnify its effects.

Geographic Distribution of Allozyme Variation in Ulmus crassifolia

We investigated the distribution of genetic variation in cedar elm, Ulmus crassifolia. This species has an unusual geographic distribution with a primary range in the south central United States (central Texas east to Mississippi) and a few isolated populations located in central Florida. Cedar elm was polymorphic at 86% of the 15 loci examined and maintained high levels of genetic diversity within populations (H, = 0.22). Over 94% of the total genetic variation was partitioned within populations (GST = 0.062), even though significant levels of variation at individual loci were detected among populations. Genetic identity between populationrs located in the primary population was high (I = 0.990), while populations located in central Florida were more divergent from each other (I = 0.923) than from populations found within the primary range (I = 0.952). The Florida populations had reduced allelic variation per locus (A = 2.07) in comparison to the primary populations (A = 2.32), even though both sets of populations had equivalent levels of genetic diversity (H, = 0.20 and 0.19, respectively). This reduction in alleles suggests that the Florida populations of U. crassifolia may have experienced a mild genetic bottleneck during their evolu- tionary history.

Allozyme Diversity and Genetic Structure in Southern Appalachian Populations of Poke Milkweed, Asclepias exaltata

We investigated levels of genetic diversity, genetic population structure, and gene flow in poke milkweed, Ascleptas exaltata. Horizontal starch-gel electrophoresis was performed on leaves collected from 846 plants in 18 populations from the southern Appalachians. Enzyme poly- morphisms were observed at 16 of the 18 loci examined and averaged 2.36 ? 0.32 (mean ? standard deviation) alleles per polymorphic locus. Mean population heterozygosity (Ho = 0.202 ? 0.036), expected heterozygosity (H, = 0.182 ? 0.023), and total gene diversity (HT = 0.235) were substantially higher than average values reported for other insect-pollinated, outcrossing herbs. Of this total genetic variation, less than 10% was partitioned among populations (GST = 0.093). Indirect estimates of the number of migrants per generation (Nm = 2.43, calculated from GST; Nm = 3.46, calculated from the frequency of 11 alleles unique to single populations) suggest that gene flow is extensive in A. exaltata. These data are consistent with the view that the comose, wind-dispersed seeds and the durable, insect-dispersed pollinia of milkweeds promote high levels of gene flow, resulting in low levels of genetic differentiation between populations.

THE RELATIONSHIP BETWEEN THE FIVE STIGMATIC CHAMBERS AND TWO OVARIES OF MILKWEED (ASCLEPIAS AMPLEXICAUUS SM.) FLOWERS: A THREE-DIMENSIONAL ASSESSMENT

To assess the spatial relationships between the five stigmatic chambers and two separate ovaries of milkweed flowers with respect to pollen tube transmission, we performed experimental cross-pollinations of Asclepias amplexicaulis Sm. and examined cleared or sectioned gynostegia of these flowers 12–24 h after pollination. Three adjacent stigmatic chambers transmit pollen tubes to one of the two separate ovaries, and the remaining two chambers transmit pollen tubes to the second ovary. Despite the potential for pollen tubes to cross over at the point of fusion of the two styles, we never observed this phenomenon in our sections. Thus, it should be possible to perform controlled pollinations of selected ovaries in milkweed flowers. We have also discovered a furrow on the upper surface of the gynostegium that enables one to identify which stigmatic chambers transmit pollen tubes to particular styles and ovaries: three stigmatic chambers transmitting to one ovary are on one side of this furrow, and the two chambers transmitting to the second ovary are on the opposite side.

Systematic relationships within Gelsemium (Loganiaceae): evidence from isozymes and cladistics

We used horizontal starch-gel electrophoresis to assess the degree of genetic diver- gence between four populations each of Gelsemium sempervirens and G. rankinii from the southeastern United States and to test the hypothesis that G. sempervirens is an autotetraploid derivative of G. rankinii. Levels of genetic variation in both taxa are somewhat higher than the mean for long-lived, woody perennials. There was no indication that the alleles of one species represent a subset of the variation present in the other nor was there any indication of tetrasomic expression in G. sempervirens. Moreover, chromosome counts from root tip squashes confirm that both species are diploid, with 2n = 16. There is, however, unusual mitotic behavior that may explain apparently erroneous counts of lower numbers by some previous workers, who also reported larger and fewer stomates in the presumably autotetraploid G. sempervirens. Sharp genetic differences between the two species (mean genetic identity, I = 0.48) parallel their morphological discontinuities in many characters, and there was no evidence of hybridization or introgression in regions of sympatry. A cladistic analysis using eight morphological characters supports the view that G. sempervirens and C. rankinii are sister species, more closely related to each other than either is to the third species in the genus, G. elegans from southeast Asia. Genetic evidence suggests that the lineage leading to G. rankinii and G. sempervirens split in the late Tertiary, about 3-3.5 million years ago. It is possible that G. rankinii adjusted to life in Coastal Plain swamps by loss of winged seeds, becoming adapted for dispersal by water, rather than wind.

ON THE MECHANISM FOR REPAIR OF CROSS-LINKED DNA IN E. coli TREATED WITH PSORALEN AND LIGHT

ABSTRACT This article describes our recent progress in studies on the mechanism of repair of cross-linked DNA in E. coli treated with psoralen and light. Some of the reactions involved in this complex repair sequence were carried out in permeabilized cells and in vitro with purified enzymes. Other studies in whole cells indicate that genetic recombination is involved in this sequence, and that successive repair events controlled by recA occur singly in time, in clusters that may correspond to domains of the folded chromosome.

The breeding system of Zephyranthes atamasco (Amaryllidaceae)

mean ? standard deviation) as those from cross-pollinations (24.7 ? 14.0). Germination was slightly lower for seeds produced from self-pollinations (62.3 ? 36.1%) as compared with seeds from cross-pollinations (77.7 ? 22.9%). Self- and cross-pollinations resulted in fruits with significantly more seeds than open-pollinations (13.4 ? 7.3). Unlike other long-styled species of Zephyranthes, Z. atamasco is fully self-compatible, and seeds produced from self-pollinations are similar in number and viability to seeds produced from cross-pollinations. Nevertheless, autogamous seed production is very low, presumably because the long styles project the receptive stigmatic surfaces well beyond the dehiscing anthers.