Lucinda A. McDade

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.

Evolutionary Patterns in Apiaceae: Inferences Based on matK Sequence Data

Phylogenetic relationships among the genera of Apiaceae are poorly understood, and the commonly employed systems of classification of this family are widely regarded as artificial. The understand- ing of evolutionary relationships in Apiaceae has been hampered by difficulties in interpreting traditional taxonomic characters, especially fruit characters. A cladistic analysis of 53 sequences from the chloroplast gene matK was used to provide a phylogenetic perspective for interpreting evolutionary patterns and relationships in Apiaceae. Results of the analysis of mnatK sequence data are in conflict with the subfamilial and tribal treatment of Cerceau-Larrival, as well as the tribal system of Drude. matK sequence data do support much of Drudes subfamilial system, suggesting subfamilies Apioideae and Saniculoideae are largely monophyletic. The saniculoid genus Lagoecia, however, is found among the apioids and should perhaps be transferred to that subfamily. Three of the apioid genera analyzed (Bupleurum, Anginon, and Heteromorpha) are placed in basally-branching clades within subfamily Apioideae. This topology, along with evidence from morphology, anatomy, geographic distribution, and insertions and deletions in matK sequences, suggests that subfamily Apioideae may have originated in southern Africa from woody, simple-leaved ancestors. Relationships among the remaining taxa of Apioideae agree largely with other recent molecular studies, and suggest that the carpological characters used to construct older systems of classification have been prone to parallel evolution.

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.

Phylogenetic relationships among Acanthaceae: evidence from noncoding trnL-trnF chloroplast DNA sequences

We used sequence data from the intron and spacer of the trnL-trnF chloroplast region to study phylogenetic relationships among Acanthaceae. This region is more variable than other chloroplast loci that have been sequenced for members of Acanthaceae (rbcL and ndhF), is more prone to length mutations, and is less homoplasious than these genes. Our results indicate that this region is likely to be useful in addressing phylogenetic questions among but not within genera in these and related plants. In terms of phylogenetic relationships, Elytraria (representing Nelsonioideae) is more distantly related to Acanthaceae sensu stricto (s.s.) than Thunbergia and Mendoncia. These last two genera are strongly supported as sister taxa. Molecular evidence does not support monophyly of Acanthaceae s.s., although there is strong morphological evidence for this relationship. There is strong support for monophyly of four major lineages within Acanthaceae s.s.: the Acanthus, Barleria, Ruellia, and Justicia lineages as here defined. The last three of these comprise a strongly supported monophyletic group, and there is weaker evidence linking the Ruellia and Justicia lineages as closest relatives. Within the Acanthus lineage, our results confirm the existence of monophyletic lineages representing Aphelandreae and Acantheae. Lastly, within the Justicia lineage, we develop initial hypotheses regarding the definition of sublineages; some of these correspond to earlier ideas, whereas others do not. All of these hypotheses need to be tested against more data.

Population-Level Variation in the Expression of Heterostyly in Three Species of Rubiaceae: Does Reciprocal Placement of Anthers and Stigmas Characterize Heterostyly?

Heterostyly (i.e., reciprocal placement of anthers and stigmas between two or three floral morphs) is hypothesized to enhance outcrossing and reduce selfing. However, few studies have documented reciprocity among individual plants; instead, mean anther and stigma heights for floral morphs are usually reported, masking interindividual variation. We measured eight floral dimensions for individuals in five populations of three heterostylous Rubiaceae. The three methods used to quantify reciprocity yielded different conclusions regarding the degree to which populations conformed to expectations for heterostylous plants. Only Psychotria poeppigiana had stigma and, to a lesser degree, anther heights in discrete classes. Variation among plants of Bouvardia ternifolia and Psychotria chiapensis yielded a continuum of anther and stigma heights across populations. Comparison of distances between stigma and anthers indicated that only flowers of B. ternifolia had, as expected, a constant value for this distance. Finally, regression relationships between anther and stigma heights and corolla length showed that only in one population each of B. ternifolia and P. poeppigiana, and in P. chiapensis, was distance between anthers and stigmas the same across the range of corolla sizes for both floral morphs. Variation among these species in expression of heterostyly was not clearly linked to phylogenetic relationship or pollinator syndromes. Two approach herkogamous (AH) species were studied for comparison. Flowers of Psychotria brachiata were consistently AH, but flowers of P. pittieri were highly variable. Determining fitness consequences of population-level variation in sexual systems requires studies linking floral morphology to pollinator behavior and pollen transfer.

Species concepts and problems in practice: insight from botanical monographs

monographs from three journals treating 1,790 species were surveyed to develop an overview of 1) species and infraspecific concepts used by monographers and 2) problems in species delimitation due to variation, hybridization, and asexual reproduction. Most monographers did not discuss species or infraspecific concepts; the majority of those who did used a morphological or taxonomic concept. Infraspecific categories, whether subspecies or varieties, were used by most monographers to delimit morphologically and geographically differentiated entities within species. About 7% of the species monographed were sufficiently variable that species delimitation was difficult and 10% were subdivided infraspecifically. About 12% were involved in hybridization, but in only about 1% was there sufficient complexity to make species delimitation problematic. These data are compared to the incidence of hybridization reported for the flora of the British Isles. About 5% of the species surveyed were hypothesized to be of hybrid origin. Asexual reproduction was a significant problem in monographs treating only two groups. These biological phenomena, partic- ularly hybridization and asexual reproduction, were distributed unevenly across the plant groups monographed, with most presenting few problems. Based on these results, it is argued that there are gaps between theoretical and practical work at the species level that should be bridged. It seems clear that monographers working with most groups need not be unduly concerned that the biological nature of the taxa they study makes them inappropriate for phylogenetic analyses or for the ap- plication of certain species concepts. On the other hand, difficult groups are equally important for understanding the patterns and processes of evolution and their special biological properties should be highlighted.

Phylogenetic Relationships among Acanthaceae: Evidence from Two Genomes

Abstract We used sequence data from the nuclear ribosomal internal transcribed spacer (nr-ITS) region, both alone and in combination with data from the intron and spacer of the trnL-trnF chloroplast (cp) region, to study phylogenetic relationships within the large tropical and subtropical family Acanthaceae. Substitution rate in the nr-ITS region is nearly twice that of the trnL-trnF cp region, and more than twice the rates of other cp loci that have been sequenced for members of Acanthaceae (i.e., rbcL, ndhF). In terms of phylogenetic relationships, the hypothesis based on ITS was largely congruent with the trnL-trnF results. Exceptions are Crossandra pungens and the two Acanthus species, which are placed enigmatically by nr-ITS data. The combined analysis provides strong support for a single hypothesis of relationships among Acanthaceae sensu stricto (s.s.) and their closest relatives. 1) Elytraria (representing Nelsonioideae) is more distantly related to Acanthaceae s.s. than Thunbergia and Mendoncia. 2) These last two genera are sister taxa and together are the sister group of Acanthaceae s.s. 3) Acanthaceae s.s. are monophyletic. 4) There are at least four major monophyletic lineages within Acanthaceae s.s.: the Acanthus, Barleria, Ruellia, and Justicia lineages. 5) These four lineages are related as follows: {Acanthus lineage [Barleria lineage (Justicia + Ruellia lineages)]}. 6) Within the Justicia lineage, there are at least five monophyletic sublineages, related as follows: {Odontonema sublineage [Stenostephanus sublineage (Henrya sublineage {Dicliptera + New World Justicia sublineages})]}. Communicating Editor: Jeff Rettig

Phylogenetic Relationships of the Mangrove Family Avicenniaceae Based on Chloroplast and Nuclear Ribosomal DNA Sequences

Abstract Chloroplast (rbcL, trnL intron, trnL-trnF spacer) and nuclear ribosomal (ITS1, 5.8S, ITS2) DNA sequences were analyzed to identify the closest terrestrial relatives of the mangrove family Avicenniaceae. These plants have been classified within or near Verbenaceae in most synoptic treatments of angiosperms. Surprisingly, Avicenniaceae were placed as part of Acanthaceae s.l. in analyses of all data sets, using both parsimony and maximum likelihood. Within Acanthaceae s.l., our analyses consistently placed Avicennia as sister to Thunbergioideae but without strong support. Constrained maximum likelihood analyses indicated that alternative placements of Avicennia near the base of Acanthaceae s.l. were not significantly less likely than the sister group relationship with Thunbergioideae. However, placement with Verbenaceae was significantly less likely, as was placement with Pedaliaceae. Morphological evidence is reviewed in this phylogenetic context, and we suggest that articulated nodes and inflorescence structure (including flowers subtended by a bract and two bracteoles) may provide synapomorphies for Avicennia and Acanthaceae s.l. We can identify no clear morphological synapomorphies linking Avicennia to Verbenaceae. Avicennia shares a number of features with each of its putative relatives that are likely to be symplesiomorphic or are of uncertain phylogenetic status. Communicating Editor: Aaron Liston

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.

POLLINATOR RELATIONSHIPS, BIOGEOGRAPHY, AND PHYLOGENETICS

ummingbirds and the flowers they visit are among the most colorful examples of mutualistic relationships between animals and flowering plants. The acrobatics, pugnacious behavior, and startling colors of many hummingbird species have won them the attention of biologists and hobbyists alike. In turn, the large and showy flowers of many of the plants pollinated by hummingbirds make them favorites of horticulturalists and well-collected by botanists. I have studied the systematics and reproductive biology of the neotropical plant genus Aphelandra (Acanthaceae). These plants have long, tubular, brightly colored (yellow, orange, red, and pink), and nectar-rich corollas that are the epitome of the hummingbird-pollinated flower (Faegri and van der Pijl 1966). From more than 100 hours spent observing visitors to Aphelandra flowers, I have documented that they are indeed pollinated by hummingbirds. Most of the plant species have strongly curved corollas that are 5.5-7 cm long (Figure la-d); these are pollinated by hermit hummingbirds (subfamily Phaethorninae), whose long, decurved bills match the