Pollination ecology and mating system of Solanum rostratum (Solanaceae) in North America

Buzz-pollination or pollination by vibration occurs in several families of angiosperms including some important commercial crops such as potatoes and tomatoes. Buzz-pollinated flowers release pollen via small pores or slits on the anther’s tip that require the use of vibrations by specialized pollinators, usually bees, to remove the pollen. Some buzz-pollinated species have elaborate floral morphologies including dimorphic anthers within the same flower (heteranthery), and mirror-image flowers (enantiostyly) where the style is reciprocally deflected to either the left or right side of the floral axis. The complex floral morphology and buzz-pollination syndrome seen in these species require a close physical interaction between the sexual organs of the flowers and the bodies of insect visitors. Despite the broad taxonomic distribution of buzz-pollination in angiosperms (more than 60 families are buzz-pollinated) relatively few studies have described the pollination ecology of these species under natural conditions. The main goal of the present work was to characterize the pollination biology, mating patterns and antagonistic interactions (e.g. pollen theft) in natural populations of a buzz-pollinated species. For this purpose, I studied Solanum rostratum (Solanaceae), a buzz-pollinated, self-compatible, annual weed with complex floral morphology (both enantiostylous and heterantherous flowers). This species usually grows in disturbed areas in its native range (Mexico) and has become invasive around the world. My research was divided into three components. First, I characterized the pollination and reproductive biology of natural populations in Mexico. I performed floral manipulations in six natural populations of S. rostratum to estimate fruit and seed set. In three of these populations, I carried out 115 hours of pollinator observations and quantified the incidence of pollinators versus pollen thieves. I also measured the efficiency of a subset of floral visitors in triggering fruit set after single visits. Second, I investigated whether morphological correspondence between the size of the pollinator’s body and floral morphology influences pollen transfer. In experimental arrays, I exposed flowers of S. rostratum that varied in the distance between their sexual organs, to bumblebees (Bombus terrestris) of different sizes, and recorded pollen deposition and fruit and seed production. Finally, I determined the mating system (i.e. the proportion of self- and cross-fertilized offspring) of natural populations in Mexico and of introduced populations in the United States of America, using newly developed microsatellite markers. My results show that S. rostratum is visited by a wide range of bees of different sizes (0.9–9.8 mm in thorax diameter), but that only a small subset of these visitors act as pollinators. Most visitors act as pollen thieves, consuming pollen while effecting little or no pollination. I also found that correspondence between a pollinator’s size and the separation of the S. rostratum sexual organs determines pollen deposition and fruit production; pollen deposition decreased when bees were small relative to the distance between the sexual organs visited the flowers. My genetic analyses show that natural populations of S. rostratum maintain a relatively high outcrossing rate (tm = 0.75 ± 0.03) across the native and introduced range. Furthermore, genetic diversity is reduced in invasive populations, but this is not accompanied by changes in mating system. My work shows that the morphological fit between the pollinator and the flowers is important in determining the dynamics of pollen transfer and fruit production in this buzz-pollinated plant. Distinguishing between pollinators and pollen thieves in buzz-pollinated plants is essential for understanding the evolution of buzz-pollination, as pollen theft could be a major selective force for these species

High incidence of pollen theft in natural populations of a buzz-pollinated plant

More than 20,000 angiosperm species possess non-dehiscent anthers that open through small pores at the anther’s tip. These flowers are visited by bees that use vibrations to remove pollen, a phenomenon known as buzz pollination. However, some floral visitors fail to transfer pollen efficiently, either through a mismatch of flower and insect size, or because they are unable to buzz-pollinate. These visitors collect pollen, but provide little or no pollination, behaving as pollen thieves. Although pollen theft is widespread in plants, few studies have quantified the incidence of pollen thieves for buzz-pollinated plants. We use observations of natural populations and floral manipulations ofSolanumrostratum(Solanaceae) to investigate the incidence of pollen theft, find morphological and behavioural differences between pollinators and thieves, measure the pollination efficiency of visitors, and characterize the reproductive ecology of this herb. We found that most visitors act as thieves, with <20% of all bees contacting the stigma. Insect visitors that regularly failed to contact the stigma (illegitimate visitors), included buzzing and non-buzzing bees, were significantly smaller, visited fewer flowers per bout, and stayed longer in each flower than (legitimate) visitors that regularly contact the stigma. Few flowers visited solely by illegitimate visitors set fruit. Our results show thatS.rostratumrequires insect visitation to set seed and natural populations experience moderate pollen limitation. We conclude that insect size, relative to the flower, is the main determinant of whether a visitor acts as a pollinator or a pollen thief inS.rostratum

Thirteen microsatellites developed by SSR-enriched pyrosequencing for Solanum rostratum (Solanaceae) and related species

Premise of the study: Microsatellite markers were developed using second-generation sequencing in Solanum rostratum as a tool to study the reproductive biology and genetic structure of this invasive species. Methods and Results: Thirteen microsatellites were successfully discovered and amplified in a single multiplexed PCR. All loci showed genetic variation in S. rostratum. Cross-amplification in five closely related taxa was successful for a subset of loci. Conclusions: The set of 13 microsatellite markers developed here provides a time-effective and cost-effective genetic tool to study the reproductive biology of S. rostratum. The demonstrated transferability of the PCR multiplex to related taxa also highlights its usefulness for evolutionary studies across Solanum sect. Androceras

Recurrent modification of floral morphology in heterantherous Solanum reveals a parallel shift in reproductive strategy

Floral morphology determines the pattern of pollen transfer within and between individuals. In hermaphroditic species, the spatial arrangement of sexual organs influences the rate of self-pollination as well as the placement of pollen in different areas of the pollinator's body. Studying the evolutionary modification of floral morphology in closely related species offers an opportunity to investigate the causes and consequences of floral variation. Here, we investigate the recurrent modification of flower morphology in three closely related pairs of taxa in Solanum section Androceras (Solanaceae), a group characterized by the presence of two morphologically distinct types of anthers in the same flower (heteranthery). We use morphometric analyses of plants grown in a common garden to characterize and compare the changes in floral morphology observed in parallel evolutionary transitions from relatively larger to smaller flowers. Our results indicate that the transition to smaller flowers is associated with a reduction in the spatial separation of anthers and stigma, changes in the allometric relationships among floral traits, shifts in pollen allocation to the two anther morphs and reduced pollen : ovule ratios. We suggest that floral modification in this group reflects parallel evolution towards increased self-fertilization and discuss potential selective scenarios that may favour this recurrent shift in floral morphology and function

Mating system in Mexican populations of the annual herb Solanum rostratum Dunal (Solanaceae)

Traditionally, annual colonising species are expected to have high rates of self-fertilisation, although recent theoretical and empirical studies have shown that cross-fertilisation can be selected for under heterogeneous pollination environments. Solanum rostratum is a self-compatible annual herb that colonises disturbed habitats. Despite the lack of physiological mechanisms to prevent self-fertilisation, pollen transfer between individuals is expected to be favoured because of its complex floral morphology. In previous studies of S. rostratum it has been shown that anther dimorphism within flowers results in precise pollen placement on the pollinator's body, and the presence of mirror-image floral morphs within plants promotes outcrossing in experimental arrays. However, the mating system of natural populations of S. rostratum has never been assessed, and thus whether it is predominantly selfing or outcrossing remains unknown. We hypothesise that floral and inflorescence morphology of S. rostratum should facilitate cross-fertilisation, making it a predominantly outcrossing despite its lack of a self-incompatibility system. To test this hypothesis, we estimated outcrossing rates by genotyping 700 individuals at 13 microsatellite loci, sampled from four populations across a 690-km transect in the species' native range. We found that populations had mean outcrossing rates of 0.70 ± 0.03, with multiple sires contributing to paternity of each progeny array (average effective number of sires = 8.97 ± 0.57). This indicates that natural populations S. rostratum have relatively high levels of outcrossing, probably facilitated by its floral and inflorescence morphology. We speculate that partial selfing in this species may be an unavoidable consequence of displaying multiple flowers at the same time (geitonogamy), as well as the result of self-pollen transfer by illegitimate visitors

Intraspecific variation of scent and its impact on pollinators' preferences

Floral scents shape plant-pollinator interactions. Although populations of the same species can vary in their floral scent, little is known about how this variation affects pollinator visitation. In this study, we compare the scents emitted by buzz-pollinated Solanum rostratum (Solanaceae) in two areas of its distribution (Mexico and USA) and investigate how these differences in scent affect pollinator preferences and attraction. We determined the variation of floral volatile compounds using hexane extraction followed by gas chromatography coupled with spectrometry. We also performed a field cage multiple-choice bioassay and a Y-tube behavioural bioassay using Bombus impatiens. We recorded 13 volatile compounds in floral extracts for plants from both ranges that varied qualitative and semi-quantitatively among populations. We found that in the field cage experiment, bumble bees visited plants from the US populations more frequently than plants from Mexican populations. However, bees showed no difference in preference between extracts from Mexican or US flowers. We conclude that although bees show differential visitation to whole plants of different regions, variation in floral extract alone does not translate into differences in preference by B. impatiens. The potential effects of variation in floral scent on the other native bee pollinators remain to be assessed. Floral scents are key in plant-pollinator interactions. These scents can vary even among populations of the same species, which influences pollinator attraction. In this study, we determined the variation in floral scents in a buzz-pollinated Solanum rostratum in two areas of its distribution (Mexico and the USA). Moreover, we investigated how these differences affect pollinator preferences and attraction of Bombus impatiens. Our main finding is that floral scents emitted by S. rostratum plants from the Mexican range differ from the USA distribution range; although we found similar preferences of bumble bees for floral extracts, bumble bees preferred to visit plants from the US populations in a field cage bioassay. These contradictory results can probably be attributed to other signals not evaluated in this study or the extraction method of floral extracts. Future studies are necessary to evaluate the potential effects of variation in floral scent on other native bee pollinators