Rachel A. Levin

Fragrance chemistry, nocturnal rhythms and pollination ''syndromes'' in Nicotiana

GC-MS analyses of nocturnal and diurnal floral volatiles from nine tobacco species (Nicotiana; Solanaceae) resulted in the identification of 125 volatiles, including mono- and sesquiterpenoids, benzenoid and aliphatic alcohols, aldehydes and esters. Fragrance chemistry was species-specific during nocturnal emissions, whereas odors emitted diurnally were less distinct. All species emitted greater amounts of fragrance at night, regardless of pollinator affinity. However, these species differed markedly in odor complexity and emission rates, even among close relatives. Species-specific differences in emission rates per flower and per unit fresh or dry flower mass were significantly correlated; fragrance differences between species were not greatly affected by different forms of standardization. Flowers of hawkmoth-pollinated species emitted nitrogenous aldoximes and benzenoid esters on nocturnal rhythms. Four Nicotiana species in section Alatae sensu strictu have flowers that emit large amounts of 1,8 cineole, with smaller amounts of monoterpene hydrocarbons and alpha-terpineol on a nocturnal rhythm. This pattern suggests the activity of a single biosynthetic enzyme (1,8 cineole synthase) with major and minor products; however, several terpene synthase enzymes could contribute to total monoterpene emissions. Our analyses, combined with other studies of tobacco volatiles, suggest that phenotypic fragrance variation in Nicotiana is shaped by pollinator- and herbivore-mediated selection, biosynthetic pathway dynamics and shared evolutionary history.

Family-level relationships of Onagraceae based on chloroplast rbcL and ndhF data

Despite intensive morphological and molecular studies of Onagraceae, relationships within the family are not fully understood. One drawback of previous analyses is limited sampling within the large tribe Onagreae. In addition, the monophyly of two species-rich genera in Onagreae, Camissonia and Oenothera, has never been adequately tested. To understand relationships within Onagraceae, test the monophyly of these two genera, and ascertain the affinities of the newly discovered genus Megacorax, we conducted parsimony and maximum likelihood analyses with rbcL and ndhF sequence data for 24 taxa representing all 17 Onagraceae genera and two outgroup Lythraceae. Results strongly support a monophyletic Onagraceae, with Ludwigia as the basal lineage and a sister-taxon relationship between Megacorax and Lopezia. Gongylocarpus is supported as sister to Epilobieae plus the rest of Onagreae, although relationships within the latter clade have limited resolution. Thus, we advocate placement of Gongylocarpus in a monogeneric tribe, Gongylocarpeae. Most relationships within Onagreae are weakly resolved, suggesting a rapid diversification of this group in western North America. Neither Camissonia nor Oenothera appears to be monophyletic; however, increased taxon sampling is needed to clarify those relationships. Morphological characters generally agree with the molecular data, providing further support for relationships.

Fragrance chemistry and pollinator affinities in Nyctaginaceae

We present results of dynamic head-space collections and GC-MS analyses of floral and vegetative fragrances for 20 species in three genera of Nyctaginaceae: Acleisanthes, Mirabilis and Selinocarpus. Most of the species included in this study are either hawkmoth or noctuid moth-pollinated. A wide variety of compounds were observed, including mono- and sesquiterpenoids, aromatics (both benzenoids and phenylpropanoids), aliphatic compounds, lactones, and nitrogen-bearing compounds. Intraspecific variation in fragrance profiles was significantly lower than interspecific variation. Each species had a unique blend of volatiles, and the fragrance of many species contained species-specific compounds. The fragrance profiles presented here are generally consistent with previous studies of fragrance in a variety of moth-pollinated angiosperms.

Relationships within tribe Lycieae (Solanaceae): paraphyly of Lycium and multiple origins of gender dimorphism.

We infer phylogenetic relationships among Lycium, Grabowskia, and the monotypic Phrodus microphyllus, using DNA sequence data from the nuclear granule-bound starch synthase gene (GBSSI, waxy) and the chloroplast region trnT-trnF. This is the first comprehensive molecular phylogenetic study of tribe Lycieae (Solanaceae). In addition to providing an understanding of evolutionary relationships, we use the phylogenetic hypotheses to frame our studies of breeding system transitions, floral and fruit evolution, and biogeographical patterns within Lycieae. Whereas Lycium is distributed worldwide, Phrodus and the majority of Grabowskia species are restricted to South America. Tribe Lycieae is strongly supported as monophyletic, but Lycium likely includes both Grabowskia and Phrodus. Results also suggest a single dispersal event from the Americas to the Old World, and frequent dispersal between North and South America. The diversity of fruit types in Lycieae is discussed in light of dispersal patterns and recent work on fruit evolution across Solanaceae. Dimorphic gender expression has been studied previously within Lycium, and results indicate that transitions in sexual expression are convergent, occurring multiple times in North America (a revised estimate from previous studies) and southern Africa.

The Systematic Utility of Floral and Vegetative Fragrance in Two Genera of Nyctaginaceae

We examined relationships between fragrance and phylogeny using a number of approaches to coding fragrance data and comparing the hierarchical information in fragrance data with the phylogenetic signal in a DNA sequence data set. We first used distance analyses to determine which coding method(s) best distinguishes species while grouping conspecifics. Results suggest that interspecific differences in fragrance composition were maximized by coding as presence/absence of fragrance compounds and biosynthetic pathways rather than when quantitative information was also included. Useful systematic information came from both compounds and pathways and from fragrance emitted by both floral and vegetative tissues. The coding methods that emerged from the distance analyses as best distinguishing species were then adapted for use in phylogenetic analysis. Although hierarchical signal among fragrance data sets was congruent, this signal was highly incongruent with the phylogenetic signal in the DNA sequence data. Notably, topologies inferred from fragrance data sets were congruent with the DNA topology only in the most distal portions (e.g., sister group pairs or closely related species that had similar fragrance profiles were often recovered by analyses of fragrance). Examination of consistency and retention indices for individual fragrance compounds and pathways as optimized onto one of the most-parsimonious trees inferred from DNA data revealed that although most compounds were homoplastic, some compounds were perfectly congruent with the DNA phylogeny. In particular, compounds and pathways found in a few taxa were less homoplastic than those found in many taxa. Pathways that synthesize few volatiles also seem to have lower homoplasy than those that produce many. Although fragrance data as a whole may not be useful in phylogeny reconstruction, these data can provide additional support for clades reconstructed with other types of characters. Factors other than phylogeny, including pollinator interactions, also likely influence fragrance composition.

Paraphyly in Tribe Onagreae: Insights into Phylogenetic Relationships of Onagraceae Based on Nuclear and Chloroplast Sequence Data

Abstract Onagraceae are a family of 17 genera in seven tribes, with the majority of species in tribes Onagreae and Epilobieae. Despite the species-richness of these two tribes, to date no phylogenetic study has been done with sufficient taxon sampling to examine relationships between and within these tribes. In this study, we used DNA sequence data from one nuclear region (ITS) and two chloroplast regions (trnL-trnF and rps16) to infer phylogenetic relationships among 93 taxa across the family, with concentrated sampling in the large tribe Onagreae. Results strongly suggest that tribe Gongylocarpeae is sister to tribes Epilobieae + Onagreae, both of which are monophyletic. Within Onagreae, Camissonia seems to be broadly paraphyletic, and Oenothera is also paraphyletic. In Oenothera there appear to be two lineages, one of which has Gaura + Stenosiphon nested within it. At the base of the Onagraceae phylogeny, we have clarified previous confusion regarding conflicting placements of Hauya and Lopezia based on nuclear versus chloroplast data. Results of these analyses are supported by morphology and suggest the need for new taxonomic delimitations, which are forthcoming.

A TALE OF TWO CONTINENTS: BAKER'S RULE AND THE MAINTENANCE OF SELF-INCOMPATIBILITY IN LYCIUM (SOLANACEAE)

Abstract Over 50 years ago, Baker (1955, 1967) suggested that self-compatible species were more likely than self-incompatible species to establish new populations on oceanic islands. His logic was straightforward and rested on the assumption that colonization was infrequent; thus, mate limitation favored the establishment of self-fertilizing individuals. In support of Bakers rule, many authors have documented high frequencies of self-compatibility on islands, and recent work has solidified the generality of Bakers ideas. The genus Lycium (Solanaceae) has ca. 80 species distributed worldwide, and phylogenetic studies suggest that Lycium originated in South America and dispersed to the Old World a single time. Previous analyses of the S-RNase gene, which controls the stylar component of self-incompatibility, have shown that gametophytically controlled self-incompatibility is ancestral within the genus, making Lycium a good model for investigating Bakers assertions concerning reproductive assurance following oceanic dispersal. Lycium is also useful for investigations of reproductive evolution, given that species vary both in sexual expression and the presence of self-incompatibility. A model for the evolution of gender dimorphism suggests that polyploidy breaks down self-incompatibility, leading to the evolution of gender dimorphism, which arises as an alternative outcrossing mechanism. There is a perfect association of dimorphic gender expression, polyploidy, and self-compatibility (vs. cosexuality, diploidy, and self-incompatibility) among North American Lycium. Although the association between ploidy level and gender expression also holds for African Lycium, to date no studies of mating systems have been initiated in Old World species. Here, using controlled pollinations, we document strong self-incompatibility in two cosexual, diploid species of African Lycium. Further, we sequence the S-RNase gene in 15 individuals from five cosexual, diploid species of African Lycium and recover 24 putative alleles. Genealogical analyses indicate reduced trans-generic diversity of S-RNases in the Old World compared to the New World. We suggest that genetic diversity at this locus was reduced as a result of a founder event, but, despite the bottleneck, self-incompatibility was maintained in the Old World. Maximum-likelihood analyses of codon substitution patterns indicate that positive Darwinian selection has been relatively strong in the Old World, suggesting the rediversification of S-RNases following a bottleneck. The present data thus provide a dramatic exception to Bakers rule, in addition to supporting a key assumption of the Miller and Venable (2000) model, namely that self-incompatibility is associated with diploidy and cosexuality.

Phylogenetic Relationships Within Nyctaginaceae Tribe Nyctagineae: Evidence from Nuclear and Chloroplast Genomes

Abstract Nyctaginaceae are a small family of mainly New World tropical and subtropical trees, shrubs, and herbs. To date phylogenetic relationships within the family have not been examined. This study provides the first phylogenetic hypothesis of relationships within Nyctaginaceae tribe Nyctagineae based on sequence data from both nuclear (ITS) and chloroplast (accD 5‘coding region and intergenic region between the rbcL and accD genes). Morphological characters are also discussed as they relate to the phylogeny inferred using molecular data. Results suggest that neither Acleisanthes nor Selinocarpus is monophyletic but that together they comprise a monophyletic lineage. The genus Mirabilis is strongly supported as monophyletic, but the monophyly of two of its sections is suspect. Morphology generally agrees with the molecular data and in some instances reinforces clades weakly supported by nuclear and chloroplast data. Further sampling will help clarify relationships of these genera within Nyctaginaceae. Communicating Editor: Aaron Liston

Floral biology of North American Oenothera sect. Lavauxia (Onagraceae): Advertisements, rewards, and extreme variation in floral depth

Abstract We studied the floral biology of five North American members of Oenothera L. sect. Lavauxia (Spach) Endl. (Onagraceae L.) in field and common greenhouse settings. Oenothera sect. Lavauxia floral morphology ranges from small, cleistogamous flowers (O. flava subsp. flava (A. Nels.) Garrett in Garrett) to some of the longest-tubed flowers in North America (O. flava subsp. taraxacoides (Wooton & Standl.) W. L. Wagner). Our goal was to compare qualitative and quantitative aspects of floral advertisement and reward among taxa in section Lavauxia. All taxa are night-blooming and self-compatible, have yellow petals with ultraviolet reflectance, and produce floral scents dominated by nitrogenous compounds and monoterpenes. Methyl nicotinate is present in the fragrances of all taxa of section Lavauxia regardless of flower size or putative mating system. Because this floral volatile is largely absent from other Oenothera species, we hypothesize that it is a synapomorphy for section Lavauxia. The rare O. acutissima W. L. Wagner, which is endemic to the Uintah Mountains, is polymorphic for odors dominated by linalool- or ocimene-derived compounds. Field observations in its type locality in northeastern Utah, U.S.A., revealed frequent floral visitation by crepuscular hawkmoths during the first 1.5 hours after anthesis, a pattern common to O. flava subsp. taraxacoides and other large-flowered Oenothera throughout western North America. Quantitative aspects of floral advertisement (flower size, scent emission) and reward (nectar volume) are dramatically reduced in putatively autogamous taxa (O. flava subsp. flava, O. triloba Nutt.), whereas qualitative aspects (flower color, scent, and nectar chemistry) remain comparable. All taxa could be distinguished through ordination of characters related to flower size, herkogamy, and scent chemistry. Extreme nectar tube length variation across the range of O. flava renders this an excellent model system for measuring the costs and mechanisms of shifts between outcrossing and autogamy.

Out of America to Africa or Asia: Inference of dispersal histories using nuclear and plastid DNA and the S-RNase self-incompatibility locus

The plant genus Lycium (Solanaceae) originated in the Americas and includes approximately 85 species that are distributed worldwide. The vast majority of Old World species occur in southern Africa and eastern Asia. In this study, we examine biogeographic relationships among Old World species using a phylogenetic approach coupled with molecular evolutionary analyses of the S-RNase self-incompatibility gene. The phylogeny inferred from nuclear granule-bound starch synthase I (GBSSI), nuclear conserved ortholog set II (COSII) marker C2_At1g24360, and plastid spacer data (trnH-pbsA, trnD(GUC)-trnT(GGU), rpl32-trnL(UAG), and ndhF-rpl32) includes a clade of eastern Asian Lycium nested within the African species, suggesting initial dispersal from the Americas to Africa, with subsequent dispersal to eastern Asia. Molecular dating estimates suggest that these dispersal events occurred relatively recently, with dispersal from the Americas to Africa approximately 3.64 Ma (95% highest posterior density [HPD]: 1.58-6.27), followed by subsequent dispersal to eastern Asia approximately 1.21 Ma (95% HPD: 0.32-2.42). In accordance, the S-RNase genealogy shows that S-RNases isolated from Old World species are restricted to four lineages, a subset of the 14 lineages including S-RNases isolated from New World Lycium species, supporting a bottleneck of S-RNase alleles concomitant with a single dispersal event from the Americas to the Old World. Furthermore, the S-RNase genealogy is also consistent with dispersal of Lycium from Africa to Asia, as eastern Asian alleles are restricted to a subset of the lineages that also include African alleles. Such a multilocus approach, including complementary data from GBSSI, COSII, plastid spacer regions, and S-RNase, is powerful for understanding dispersal histories of closely related species.