A reevaluation of the use of rhizome scars to age plants of Trillium erectum (Melanthiaceae)1.

PREMISE OF STUDY: Herbaceous perennials are important long-lived plants in North American forests. Trillium has been used as a model organism to examine the effects of ecological processes on age structure in herbaceous forest perennials. Here, the methods of aging Trillium rhizomes are critically examined. • METHODS: Rhizomes of seedlings, single-bract plants, three-bract nonflowering plants, and flowering plants of Trillium erectum were examined. The patterns of cataphyll and scape scars on rhizomes were examined with respect to demographic category. • KEY RESULTS: Trillium rhizomes produce two cataphyll scars per year on single-bract, three-bract nonflowering, and flowering plants. Scape scars were only evident on rhizomes of three-leaved nonflowering and flowering plants. The percentage of intact rhizomes ranged from 10-67% for three-bract nonflowering plants, and 0-51% for flowering plants. Rhizomes in all demographic categories had evidence of recessing tissues from the oldest portion of the rhizome indicating that accurate age estimates are not possible on many plants. • CONCLUSIONS: Accepted methods of aging Trillium rhizomes have significant drawbacks. The primary problem is that rhizomes rot from the oldest portions in all demographic categories. A second problem is that plants producing multiple scape scars in a given year could mistakenly be counted as multiple years. Finally, confusing terminology and misrepresentations in Trillium literature suggests that many previous studies did not correctly determine age. Given the challenges of aging Trillium, we suggest that future studies use rhizome aging to study early demographic stages only and the ecological processes that influence their growth.

Hybrid bridges to gene flow: a case study in milkweeds (Asclepias).

Natural hybridization occurs throughout areas of sympatry for the North American milkweeds Asclepias exaltata and A. syriaca (Asclepiadaceae), even though the formation of F1 hybrid seed is a rare event. For introgressive hybridization to proceed, F1 and advanced hybrids must be released from reproductive barriers and successfully mate with one or both parental species. I investigated the mating system of natural hybrids between A. exaltata and A. syriaca in three populations in Shenandoah National Park, Virginia. Allozyme data and a maximum-likelihood procedure were used to estimate the frequency of six genotypic classes (parentals, F1, F2, and backcrosses) of the hybridizing populations, the pollinia received by hybrid plants, and the paternal parents of seeds produced by hybrids. F1 hybrids, backcross A. syriaca, and parental A. syriaca individuals were common in three hybrid populations. Even though self-pollinations and interhybrid pollinations were common, F2 seed production and the occurrence of F2 individuals were rare in hybrid populations. Hybrid plants received more pollen from A. syriaca than A. exaltata, which resulted in the production of more backcross-A. syriaca seed than backcross-A. exaltata seed. Asclepias exaltata was rare in the hybrid populations, but A. exaltata pollinia were received by hybrids and this species sired between 15% and 36% of the seeds produced on hybrids. The potential for introgression with A. exaltata populations is lower because this species is unsuccessful as the maternal parent in interspecific and backcross hand-pollinations. The asymetry of hybridization with A. syriaca as the maternal parent is further supported by the incorporation of maternally inherited chloroplast DNA markers in hybrids. Hybrid milkweeds frequently backcross with both parental species and may be released from the reproductive barriers that limit the formation of F1 hybrids in natural populations. The direction of interspecific gene flow and introgression in milkweeds is influenced by the reproductive biology of hybrids, the constituency of the surrounding population, and failure of some crosses to produce seeds. Finally, introgressive hybridization remains an important evolutionary force even when the initial formation of F1 hybrids in natural populations is rare.

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.

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 (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.

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.