Looking for non-hermaphrodite cacti: multidisciplinary studies in Gymnocalycium bruchii endemic to central Argentina.

KEY MESSAGE: Through a multidisciplinary study we found that Gymnocalycium bruchii, an endemic cactus from central Argentina, acts as a dioecious species, which is the first record in this genus. Cactaceae species are typically hermaphroditic; however, about 2% have other different reproductive systems. These non-hermaphroditic species may develop sexual dimorphism in flowers or other reproductive, vegetative or ecological traits, besides a specific breeding system and floral ontogeny. Therefore, multidisciplinary research is necessary to fully understand reproduction in those species. For this purpose, we studied Gymnocalicium bruchii, a globose cactus endemic to central Argentina that is presumably dioecious or gynodioecious. We made observations in two natural and two cultivated populations. We made morphological observations of plants and flowers, and performed quantitative analyses to determine the sex ratio, size of plants and flowers, flower production, fruiting, among other variables. We performed hand-pollination, self-fertilization and free-pollination tests to determine the breeding system. Finally, we studied the anatomy and ontogeny of the reproductive organs using permanent histological slides of flower morphs at different stages. Our results confirm that Gymnocalicium bruchii is a dioecious species. Female flowers have atrophied anthers and a functional gynoecium that produces fruits and seeds. Male flowers are bigger and have a functional androecium but a sterile gynoecium. In the cultivated population, the sex ratio was 1/1, whereas the number of male individuals was higher in both natural populations. Pollination tests corroborated dioecy. Ontogenetic studies revealed that in female flowers the anthers collapse before microspore maturation, while in male flowers the gynoecium shows normal development of the ovary, style, stigma, and ovules; however, the latter are never fertilized.

Determination of amino acid content, fatty acid profiles, and phenolic compounds in non-conventional edible fruits of seven species of palm trees (Arecaceae) native to the southern half of South America.

The fruits of palm trees (Arecaceae) are a very important food source for fauna and for humans, despite the still incipient role of these fruits in conventional diet. Seven palm tree species (plus one variety) that are native to the southern half of South America located east of the Andes (Argentina, Paraguay, Uruguay, and the southern states of Brazil) were studied. For the first time, the fatty acid profiles of palm seeds and the content of both amino acids and phenolic compounds present in the pulp (yellowish fleshy mesocarps) of the fruits were determined. High-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was used to screen and quantify thirty-two phenols and HPLC was used to determine the amino acid contents and fatty acids profiles of the species. Twenty-five percent of the fatty acids in the seeds of B. lallemantii, Butia odorata (var. pulposa), B. witeckii, and Syagrus romanzoffiana were found to be unsaturated. One species (B. lallemantii) had a high and well-balanced amino acid content (28,900 mg/kg), while six species (B. odorata, B. catarinensis, B. witeckii, Euterpe edulis, and Syagrus romanzoffiana) contained more than 14,000 mg/kg of amino acids. A high content of the phenolic compounds ferulic acid, luteolin, quercetin-3-rutinoside, isoquercetin, and isorhamnetin were found in many species, most notably in B. odorata, B. catarinensis, and B. witeckii.

Müllerian mimicry between oil-producing orchids and Malpighiaceae? An old hypothesis finally tested.

The concept of mimicry has been developed for animals, but it also applies to plants. Plant species may be Müllerian mimics if they have similar reproductive traits and offer similar rewards to the pollinators. Several Oncidiinae orchids offer floral oils to their pollinators and have been suggested to form a Müllerian complex with species of Malpighiaceae. We provide a test of this hypothesis using Gomesa flexuosa (Orchidaceae) and Janusia guaranitica (Malpighiaceae), which are sympatric and phenologically synchronous, secrete the same floral resource (oils), and show similar flower morphology. We exposed individuals of Gomesa near and far from individuals of Janusia and monitored floral visitation. Both species were exclusively pollinated by oil-collecting bees, sharing Centris trigonoides as a pollinator. Nevertheless, the probability of bee visitation, number of contact approximations, and number of visits to flowers of Gomesa were similar when individuals were near and far from Janusia. These findings do not support the Müllerian mimicry hypothesis in these two species. Their resemblances can be better explained by the "exploitation of perceptual biases" hypothesis. According to this hypothesis, pre-existing traits in Oncidiinae orchids (e.g. colour, shape, rewards) may coopt oil-collecting bees that usually search for rewards in Malpighiaceae species with similar flower traits.

Storage of orchid pollinia with varying lipid thermal fingerprints.

Orchid pollinia have the potential to make a valuable contribution to current techniques of germplasm storage and assisted reproduction, yet information regarding their preservation and their ability to remain viable over time is currently limited. Dactylorhiza fuchsii and Disa uniflora were used as models for investigating potential techniques for storing orchid pollinia. Initially, freshly harvested pollinia of Dact. fuchsii were incubated at 25 °C and 100% RH (relative humidity) for up to 7 days and germinated in vitro. For pollinia from both species, moisture sorption isotherms were constructed and thermal fingerprints generated using differential scanning calorimetry (DSC). Pollinia were stored at three temperatures (5, - 18 and - 196 °C) after equilibration at four different RHs (5, 33, 50 and 75%) and germinated. The isotherms and DSC results varied between species. Compared with D. uniflora, pollinia of Dact. fuchsii consistently equilibrated at higher moisture content (MC) for each RH, had less detectable lipids by DSC and had shorter lifespans, remaining viable after 3-4 months only at - 20 and - 196 °C and at low RH (5 and 33%). Both species' pollinia stored well at - 20 °C and - 196 °C, although there was some evidence of a small loss of viability under cryopreservation. In conclusion, pollen of these two species can be stored successfully for at least 3-4 months, and to maximize the pre-storage quality, it is recommended that fresh pollen is collected from flowers just prior to anthesis.

Taxonomic notes on Lyroglossa and Pteroglossa (Orchidaceae: Spiranthinae): two new generic records for the flora of Rio Grande do Sul.

Lyroglossa and Pteroglossa (Orchidaceae: Spiranthinae) are first recorded for Rio Grande do Sul. Descriptions, illustrations, taxonomic notes and distribution are presented. We also provide an artificial key to distinguish the genera of the "Stenorrhynchos Clade" reported so far in Rio Grande do Sul.

Floral features, pollination biology and breeding system of Chloraea membranacea Lindl. (Orchidaceae: Chloraeinae).

BACKGROUND AND AIMS: The pollination biology of very few Chloraeinae orchids has been studied to date, and most of these studies have focused on breeding systems and fruiting success. Chloraea membranacea Lindl. is one of the few non-Andean species in this group, and the aim of the present contribution is to elucidate the pollination biology, functional floral morphology and breeding system in native populations of this species from Argentina (Buenos Aires) and Brazil (Rio Grande do Sul State). METHODS: Floral features were examined using light microscopy, and scanning and transmission electron microscopy. The breeding system was studied by means of controlled pollinations applied to plants, either bagged in the field or cultivated in a glasshouse. Pollination observations were made on natural populations, and pollinator behaviour was recorded by means of photography and video. KEY RESULTS: Both Argentinean and Brazilian plants were very consistent regarding all studied features. Flowers are nectarless but scented and anatomical analysis indicates that the dark, clavate projections on the adaxial labellar surface are osmophores (scent-producing glands). The plants are self-compatible but pollinator-dependent. The fruit-set obtained through cross-pollination and manual self-pollination was almost identical. The main pollinators are male and female Halictidae bees that withdraw the pollinarium when leaving the flower. Remarkably, the bees tend to visit more than one flower per inflorescence, thus promoting self-pollination (geitonogamy). Fruiting success in Brazilian plants reached 60·78 % in 2010 and 46 % in 2011. Some pollinarium-laden female bees were observed transferring pollen from the carried pollinarium to their hind legs. The use of pollen by pollinators is a rare record for Orchidaceae in general. CONCLUSIONS: Chloraea membrancea is pollinated by deceit. Together, self-compatibility, pollinarium texture, pollinator abundance and behaviour may account for the observed high fruiting success. It is suggested that a reappraisal and re-analysis of important flower features in Chloraeinae orchids is necessary.

A comparative survey of floral characters in Capanemia Barb. Rodr. (Orchidaceae: Oncidiinae).

BACKGROUND AND AIMS: Capanemia Barb. Rodr. comprises seven species that mostly inhabit the Brazilian Atlantic Rain Forest domain. The genus currently consists of two sections: Capanemia Cogn. and Planifolia Pabst, distinguished on the basis of leaf shape. We compare the floral morphology and anatomy of all species to determine whether separation into sections is supported by floral characters. METHODS: Both fresh flowers and herbarium specimens were investigated, and column and pollinarium features, together with the presence or absence of floral rewards, recorded. Anatomical features were examined using both light microscopy and scanning electron microscopy. KEY RESULTS AND CONCLUSIONS: With the sole exception of Capanemia therezae, all species shared a distinctive set of floral characters. Flowers were mostly white or yellowish-white and fragrant, and column wings were positioned parallel to the labellum, concealing the stigmatic cavity. Pollinaria had proportionally long tegular stipes and clavate to reniform pollinia, whereas the labellum possessed a conspicuous indument of trichomes, but was devoid of nectar or any other secretion that might function as a food-reward. Capanemia therezae, however, was exceptional in having greenish, unscented flowers with short, rounded and divergent column wings and an exposed stigmatic cavity. Its pollinaria had proportionally short tegular stipes and round pollinia, whereas the labellum lacked trichomes. Droplets of nectar were evident on the adaxial surface of the labellum, adjacent to the callus. Floral features did not support the currently accepted sectional division of Capanemia. If ongoing phylogenetic studies demonstrate that both sections are indeed monophyletic, then these taxa should be distinguished solely on the basis of foliar features.

Evaluation of anti-estrogenic or estrogenic activities of aqueous root extracts of Gunnera manicata L / Avaliação das atividades anti-estrogênicos ou estrogênica de raiz aquosa extraida de Gunnera manicata L

Gunnera perpensa (Gunneraceae) is an African plant widely used in traditional medicine. This species is known for its activity involving the female reproductive system, such as inducing or increasing labor, treating female infertility, expelling the placenta and/or preventing post-partum hemorrhage. These properties are probably due to (z)-venusol, a majoritary compound, and its action in conjunction with substances in the whole extract and other natural products. In southern Brazil, a native species Gunnera manicata L. that also belongs to Gunneraceae can be found. In spite of the traditional use of G. perpensa, there is no pharmacological and phytochemical information regarding the South American Gunnera species. Therefore, the aim of this study was to investigate the activity of Brazilian G. manicata aqueous extracts on the reproductive system of immature female Wistar rats through a uterotrophic assay and to verify the presence of (z)-venusol by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Data were analyzed by analysis of variance (ANOVA) and Bonferroni´s post-hoc test (p< 0.01). Results obtained shown that G. manicata extracts did not present in vivo anti or estrogenic activity. Furthermore, (z)-venusol compound was not found. This study represents the first preliminary screening done on the South American G. manicata species.

Gunnera perpensa (Gunneraceae) é uma planta de origem africana extensamente utilizada na medicina tradicional do país. Esta espécie é conhecida por suas atividades no sistema reprodutor feminino, como indução ou aumento do trabalho de parto, tratamento da infertilidade em mulheres, expulsão da placenta e/ou impedimento de hemorragia pós-parto. Tais atividades devem-se, provavelmente, ao sinergismo existente entre o (z)-venusol, composto majoritário, e outros compostos presentes na planta. No sul do Brasil, encontra-se uma espécie nativa, Gunnera manicata L., pertencente à família Gunneraceae. Apesar do uso tradicional de G. perpensa, não há informações farmacológicas e fitoquímicas a respeito da espécie sul Americana de Gunnera. Assim, o objetivo deste estudo foi investigar a atividade de extratos aquosos da espécie brasileira G. manicata no sistema reprodutor de ratas Wistar imaturas através de ensaio uterotrófico e verificar a presença do composto (z)-venusol utilizando-se cromatografia líquida acoplada a espectrômetro de massas em tandem (CL-EM/EM). Para a análise estatística, utilizou-se ANOVA/Bonferroni (p<0,01). Os resultados obtidos demonstraram que os extratos de G. manicata testados não apresentaram atividade anti ou estrogênica in vivo. Na análise química não foi verificada a presença do composto (z)-venusol. Este estudo representa o primeiro screening realizado com a espécie sul-americana G. manicata.

Morfología florll y polinización de orquídeas: el segundo libro de charles darwin / Floral Morphology and Pollination in Orchidaceae: Charles Darwin s Second Book

El segundo libro de Darwin fue íntegramente dedicado a la morfología floral y polinización de diversos grupos de orquídeas de regiones templadas y tropicales. Este libro fue publicado en 1862 y parece haber sido concebido como una fuente de pruebas o un complemento para ideas sugeridas en El origen de las especies, en especial la noción sobre las ventajas del cruzamiento entre individuos diferentes, aunque sean hermafroditas (como es el caso de las orquídeas). La gran diversidad de morfologías florales y las diversas estrategias reproductivas que promueven la polinización cruzada en Orchidaceae fascinaron a Darwin, quien utilizó a este grupo de plantas como modelo para apoyar sus ideas. Darwin describió por primera vez y de modo impecable estrategias reproductivas como la protandria en orquídeas terrestres y la producción de flores imperfectas (unisexuales) en Catasetum, entre muchas otras contribuciones. Se analizan las ideas y propuestas de Darwin en este libro a la luz de nuestros conocimientos actuales y se muestran en gran parte correctas y vigentes.

Darwin s second book was totally dedicated to the floral functional morphology and pollination of temperate and tropical orchids. This book was published in 1862 and was likely conceived as an assemblage of evidence supporting ideas that were proposed in -On The Origin of The Species-; namely, the advantages of the intercrossing between different coespecific individuals, even if they are hermaphrodite (like the orchids). The great floral diversity and the outstanding number of reproductive strategies that promote cross-pollination in Orchidaceae fascinated Darwin who, in turn, used this plant group as a model to support his ideas. Darwin described for the first time and in a very accurate way, orchid reproductive strategies that clearly promote cross-pollination, such as protandry in terrestrial orchids and the production of unisexual flowers in Catasetum, among many other important contributions. Darwin s ideas and proposals regarding Orchidaceae are analyzed on the light of our present knowledge and prove to be mostly correct and valid.

Stingless bees: chemical differences and potential functions in Nannotrigona testaceicornis and Plebeia droryana males and workers.

Cuticular wax, abdominal and cephalic extracts of foraging workers and males of Nannotrigona testaceicornis and Plebeia droryana, from the "Aretuzina" farm in São Simão, SP, Brazil, were analyzed by GC-MS. The principal constituents were hydrocarbons, terpenes, aldehydes, esters, steroids, alcohols, and fatty acids. Interspecific differences for both cuticular wax and cephalic extracts were found. The composition of cuticular wax and cephalic extracts was similar at the intraspecific level, with minor component differences between males and workers. Abdominal extracts differentiated sexes (male and worker) at the intraspecific and interspecific levels. The main chemical components in abdominal extracts of N. testaceicornis workers and males were geranylgeranyl acetate and (Z)-9-nonacosene, respectively. The principal components of abdominal extracts from P. droryana workers and males were tetradecanal and unsaturated fatty acids (linoleic and linolenic acids), respectively. A secondary alcohol, (S)-2-nonanol, was detected in Plebeia droryana males only, but not in workers. Preliminary field experiments showed that (S)-(+)-2-heptanol and (S)-(+)-2-heptanol/(S)-(+)-2-nonanol (1:1) attracted workers of P. droryana, N. testaceicornis,and Frieseomelitta silvestrii. However, males did not respond suggesting that these compounds do not function as alarm or recruitment pheromones. In addition, racemic mixtures were inactive.

Investigação da presença de efedrinas em Ephedra tweediana Fisch & C.A. Meyer e em E. triandra Tul. (Ephedraceae) coletadas em Porto Alegre/RS / Investigation of the presence of ephedrines in Ephedra tweediana Fisch & C.A. Meyer and E. triandra Tul. (Ephedraceae) collected in Porto Alegre/RS

Amostras de Ephedra tweediana Fisch & C.A. Meyer, coletadas de populações nativas da Reserva Biológica do Lami José Lutzenberger (Porto Alegre, RS, Brasil), e amostras de Ephedra triandra Tul., obtidas de plantas cultivadas em Porto Alegre/RS, foram extraídas com acetona, derivatizadas com ciclohexanona e analisadas por CG/EM. Para verificação da eficiência da metodologia, além das amostras de Ephedra tweediana e E. triandra, foram analisadas cinco amostras comerciais de Ephedra, de procedências distintas, cedidas por farmácias de manipulação locais. Os resultados encontrados indicam a ausência de efedrinas em Ephedra tweediana e E. triandra e presença de efedrina e/ou pseudoefedrina nas amostras comerciais.

Samples of Ephedra tweediana, collected from native populations occurring in the Reserva Biológica do Lami José Lutzenberger (Porto Alegre, RS, Brazil), and from cultivated plants of Ephedra triandra were submitted to extraction with acetone, derivatized with cyclohexanone and analyzed by GC/MS. In order to verify the efficiency of the methodology, besides Ephedra tweediana and E. triandra, samples of five commercial Ephedra extracts were analyzed, from distinct origins, get up from local drugstores. The results showed the absence of ephedrines in Ephedra tweediana and E. triandra, and the presence of ephedrine and/or pseudoephedrine in commercial samples.

Molecular phylogeny of the neotropical genus Christensonella (Orchidaceae, Maxillariinae): species delimitation and insights into chromosome evolution.

BACKGROUND AND AIMS: Species' boundaries applied within Christensonella have varied due to the continuous pattern of variation and mosaic distribution of diagnostic characters. The main goals of this study were to revise the species' delimitation and propose a more stable classification for this genus. In order to achieve these aims phylogenetic relationships were inferred using DNA sequence data and cytological diversity within Christensonella was examined based on chromosome counts and heterochromatin patterns. The results presented describe sets of diagnostic morphological characters that can be used for species' identification. METHODS: Phylogenetic studies were based on sequence data of nuclear and plastid regions, analysed using maximum parsimony and maximum likelihood criteria. Cytogenetic observations of mitotic cells were conducted using CMA and DAPI fluorochromes. KEY RESULTS: Six of 21 currently accepted species were recovered. The results also support recognition of the 'C. pumila' clade as a single species. Molecular phylogenetic relationships within the 'C. acicularis-C. madida' and 'C. ferdinandiana-C. neowiedii' species' complexes were not resolved and require further study. Deeper relationships were incongruent between plastid and nuclear trees, but with no strong bootstrap support for either, except for the position of C. vernicosa. Cytogenetic data indicated chromosome numbers of 2n = 36, 38 and 76, and with substantial variation in the presence and location of CMA/DAPI heterochromatin bands. CONCLUSIONS: The recognition of ten species of Christensonella is proposed according to the molecular and cytogenetic patterns observed. In addition, diagnostic morphological characters are presented for each recognized species. Banding patterns and chromosome counts suggest the occurrence of centric fusion/fission events, especially for C. ferdinandiana. The results suggest that 2n = 36 karyotypes evolved from 2n = 38 through descendent dysploidy. Patterns of heterochromatin distribution and other karyotypic data proved to be a valuable source of information to understand evolutionary patterns within Maxillariinae orchids.

Dating the origin of the Orchidaceae from a fossil orchid with its pollinator.

Since the time of Darwin, evolutionary biologists have been fascinated by the spectacular adaptations to insect pollination exhibited by orchids. However, despite being the most diverse plant family on Earth, the Orchidaceae lack a definitive fossil record and thus many aspects of their evolutionary history remain obscure. Here we report an exquisitely preserved orchid pollinarium (of Meliorchis caribea gen. et sp. nov.) attached to the mesoscutellum of an extinct stingless bee, Proplebeia dominicana, recovered from Miocene amber in the Dominican Republic, that is 15-20 million years (Myr) old. This discovery constitutes both the first unambiguous fossil of Orchidaceae and an unprecedented direct fossil observation of a plant-pollinator interaction. By applying cladistic methods to a morphological character matrix, we resolve the phylogenetic position of M. caribea within the extant subtribe Goodyerinae (subfamily Orchidoideae). We use the ages of other fossil monocots and M. caribea to calibrate a molecular phylogenetic tree of the Orchidaceae. Our results indicate that the most recent common ancestor of extant orchids lived in the Late Cretaceous (76-84 Myr ago), and also suggest that the dramatic radiation of orchids began shortly after the mass extinctions at the K/T boundary. These results further support the hypothesis of an ancient origin for Orchidaceae.

Molecular phylogenetics of Maxillaria and related genera (Orchidaceae: Cymbidieae) based on combined molecular data sets.

The orchid genus Maxillaria is one of the largest and most common of neotropical orchid genera, but its current generic boundaries and relationships have long been regarded as artificial. Phylogenetic relationships within subtribe Maxillariinae sensu Dressler (1993) with emphasis on Maxillaria s.l. were inferred using parsimony analyses of individual and combined DNA sequence data. We analyzed a combined matrix of nrITS DNA, the plastid matK gene and flanking trnK intron, and the plastid atpB-rbcL intergenic spacer for 619 individuals representing ca. 354 species. The plastid rpoC1 gene (ca. 2600 bp) was sequenced for 84 selected species and combined in a more limited analysis with the other data sets to provide greater resolution. In a well-resolved, supported consensus, most clades were present in more than one individual analysis. All the currently recognized minor genera of "core" Maxillariinae (Anthosiphon, Chrysocycnis, Cryptocentrum, Cyrtidiorchis, Mormolyca, Pityphyllum, and Trigonidium) are embedded within a polyphyletic Maxillaria s.l. Our results support the recognition of a more restricted Maxillaria, of some previously published segregate genera (Brasiliorchis, Camaridium, Christensonella, Heterotaxis, Ornithidium, Sauvetrea), and of several novel clades at the generic level. These revised monophyletic generic concepts should minimize further nomenclatural changes, encourage monographic studies, and facilitate more focused analyses of character evolution within Maxillariinae.

Pollination by sexual mimicry in Mormolyca ringens: a floral chemistry that remarkably matches the pheromones of virgin queens of Scaptotrigona sp.

The chemical composition of some volatile (2-heptanol) and nonvolatile constituents (a homologous 9-alkene/alkane series) of Mormolyca ringens flowers and Scaptotrigona sp. queen waxes (homologous 9-alkene/alkane series) and cephalic extracts (homologous series of 2-alkanols, including 2-heptanol) involved with the pseudocopulation or sexual mimicry in Orchidaceae pollination is compared. The similarity in chemical composition of flowers and insects is assigned to the chemically induced copulatory activity in Scaptotrigona males.

The chemistry of pollination in selected Brazilian Maxillariinae orchids: floral rewards and fragrance.

We report the chemical composition of the floral rewards and the fragrance of 10 Maxillariinae (Orchidaceae) species. The species that offer rewards (labellar secretions) are usually scentless, the rewards being collected by bees. Chemical analyses revealed that the major chemical class of compounds present in the labellar secretions are triterpenoids. The rewardless Maxillariinae flowers were usually scented, and chemical analyses of their volatiles revealed that they were composed of mono and sesquiterpenoids.

Sexual mimicry in Mormolyca ringens (Lindl.) Schltr. (Orchidaceae: Maxillariinae).

BACKGROUND AND AIMS: Pollination through sexual mimicry, also known as pseudocopulation, has been suggested to occur in some genera of the Neotropical orchid subtribe Maxillariinae. However, it has been demonstrated so far only for Trigonidium obtusum. This study reports and illustrates pollination through sexual mimicry in Mormolyca ringens. METHODS: A total of 70 h were dedicated to the observation of flowers and pollinator behaviour, which was photographically recorded. Flower features involved in pollinator attraction were studied using a stereomicroscope and by SEM analyses. Preliminary observations on the plant breeding system were made by manually self-pollinating flowers. The chemical composition of the fragrance volatiles was determined by GC/MS analysis. KEY RESULTS: The flower features of M. ringens parallel those of other pseudocopulatory flowers. The labellum shape and indument are reminiscent of an insect. Sexually excited drones of Nannotrigona testaceicornis and Scaptotrigona sp. (both in the Apidae: Meliponini) attempt copulation with the labellum and pollinate the flower in the process. In both bee species, the pollinarium is attached to the scutellum. Pollinator behaviour may promote some degree of self-pollination, but preliminary observations indicate that M. ringens flowers are self-incompatible. Flowers are produced all the year round, which ties in with the production of bee males several times a year. The phylogenetic relationships of M. ringens are discussed and a number of morphological and phenological features supporting them are reported. CONCLUSIONS: It is expected that further research could bring to light whether other Maxillariinae species are also pollinated through sexual mimicry. When a definitive and robust phylogeny of this subtribe is available, it should be possible to determine how many times pseudocopulation evolved and its possible evolutionary history.

Pollinarium morphology and floral rewards in Brazilian Maxillariinae (Orchidaceae).

BACKGROUND AND AIMS: There is strong support for the monophyly of the orchid subtribe Maxillariinae s.l., yet generic boundaries within it are unsatisfactory and need re-evaluation. In an effort to assemble sets of morphological characters to distinguish major clades within this subtribe, the pollinarium morphology and floral rewards of representative Brazilian species of this subtribe were studied. METHODS: The study was based on fresh material from 60 species and seven genera obtained from cultivated specimens. Variation of pollinarium structure and floral rewards was assessed using a stereomicroscope and by SEM analysis. KEY RESULTS: Four morphological types of pollinaria are described. Type 1 appears to be the most widespread and is characterized by a well-developed tegula. Type 2 lacks a stipe and the pollinia are attached directly to the viscidium. Type 3 also lacks a stipe, and the viscidium is rigid and dark. In Type 4, the stipe consists of the whole median rostelar portion and, so far, is known only from Maxillaria uncata. Nectar, trichomes, wax-like and resin-like secretions are described as flower rewards for different groups of species within the genus Maxillaria. Data on the biomechanics and pollination biology are also discussed and illustrated. In Maxillariinae flowers with arcuate viscidia, the pollinaria are deposited on the scuttellum of their Hymenopteran pollinators. In contrast, some flowers with rounded to rectangular, pad-like viscidia fix their pollinaria on the face of their pollinators. CONCLUSIONS: Pollinarium morphology and floral features related to pollination in Brazilian Maxillariinae are more diverse than previously suggested. It is hoped that the data presented herein, together with other data sources such as vegetative traits and molecular tools, will be helpful in redefining and diagnosing clades within the subtribe Maxillariinae.

Notes on the pollination biology of Notylia nemorosa (Orchidaceae): do pollinators necessarily promote cross pollination?

The pollination biology of Notylia nemorosa was elucidated from field and ex situ observations. Field observations were carried out in Minas Gerais, southeastern Brazil, where this orchid is pollinated by males of Eulaema nigrita(Euglossini). Plants cultivated in Campinas (São Paulo State, southeastern Brazil) were eventually pollinated by males of Eulaema nigrita and Euglossa melanothricha(Euglossini). In both cases, Euglossini males collect aromatic compounds produced on the surface of the lip, fix the pollinaria on the dorsal surface of the labrum and perform pollination. This species is protandrous. Recently opened flowers present their stigmatic surfaces blocked and thus flowers act only as pollen donors. After 2-3 days the viscidium dries out and pollinaria cannot be removed anymore. Then, a narrow slit opens in the stigmatic cavity, allowing pollen deposition. Experiments performed with cultivated plants suggest that this orchid species is strongly, though not completely self-incompatible. Pollination experiments showed that bees tend to stay several minutes in each inflorescence. This behavior may promote some degree of geitonogamous pollination because flowers in male and female phases coexist in the same inflorescence. The combination of self-incompatibility with this specific pollinator behavior may explain the rarity of fruits in several neotropical reward-offering Orchidaceae.

The pollination mechanism in Trigonidium obtusum Lindl (Orchidaceae: Maxillariinae): sexual mimicry and trap-flowers.

The pollination process in Trigonidium obtusum Lindl. (Epidendroideae: Maxillariinae) is documented. The flowers are pollinated by sexually excited drones of Plebeia droryana (Meliponinae). When attempting to copulate either with sepals or petals, these bees slip on the waxy perianth surface and become trapped in the funnel-like flower tube. Bees trying to escape from the flowers may instead access the space between the column and lip, fixing the pollinarium on their scutellum. Pollinarium-bearing bees may pollinate the flowers when repeating the above-mentioned steps, leaving pollinia on the concave stigmatic surface, thus effecting pollination. Recently removed pollinaria are too broad to enter the stigma but they begin to dehydrate and within 40 min of removal are small enough to fit the stigmatic cavity. This mechanism prevents insect-mediated self-pollination and promotes cross-pollination. Preliminary evidence based on experiments with cultivated plants suggests that they are self-compatible but that fruit set is pollinator-dependent. The data obtained are discussed in a phylogenetic context. It is suggested that the pseudocopulatory syndrome in Trigonidium could have evolved from rewardless (food advertising) ancestors. Pseudocopulation in the context of the long flowering period of this orchid species (about 7 months) is understandable since the eusocial Plebeia bees produce fertile individuals several times a year.