Calycinal secretory structures in Calolisianthus pedunculatus (Cham. & Schltdl) Gilg (Gentianaceae): anatomy, histochemistry, and functional aspects.

Secretory structures were little studied in Gentianaceae. Glandular areas on the calyx dorsal region are commonly reported for Helieae species, the main tribe of Gentianaceae. So, the elucidation of nature of glandular areas is particularly relevant. Trichomes secreting mucilage, interpreted as colleters, are reported only for the sepals of Gentianinae species. We aimed to anatomically characterize and identify the nature of the calycinal secretory structures in Calolisianthus pedunculatus. Samples from floral buds, flowers, and fruits were collected, fixed, and processed following usual procedures for light and scanning electron microscopies. Histochemical tests were performed to determine the nature of the secretion. Glucose, fructose, and sucrose were measured with an ELISA reader. Colleters occur on the sepal ventral region and are composed of a multicellular secretory head and a stalk. These structures secrete polysaccharides and proteins, and the secretion is probably released through cuticle microchannels. Nectaries, on the other hand, occur on the sepal dorsal region. They are formed by 3-5 cells arranged in rosettes circling a central cell or pore. These structures also secrete polysaccharides (mainly fructose), lipids, and proteins. The identification of the secretory structures in the sepals of Calolisianthus pedunculatus highlights the importance of anatomical studies in this family. The interpretation of the glandular areas on the calyx of the Helieae species as nectaries has been proven, as well as the confirmation of colleters as common structures in the sepals of Gentianaceae. Besides the taxonomic and phylogenetic importance of nectars and colleters, we highlight the importance of the secretion for the protection of floral buds against dehydration.

Hollow Internodes Permit a Neotropical Understory Plant to Shelter Multiple Mutualistic Ant Species, Obtaining Protection and Nutrient Provisioning (Myrmecotrophy).

The Neotropical understory plant Tachia guianensis (Gentianaceae)-known to shelter the colonies of several ant species in its hollow trunks and branches-does not provide them with food rewards (e.g., extrafloral nectar). We tested whether these ants are opportunistic nesters or whether mutualistic relationships exist as for myrmecophytes or plants sheltering ant colonies in specialized hollow structures in exchange for protection from enemies and/or nutrient provisioning (myrmecotrophy). We noted 37 ant species sheltering inside T. guianensis internodes, three of them accounting for 43.5% of the cases. They protect their host plants from leaf-cutting ant defoliation and termite damage because individuals devoid of associated ants suffered significantly more attacks. Using the stable isotope 15N, we experimentally showed that the tested ant species furnish their host plants with nutrients. Therefore, a mutualism exists. However, because it is associated with numerous ant species, T. guianensis can be considered a nonspecialized myrmecophyte.

Plasticity of floral longevity and floral display in the self-compatible biennial Sabatia angularis (Gentianaceae): untangling the role of multiple components of pollination.

BACKGROUND AND AIMS: Plasticity of floral traits in response to pollination can enable plants to maximize opportunities for pollen import and export under poor pollination conditions, while minimizing costs under favourable ones. Both floral longevity and display are key traits influencing pollination. While pollination-induced flower wilting is widely documented, we lack an understanding of the multifactorial complexity of this response, including the influence of other pollination components, costs of extended longevity and subsequent impacts on floral display. METHODS: Plasticity of floral longevity was experimentally evaluated in Sabatia angularis in response to multiple pollination factors: pollen addition, removal, and source (self, single-donor outcross, multiple-donor outcross) and timing of pollination. Effects of pollen quantity were further evaluated by exploiting variation in autonomous self-pollen deposition. Delayed pollination costs were tested comparing seed set from early versus late pollinations. Finally, I compared floral display metrics (peak floral display, time to peak flower, flowering duration, mean flowering rate) between experimentally pollinated and control plants. KEY RESULTS: Floral longevity was highly plastic in response to pollen addition and its timing, and the response was dose-dependent but insensitive to pollen source. Pollen removal tended to extend floral longevity, but only insofar as it precluded pollination-induced wilting via autonomous self-pollination. Under delayed pollination, the wilting response was faster and no cost was detected. Pollination further led to reduced peak floral displays and condensed flowering periods. CONCLUSIONS: Floral longevity and display plasticity could optimize fitness in S. angularis, a species prone to pollen limitation and high inbreeding depression. Under pollinator scarcity, extended floral longevities offer greater opportunities for pollen receipt and export at no cost to seed set, reproductive assurance via autonomous self-pollination and larger, more attractive floral displays. Under high pollinator availability, shortened longevities lead to smaller displays that should lower the risk of geitonogamy.

Florivory Modulates the Seed Number-Seed Weight Relationship in Halenia elliptica (Gentianaceae).

Generally, plant reproductive success might be affected negatively by florivory, and the effects may vary depending on the timing and intensity of florivory. To clarify the impacts of florivory by the sawfly larvae (Tenthredinidae) on seed production of Halenia elliptica D. Don, we simulated florivory by removing different proportion of flowers at three reproductive stages in this alpine herb and then examined the seed number per fruit, the seed weight, and the seed mass per fruit of the remaining flowers. Seed number per fruit reduced significantly when flowers were removed at flowering and fruiting stages or when 15% and 60% of flowers were removed. However, seed weight increased significantly after flowers were removed, independent of treatments of reproductive stage and proportion. There was a similar seed mass per fruit between the plants subjected to simulation of florivory and control. The results indicated that florivory modulated the seed number-seed weight relationship in this alpine species. Our study suggested that selective seed abortion and resource reallocation within fruits may ensure fewer but larger seeds, which were expected to be adaptive in the harsh environments.

Extrafloral nectaries in neotropical Gentianaceae: occurrence, distribution patterns, and anatomical characterization.

PREMISE OF THE STUDY: Extrafloral nectaries (EFNs) are structures that secrete nectar and protect plants against herbivores and pathogens. In Gentianaceae, these structures have been described in species of Calolisianthus, Fagraea, and Anthocleista and are important morphological markers for taxonomic and phylogenetic studies. To establish a foundation for further studies, we investigated the occurrence, distribution patterns, and anatomy of EFNs on leaves of 27 species belonging to 13 genera and three tribes of neotropical Gentianaceae. • METHODS: Leaf samples were diaphanized, stained with basic fuchsin, and mounted in glycerinated gelatin. Cross sections were obtained from material embedded in methacrylate or paraffin, stained, and mounted in Permount. Polysaccharides were histochemically stained with periodic acid-Schiff stain. Samples were also examined with scanning electron microscopy. • KEY RESULTS: Unusual EFNs, visible only with light microscopy, were formed of modified epidermal cells. Each EFN consisted of 2-5 secretory cells encircling a central cell. The EFNs varied in size and in the shape and arrangement of the adjacent cells surrounding the secretory cells. EFNs occurred in all analyzed species as isolated units distributed throughout the leaf blade or as aggregates; aggregates were generally visible to the naked eye. Based on their occurrence as aggregates or isolated units and on their location on the leaf blade, six distribution patterns were identified. • CONCLUSIONS: This is the first comprehensive study of EFNs on the leaves of neotropical Gentianaceae. The data suggested that NEFs evolved from isolated units for NEFs in aggregates. The results represent a new source of data for future ecological, systematic, and phylogenetic studies in Gentianaceae.

Ancestral and monophyletic presence of diplostigmaty in Sebaea (Gentianaceae) and its potential role as a morphological mixed mating strategy.

* Diplostigmaty, the presence of a primary (apical) stigma and secondary (mid-stylar) stigmas along the style, is only known from the genus Sebaea (Gentianaceae). Early work indicated that the secondary stigmas provide a mechanism of autogamy, suggesting that it might ensure reproductive assurance. * Here, we test the monophyly of this unique morphological trait. Using Bayesian methods, we infer a nuclear DNA phylogeny for 96 accessions, including c. 50% of the species from the genus Sebaea. With this phylogeny, we infer the distribution of ancestral states on critical nodes using parsimony and likelihood methods. * The inferred nrDNA phylogeny shows that the genus Sebaea is divided in two statistically well-supported clades, A and B, consistent with recent estimates. The most recent ancestor (MRCA) of clade A, except the most basal species (Sebaea pusilla), is resolved as diplostigmatic. No reversal to a single stigma is observed within this clade. * We suggest that diplostigmaty is evolutionarily stable through time. We also discuss why this reproductive system is not found elsewhere than in Gentianaceae and the potential advantage of diplostigmaty as a stable mixed mating strategy.

The polyphyletic genus Sebaea (Gentianaceae): a step forward in understanding the morphological and karyological evolution of the Exaceae.

Within the Gentianaceae-Exaceae, the most species-rich genus Sebaea has received very little attention in terms of phylogenetic or karyological investigations. As a result, the exact number of species remains vague and the relationships with the other members of the Exaceae poorly understood. In this paper, we provide the first comprehensive phylogeny of the Exaceae including most Sebaea species known so far based on four cpDNA sequence regions. In addition, morphological and karyological characters were mapped on the inferred phylogenetic trees to detect possible non-molecular synapomorphies. Our results reveal the paraphyly of Sebaea and highlight new generic relationships within the Exaceae. Sebaea pusilla (lineage S1--Lagenias) forms a highly supported and early diverging clade with Sebaeas.str. (clade S2 -Sebaea). A third clade of the former Sebaea s.l. (clade S3--Exochaenium) contains exclusively tropical African species, and is sister with a large clade containing all the remaining genera of Exaceae. Within the latter, the proposed sister relationships between the recently described Klackenbergia and Ornichia are highly supported. Optimization of several morphological characters onto the inferred phylogenetic trees reveals several synapomorphies for most highly supported clades. In particular, lineage S1 (Lagenias) is supported by medifixed anthers that are inserted at the base of the corolla tube and cubical seeds with polygonal testa cells; clade S2 (Sebaea) is supported by both the presence of secondary stigmas along the style and ridged seeds with rectangular testa cells arranged in row; clade S3 (Exochaenium) is supported by its particular gynoecium (stylar polymorphism and clavate, papillose stigma). Finally, karyological reconstructions suggest a basal number of x=7 for the Exaceae and several episodes of dysploidy leading to x=8 and 9.

Application of microscopy in authentication of traditional Tibetan medicinal plant Halenia elliptica.

Halenia elliptica D. Don, a popularly used ethnodrug from Qinghai-Tibetan plateau, was studied to reveal the indispensable morphoanatomic details. The fixed, sectioned, and stained plant materials as well as the epidermis, powder, and maceration materials were studied using light microscope according to the usual microscopic techniques. The results of the microscopic features were systematic described and illustrated. In the root, an endodermal cell was divided into 8-16-22 and 38-50-62 daughter cells in transverse section and in face view, respectively, and 9-11-13 phloem strands were present in primary structure; in the stem, stone cells were observed in the cortex, pericycle, and external phloem while 17-19-21 internal phloem strands were present in an incontinuous ring; in the pedicel, 8-10-12 internal phloem strands were observed to form an incontinuous ring; anisocytic and anomocytic stomata were present in leaf and sepal epidermis; pollen grain was with three germinal apertures and furrows; a few tracheids, a large number of spiral vessels, and various fibers were observed. Also, semiquantitative and quantitative micrographic parameter tables were simultaneously presented. Further, the key authentication parameters were concluded. The study indicated that light microscopy and related techniques could be unambiguously applied to the authentication of Halenia elliptica.

The diversification of Halenia (Gentianaceae): ecological opportunity versus key innovation.

The plant genus Halenia (Gentianaceae) consists of herbs growing in temperate and tropical alpine habitats and most species possess flowers in which nectar is produced in spurs. This probably helps reward only specialized long-tongued pollinators, and a narrow pollinator/flower relationship is thought to accelerate diversification rates (a key innovation). To test the pattern of diversification of Halenia against the unspurred sister group we reconstructed phylogenetic relationships among 22 species plus outgroups using nuclear ITS and chloroplast rpl16 intron sequence data. We show that Halenia originated in East Asia and migrated via North America into Central America. From there, it colonized South America three times independently, probably within the last million years. Significant changes in diversification rates were found during the evolution of Halenia using a sister group method, a likelihood method, and a diversity-through-time plot. In contrast to other studies, we could not observe a direct speciation rate effect of the evolution of nectar spurs in comparison with the unspurred sister group of Halenia. Rather, increases in diversification occurred following the colonization of Central and South America by spurred progenitor taxa. This later switch in diversification may have resulted from the availability of new geographical and ecological opportunities, or from the availability of more and different pollinators in these regions. Following the latter hypothesis, the nectar spurs were a preadaption and functioned as a key innovation only in this new biotic environment. After an initial rapid increase, a reduction in diversification rate was observed in Central America, probably illustrating density dependence of speciation rates. Finally, we found preliminary evidence for the key innovation hypothesis in geologically young spurred and unspurred lineages of Halenia in South America.