Post-pollination mechanisms in Nicotiana longiflora and N. plumbaginifolia: pollen tube growth rate, offspring paternity and hybridization.

In natural populations where interfertile species coexist, conspecific and heterospecific pollen can be delivered to the stigmas. Post-pollination mechanisms might determine the seed siring success of different pollen donors within species as well as the chances for hybridization between species. Nicotiana longiflora and N. plumbaginifolia occur in sympatry in Northwest Argentina, where they have overlapping flowering seasons and share floral visitors. We explored (1) pollen tube growth rates for outcross versus self pollen in single-donor pollinations; (2) siring success of self versus outcross pollen donors in competitive pollinations, and (3) possibilities for hybridization by performing two- (outcross conspecific vs. heterospecific) and three-pollen donor (self vs. outcross vs. heterospecific) crosses. In N. longiflora, both pollen tube growth rate and siring success favored outcross pollen over self pollen and strong rejection of heterospecific pollen. In N. plumbaginifolia, pollen tube growth rate was similar for self and outcross pollen, self pollen sired similar numbers of offspring than outcross pollen and heterospecific pollen sired roughly the same number of progeny than self pollen. Results suggest that in natural sympatric populations, interspecific crosses would likely lead to unidirectional hybridization with N. plumbaginifolia as the seed parent.

Heritability and correlation structure of nectar and floral morphology traits in Nicotiana alata.

The heritability and genetic basis of nectar traits have been rarely studied in the field, where plants are exposed to environmental factors that could mask underlying genetic effects. Heritabilities and variance components were estimated for nectar and morphological traits of Nicotiana alata, using a partial diallel design. The main experiment was conducted in a Missouri experimental garden using a randomized block design with three plant density treatments, whereas a smaller experiment was conducted near native Brazil habitat to compare the environmental variance in traits between Missouri and Brazil. Significant heritability was detected for nectar volume and energy content, and for corolla tube length. Phenotypic correlations were significant between all traits investigated, whereas significant genetic correlations were only found between nectar volume and energy and between corolla limb width and mouth diameter. There were no significant family-by-density interactions detected in the Missouri field environment. All traits differed significantly between Missouri and Brazil environments, but significant genetic by environment (G x E) interactions between Missouri and Brazil were detected for only one trait. This study shows that nectar traits can be heritable despite considerable environmental variation.

Phylogenetic fragrance patterns in Nicotiana sections Alatae and Suaveolentes.

We analyzed floral volatiles from eight tobacco species (Nicotiana; Solanaceae) including newly discovered Brazilian taxa (Nicotiana mutabilis and "Rastroensis") in section Alatae. Eighty-four compounds were found, including mono- and sesquiterpenoids, nitrogenous compounds, benzenoid and aliphatic alcohols, aldehydes and esters. Floral scent from recent accessions of Nicotiana alata, Nicotiana bonariensis and Nicotiana langsdorffii differed from previously published data, suggesting intraspecific variation in scent composition at the level of biosynthetic class. Newly discovered taxa in Alatae, like their relatives, emit large amounts of 1,8-cineole and smaller amounts of monoterpenes on a nocturnal rhythm, constituting a chemical synapomorphy for this lineage. Fragrance data from three species of Nicotiana sect. Suaveolentes, the sister group of Alatae, (two Australian species: N. cavicola, N. ingulba; one African species: N. africana), were compared to previously reported data from their close relative, N. suaveolens. Like N. suaveolens, N. cavicola and N. ingulba emit fragrances dominated by benzenoids and phenylpropanoids, whereas the flowers of N. africana lacked a distinct floral scent and instead emitted only small amounts of an aliphatic methyl ester from foliage. Interestingly, this ester also is emitted from foliage of N. longiflora and N. plumbaginifolia (both in section Alatae s.l.), which share a common ancestor with N. africana. This result, combined with the synapomorphic pattern of 1,8 cineole emission in Alatae s.s., suggests that phylogenetic signal explains a major component of fragrance composition among tobacco species in sections Alatae and Suaveolentes. At the intraspecific level, interpopulational scent variation is widespread in sect. Alatae, and may reflect edaphic specialization, introgression, local pollinator shifts, genetic drift or artificial selection in cultivation. Further studies with genetically and geographically well-defined populations are needed to distinguish between these possibilities.

Pollinator preferences for Nicotiana alata, N. forgetiana, and their F1 hybrids.

The role of pollinators in plant speciation and maintenance of species boundaries is dubious because most plant species are visited by several types of pollinators, and most pollinator species visit several species of plants. We investigated pollinator preferences and their efficacy as ethological isolation mechanisms between two interfertile species, Nicotiana alata and N. forgetiana and their F1 hybrids. Hawkmoths pollinate N. alata, while primarily hummingbirds and occasionally small hawkmoths visit N. forgetiana. F1 hybrids are easily produced in the greenhouse and although the species grow in similar habitats, hybrids have not been found in nature. In Rio Grande do Sul, Brazil, near where both species are found, experimental plots were studied containing both species, and both species plus F1 hybrids. In the mixed-species plots, hawkmoths showed a strong preference for N. alata. Hummingbirds were less common and only visited N. forgetiana. Hybrid seed was produced but plants made significantly fewer hybrid offspring than predicted by the frequency of interspecific pollinator movements. Nicotiana forgetiana was the seed parent of 97% of the F1 offspring, suggesting an asymmetry in pollen delivery or postpollination processes. In plots containing F1 hybrids plus both parental species, hawkmoths preferred N. alata and undervisited the other two phenotypes, except that in the third plot they visited hybrids in proportion to the hybrid frequency. Hummingbirds strongly preferred N. forgetiana in all plots but also visited F1 hybrids in proportion to their frequency in the third plot. Overall, F1 hybrids were well pollinated and were frequently visited immediately before or after one of the parental species. Thus hybrids could facilitate gene flow between the parental species. We conclude that pollinator discrimination among species is strong but is an imperfect isolation mechanism, especially if hybrids are present.

Nectar traits in Nicotiana section Alatae (Solanaceae) in relation to floral traits, pollinators, and mating system.

Nicotiana section Alatae exhibits great diversity among species in floral morphology, mating system, and predominant pollinators. As a first step towards estimating nectar's role in floral evolution, we studied nectar traits to determine whether they vary in association with predominant pollinators and mating system. Daily phenology determines when nectar becomes available to pollinators and differed between hummingbird- and moth-pollinated species. Nectar volume and concentration varied significantly among most species and pollinator groups, but were inversely correlated, so that total energy was similar among most species. In general, nectar volume was positively correlated with corolla length. The autogamous species, N. plumbaginifolia, had a nectar volume that matched expectations based on corolla length, but with lower concentration and total energy than predicted by corolla length, while nectar volume was lower than predicted by corolla length in the autogamous population of N. longiflora. Sugar and amino acid components (determined through HPLC) were similar among species, although differences did exist. The nectar of most species was sucrose-dominant, but the autogamous N. plumbaginifolia had nectar that contained similar proportions of sucrose, glucose, and fructose. Many nectar traits varied in association with the predominant pollinators and, in some cases, with the mating system.

INBREEDING EFFECTS IN CLARKIA TEMBLORIENSIS (ONAGRACEAE) POPULATIONS WITH DIFFERENT NATURAL OUTCROSSING RATES.

Inbreeding depression is commonly observed in natural populations. The deleterious effects of forced inbreeding are often thought to be less pronounced in populations with self-pollinating mating systems than in primarily outcrossing populations. We tested this hypothesis by comparing the performance of plants produced by artificial self- and cross-pollination from three populations whose outcrossing rate estimates were 0.03, 0.26, and 0.58. Outcrossing rates and inbreeding coefficients were estimated using isozyme polymorphisms as genetic markers. Analysis of F statistics suggests that biparental inbreeding as well as self-fertilization contribute to the level of homozygosity in the seed crop. Biparental inbreeding will reduce the heterozygosity of progeny produced by outcrossing, relative to random outcrossing expectations, and hence will reduce the effects of outcrossing versus self-fertilization. Heterotic selection may increase the average heterozygosity during the life history. Selfed and outcrossed seeds from all three populations were equally likely to germinate and survive to reproduce. However, inbreeding depression was observed in fecundity traits of plants surviving to reproduction in all three populations. Even in the population whose natural self-fertilization rate was 97%, plants grown from seed produced by self-pollination produced fewer fruits and less total seed weight than plants grown from outcrossed seed. There was no detectable inbreeding depression in estimated lifetime fitness. Inbreeding effects for all reproductive yield characters were most severe in the accession from the most outcrossing population and least severe in the accession from the most self-fertilizing population.

GENETIC AND ENVIRONMENTAL VARIATION IN FLORAL TRAITS AFFECTING OUTCROSSING RATE IN CLARKIA TEMBLORIENSIS (ONAGRACEAE).

Clarkia tembloriensis exhibits a wide range of variation among its natural populations in outcrossing rate and in separation of male and female function in space (anther-stigma separation or herkogamy) and in time (protandry). Here we show that outcrossing rate is highly correlated with protandry and anther-stigma separation. Both genetic and environmental variation contribute to inter- and intrapopulation variation in protandry and anther-stigma separation. Interpopulation differentiation for protandry and anther-stigma separation was found to be polygenic. Genetic variation for protandry and anther-stigma separation within populations was demonstrated by a significant among-family variance in two populations with contrasting breeding systems. Environmental effects on the expression of mating system traits were manifested in two ways. First, significant variation among lathhouse benches suggests that small-scale environmental heterogeneity may affect the development of floral traits. Second, protandry was shortened under hot summer conditions. Hence, hotter and drier habitats, typical of the more self-pollinating populations of C. tembloriensis, can promote self-pollination purely through environmental effects.

A LIFE-HISTORY BASED STUDY OF POPULATION GENETIC STRUCTURE: SEED BANK TO ADULTS IN PLANTAGO LANCEOLATA.

We explored the extent to which the soil seed bank differed genetically and spatially in comparison to two actively growing stages in a natural population of Plantago lanceolata. All seed-bank seeds, seedlings, and adults of P. lanceolata within eight subunits in a larger population were mapped, subjected to starch gel electrophoresis, and allozyme analysis in 1988. Gel electrophoresis was also used to estimate the mating system in two years, 1986 and 1988. The spatial distributions of seeds, seedlings, and adults were highly coincident. Allele frequencies of the dormant seeds differed significantly from those of the adults for four of the five polymorphic loci. In addition, a comparison of the genotype frequencies of the three life-history stages indicated that the seed bank had an excess of homozygotes. Homozygosity, relative to Hardy-Weinberg expectations, decreased during the life cycle (for seed bank, seedlings, and adults respectively: Fit = 0.19, 0.09, 0.01; Fis = 0.14, 0.04, -0.12). Spatial genetic differentiation increased sixfold during the life cycle: (for seed bank, seedling and adults: Fs1 ∗∗∗ = 0.02, 0.05, 0.12). The apparent selfing rate was 0.01 in 1986 and 0.09 in 1988. These selfing rates are not large enough to account for the elevated homozygosity of the seed bank. Inbreeding depression, overdominance for fitness, and a "temporal Wahlund's effect" are discussed as possible mechanisms that could generate high homozygosity in the seed bank, relative to later life-history stages. In Plantago lanceolata, the influence of the mating system and the "genetic memory" of the seed bank are obscured by the time plants reach the reproductive stage.