Horsenettle, Solanum carolinense L. (subgenus Leptostemonum, Solanaceae), is a self-incompatible perennial herb native to the southeastern United States. It reproduces both sexually (seeds), and clonally through spreading roots. Individual plants produce both staminate (male) and perfect (hermaphroditic) flowers. Perfect flowers are usually borne at the base of the inflorescence and staminate near the top (Solomon 1985). Staminate flowers in S. carolinense have reduced styles that do not extend beyond the anthers, reduced ovaries, and are incapable of setting fruit even when they are artificially pollinated (Solomon 1985). S. carolinense produces pollen as the only reward to pollinators and is buzz-pollinated by large bees. Individual fruits have an average of 160 seeds (Elle 1999).
TESTING THE RESOURCE REALLOCATION HYPOTHESIS
A complete test of the resource reallocation hypothesis requires determining (1) whether resources are saved by producing staminate flowers; and (2) whether resource savings translates into an increase in one or more fitness components. We examined whether resource savings occur by asking whether staminate flowers are smaller than perfect flowers at the same position on an inflorescence. Although previous reports on this species indicate that, on average, staminate flowers are smaller (Solomon 1986), these studies do not account for the fact that gender and inflorescence position are confounded. It is thus possible that staminate flowers are smaller than perfect flowers simply because they tend to be produced on the distal end inflorescences where even perfect flowers are generally smaller (Diggle and Miller 2004). We asked whether saved resources translate into increases in other fitness components by asking whether there is a trade-off, manifested by a genetic correlation (Charlesworth and Morgan 1991), between staminate flower production and various fitness components.
To assess resource savings, we compared various measures of the sizes of perfect and staminate flowers corrected for inflorescence position. A total of 150 plants (consisting of clonally propagating 10 ramets from each of 15 genets collected from an abandoned field near Duke Forest, Durham, NC) were grown in a greenhouse at Duke University. Soon after flowering began, flowers were collected thrice-weekly from all individuals. To stimulate the production of staminate flowers (Diggle 1991b) a randomly chosen 25% of perfect flowers were pollinated during each census. This pollination level results in a similar level of fruit set as that observed in natural populations (Wise and Cummins 2002). All other flowers were collected and fixed in a 3:1 ethanol:acetic acid solution for subsequent measurements. During collection, the rank position from the base of the inflorescence was recorded for each flower.
Sizes of preserved flowers were measured using a digital caliper and a Leica MZ6 dissecting microscope equipped with a ruled objective. Eight different measurements were taken from each flower: (1) petal length, (2) petal width, (3) anther length, (4) anther width, (5) style length, (6) stigma width, (7) ovary length, and (8) ovary width. Measurements 1–4 were taken from a single petal or anther chosen at random. In addition, a subset of 45 randomly chosen flowers of each morph were subsequently used to determine pollen number and size using the protocols described by Solomon (1986). Four replicate measurements were made for each flower in a hemacytometer, and the mean was used in statistical analyses.
To distinguish statistically between positional and gender effects on floral size, we fitted each of the eight size characters using linear mixed-effects models (LME) with gender and flower rank as well as flower rank2 and flower rank2* gender as fixed effects, and inflorescence rank, ramet, and genet as random effects using the routine “PROC MIXED” (SAS 2005). Significance of fixed effects was estimated using Type III sum-of-squares. Model selection for fixed effects was done through stepwise deletion of non-significant quadratic (flower rank2) and higher order interactions (flower rank2× gender) according to the marginality principle (Zar 1974; Fox 1997).
To determine whether resources saved in the production of staminate flowers were allocated to other fitness-enhancing functions, we estimated the genetic correlations between, on one hand, the proportion of flowers that were staminate (PSF), and on the other hand, flower production, and seed production in the field. Such genetic correlations are commonly used to assess allocation trade-offs (e.g., Mazer and Delesalle 1998; Charlesworth and Morgan 1991; Ågren and Schemske 1995). Similar correlations were estimated previously for PSF and above-ground biomass, survival, and ramet production by Elle (1999).
To generate experimental plants, 136 fruits were collected haphazardly (one fruit per plant) from an abandoned field near Duke Forest, Durham, North Carolina. Six seeds per fruit were germinated and grown in 1.68 L pots containing Farfard 3P soil mix (Farfard, Agwam, MA) arranged in six spatial blocks under a 14 h daylight regime. Plants from the greenhouse were crossed in a North Carolina II design (Lynch and Walsh 1998), to produce 30 paternal half sib families, each with contributions from 10 female parents (270 full-sib families total). In the summer of 2003, seeds from these crosses were germinated in the greenhouse and, after approximately 5 weeks, transplanted to two experimental field populations in a field containing native S. carolinense. Two seedlings from each of 80 full-sib families chosen to encompass the 30 paternal half-sib families were randomly assigned to each of two spatial blocks within each population, for a total of 320 seeds per population.
Plants were censused during the summer of 2004. Because of clonal reproduction in the previous year, more than one ramet per plant was present in 2004. Of these, we allowed only one ramet to flower and on alternate days recorded the number and gender of all flowers produced. Fruits were collected after the first frost of the year and maximum diameter (d) of each fruit was measured to estimate the number of seeds produced (s) using the relationship s= 70.1 − 23.0d+ 2.18d2− 0.0415d3 (n= 114 fruits). Wise (2003) demonstrated that this relationship explains 90% of the variation in seed number in S. carolinense. Genetic correlations were estimated by calculating both full- and half-sib family means (Falconer and MacKay 1996).
TESTING THE INCREASED POLLEN DONATION HYPOTHESIS
The primary question we addressed in this experiment was whether pistil reduction in staminate flowers affects male success. One approach to addressing this issue would be simply to compare the male success of naturally produced staminate and perfect flowers. However, an experiment of this type would necessarily confound the effects of flower gender with the effects of flower position and size (Diggle 2003; this study), because perfect flowers tend to be produced lower on an inflorescence and, as a result, tend to be larger. To avoid this confounding, we instead chose to “create” staminate flowers from perfect ones by removing the style and stigma. We then assessed the relative siring success of these plants and plants with perfect flowers using arrays of potted plants in the field.
Plants for experimental arrays were generated from nine genets from the eastern United States that had specific S-locus genotypes, which served as markers for paternity analysis. Each genet was clonally replicated to produce between six and 12 ramets per genet, which were then grown in 1.68 L pots in Farfard 4P soil mix in the greenhouse. This set of plants formed the base pool from which we drew to establish experimental trials. Each experimental trial consisted of exposing an array of plants to pollinators in a garden outside the Duke Biology Greenhouses for a single day. Although the average life-span of individual flowers in the field is about 2 days (unpubl. results), experimental flowers exposed to pollinators for a single day were heavily visited and achieved very high fruit set. Each array consisted of 22 plants arranged in a polygonal grid and exposed to natural pollinators for one day, after which they were returned to the greenhouse to allow seeds to mature. No naturally occurring flowering S. carolinense were observed within 500 m.
Each array consisted of eight pollen recipients with between one and six unmanipulated flowers, seven pollen donors with three perfect flowers each, and seven pollen donors with three artificially created “staminate” flowers each. We chose to use staminate-only plants, rather than plants that had both staminate and perfect flowers, to minimize pollen–pistil interference among flowers in staminate plants, and thus maximize our ability to detect siring differences among staminate and perfect flowers.
To create a staminate flower, the pistil was removed at its base from the original perfect flower. In addition, to avoid biases in pollinators' behavior due to touching the flowers, flowers in the perfect treatment were also manipulated by the experimenter but the pistil was left intact. Plants of the three categories were placed in alternating positions within the grid.
To assess the relative male success of perfect and staminate flowers, we used different S-locus genotypes for the two types of flowers on any given day. Which ramets were used was dictated by availability of open flowers. On average each experimental trial included 5.16 genets allocated among the three treatments. Genotypes for recipients were chosen to be fully compatible with both types of donors. To control for effects of genetic background, trials were run in pairs. In each pair, the same genotypes were used for pollen recipients, with the genotypes of the perfect and staminate plants switched between trials.
To elucidate paternity, seeds were extracted from the fruits produced by recipient plants and a random sample of two to four seeds per fruit were germinated. Each recipient plant produced on average 1.76 fruits per trial (14 fruits on average per trial, 169 fruits total). Genomic DNA was extracted from leaf tissue using a modified CTAB protocol (Varadarajan and Prakash 1991) and used as template for separate PCR reactions using allele-specific primers for each possible paternal allele. In total 504 seeds were genotyped. These primers were developed by Y.-Q Lu and are listed in the Appendix (Table A1). Products were scored on 1% agarose gels stained in ethidium bromide. In a few cases more than one paternal allele was amplified, and the DNA sample was considered to be contaminated and that offspring excluded from the analysis.
Relative siring success of perfect and staminate flowers was compared using a standard likelihood approach (e.g., Ritland 1990) in which we assumed that the probability that a particular seed was sired by pollen from a staminate plant was ρ. Under the null hypothesis of equal siring success, the expected value of ρ is 0.5 because the arrays contained equal numbers of perfect and staminate flowers. The significance of deviation from this expectation was evaluated by determining whether the support for the observed value of ρ was more than two log-likelihood units greater than support for the expected value (Edwards 1992; see e.g., Fry and Rausher 1997).
To determine whether there are differences in pollinator visitation rates to the two floral morphs, we conducted pollinator observations in our experimental arrays on six days. Observation periods of 15 min were conducted between 6:15 and 9:00, after which visitation rates dropped markedly due to high temperatures and low pollen availability. For each pollinator entering the array we recorded its identity, the number and gender of flowers and plants visited, the duration of each floral visit, and the sequence of visitation, until it left the array. We considered true visits to be only visits lasting 1 sec or longer (shorter visits are highly unlikely to result in pollen removal in this buzz-pollinated species, M. Vallejo-Marín pers. obs.).
Because preliminary analyses indicated that the proportion of visits to staminate plants in our array experiment did not differ among trials (likelihood ratio test, χ2= 4.68, df = 5, P= 0.456), visits were pooled across all trials for subsequent analyses. Under the null hypothesis of no pollinator preference for staminate plants, visits to staminate plants should constitute half of all visits to donor plants. Deviation from this expectation was tested using a standard likelihood approach (Zar 1974) assuming that the probability of visiting a staminate plant can be modeled as a binomial probability (e.g., Jones 1997).
Number of flowers visited per plant and duration of visits per flower were compared among treatments using analysis of variance with flower gender as the main effect. All analyses were carried out using the statistical package R version 2.1.1.