Phylogeny and floral biology of South American Pogoniinae, and taxonomic revision and analysis of the epicuticular waxes of the genus Cleistes Rich. ex Lindl. (Orchidaceae)

Orientador: Marlies SazimaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Neste trabalho foi realizada a revisão taxonômica, o estudo químico e micromorfológico das ceras epicuticulares, bem como estudados os processos de polinização e os polinizadores de representantes sul-americanos do gênero Cleistes. Foi, também, elaborada uma hipótese filogenética da subtribo Pogoniinae com base em caracteres morfológicos e moleculares, através do seqüenciamento das regiões ITS (nrDNA), e rps16 e trnL-F (cpDNA). O gênero Cleistes apresenta 17 espécies distribuídas entre as Américas Central e do Sul, ocorrendo principalmente em regiões de cerrado do Brasil central. Três dessas espécies foram descritas durante a elaboração do presente trabalho e estão aqui apresentadas. Através do estudo da hipótese filogenética de Pogoniinae foi verificado que essa subtribo apresenta dois clados bem sustentados. Um clado é norte-americano-asiático, e inclui os gêneros Pogonia, Isotria e as espécies norte-americanas de Cleistes. O outro é centro-sul-americano e inclui as espécies de Cleistes distribuídas entre as Américas Central e do Sul. Com o desenvolvimento do presente estudo, incluindo a maioria das espécies de Cleistes nas análises, pode ser demonstrado que esse gênero é parafilético, concordando com os dados publicados por Cameron & Chase (1999). As espécies norte-americanas de Cleistes estão mais relacionadas com os gêneros norte-americano-asiáticos Isotria e Pogonia do que com as espécies centro-sul-americanas de Cleistes. A subtribo Pogoniinae também é parafilética. O saprofítico gênero Pogoniopsis está mais relacionado com representantes dos gêneros Galeola e Cyrtosia (Galeolinae) do que com os demais gêneros pertencentes à subtribo Pogoniinae. Duckeella, um gênero endêmico da Amazônia e irmão das Pogoniinae, não apresenta os caracteres sinapomórficos que define essa subtribo, devendo ser transferido para a subtribo Duckeellinae. Espécies de Cleistes apresentam micromorfologia das ceras epicuticulares existentes nas folhas variando desde lisas, com grânulos, ou até formando placas. As ceras epicuticulares de Cleistes são constituídas principalmente por álcoois, ácidos e ésteres. A composição química das ceras epicuticulares das folhas de espécies de Cleistes é muito variável entre espécies, podendo ser usada na delimitação específica dentro do gênero. Espécies de Cleistes, assim como as demais Pogoniinae, são polinizadas principalmente por abelhas, embora C. libonii apresente beija-flores do gênero Phaethornis como co-polinizadores. Espécies sul-americanas de Cleistes apresentam picos de floração, em que todos os botões maduros de cada planta abrem simultaneamente no mesmo dia. As flores das espécies estudadas são pouco duráveis (geralmente um dia), e oferecem néctar aos polinizadores. O néctar é produzido em nectários glandulares da base do labelo das flores. Para os gêneros norte-americano-asiáticos esses nectários estão ausentes e as abelhas são atraídas às flores por engano. Esse estudo sustenta a hipótese de evolução de flores de engano para flores de néctarAbstract: The taxonomic revision, the micromorphological and chemical study of the epicuticular waxes, as well as the pollination processes and pollinators of the genus Cleistes were reported. A phylogenetic inference within Cleistes and among genera of subtribe Pogoniinae was also established, based on morphology and nrDNA (ITS) and cpDNA (trnL-F and rps16) sequence data, and using maximum parsimony. The genus Cleistes includes 17 species distributed among Central and South America, occurring mainly in ¿cerrado¿ areas of Central Brazil. Three of these species are new and were described in the present study. The phylogenetic study of Pogoniinae showed that this subtribe presents two well-supported clades. One clade is North-American-Asiatic and includes the genera Isotria, Pogonia and the North-American species of Cleistes. The other clade is Central-South-American and includes the species of Cleistes occurring in Central and South America. The genus Cleistes, in agreement to Cameron & Chase (1999), is paraphyletic. The North American species of Cleistes are more related to the North American-Asiatic genera Isotria and Pogonia than to the remaining species of Cleistes. The subtribe Pogoniinae is also paraphyletic. The saprophytic genus Pogoniopsis is more related with Galeola and Cyrtosia (Galeolinae) than to the remaining genera currently recognized within subtribe Pogoniinae. The Amazonian genus Duckeella, sister of all remainder of Pogoniinae, lacks the synapomorphic characters that define this subtribe, and should be transferred to the subtribe Duckeellinae. Species of the genus Cleistes present micromorphology of the epicuticular waxes of the leaves varying from a singular film to forming granules or platelets. The epicuticular waxes in Cleistes are constituted mainly by alcohols, acids and esters. The chemical compounds of epicuticular waxes of Cleistes were very variable among species, and may be used for specific delimitation within the genus. Species of Cleistes, as for remaining Pogoniinae, are pollinated mainly by bees, although C. libonii is co-pollinated by hermit hummingbirds. The South American species of Cleistes presents flowering peaks, in which all mature buds of each plant flowering simultaneously on the same day. The studied species produced short-lived flowers (generally one day), which offer nectar as reward produced by two nectariferous glands on the basis of the lip. For the North American-Asiatic genera these nectariferous glands are absent and the floral visitors are attracted by deceit. This study supports the evolution of deceptive flowers to nectar flowersDoutoradoBiologia VegetalDoutor em Biologia Vegeta

A new remarkable Vanilla Mill. (Orchidaceae) species endemic to the Espinhaço Range, Brazil: its phylogenetic position and evolutionary relationships among Neotropical congeners

During surveys conducted on Neotropical Vanilla, a new endemic species was found in the Brazilian campos rupestres of the Espinhaço Range. Here, this new remarkable Vanilla species, namely V. rupicola Pansarin & E.L.F. Menezes, is described and illustrated. A phylogeny for Vanilla is presented and the relationships between Neotropical species are discussed. The position of V. rupicola among Neotropical Vanilla is discussed within an evolutionary context. Vanilla rupicola is recognized by its rupicolous habit, its reptant stems, and its sessile and rounded leaves. This remarkable new taxon emerges in a clade that includes V. appendiculata Rolfe and V. hartii Rolfe. Vegetative and floral features support a close relationship between V. rupicola and sister taxa, mainly regarding the apical inflorescence (V. appendiculata), the type of appendages of the central crest of the labellum, and the labellar color pattern. Phylogenetic inference suggests that the circumscription of Neotropical Vanilla groups needs revision

A light in the shadow: the use of Lucifer Yellow technique to demonstrate nectar reabsorption

Abstract Background Nectar reabsorption is a widely known phenomenon, related to the strategy of resource-recovery and also to maintain the nectar homeostasis at the nectary. The method currently performed to demonstrate nectar being reabsorbed involves the use of radioactive tracers applied to the nectary. Although this method works perfectly, it is complex and requires specific supplies and equipment. Therefore, here we propose an efficient method to obtain a visual demonstration of nectar reabsorption, adapting the use of Lucifer Yellow CH (LYCH), a fluorescent membrane-impermeable dye that can enter the vacuole by endocytosis. Results We applied a LYCH solution to the floral nectary (FN) of Cucurbita pepo L., which is a species known for its ability of nectar reabsorption, and to the extrafloral nectary (EFN) of Passiflora edulis Sims which does not reabsorb the secreted nectar. In all tests performed, we observed that LYCH stained the nectary tissues differentially according to the reabsorption ability of the nectary. The treated FN of C. pepo presented a concentrated fluorescence at the epidermis that decreased at the deeper nectary parenchyma, until reaching the vascular bundles, indicating nectar reabsorption in the flowers of the species. In contrast, treated EFN of P. edulis presented fluorescence only at the cuticle surface, indicating that nectar is not reabsorbed by that particular tissue. Conclusion LYCH is an efficient marker to demonstrate nectar reabsorption.The authors thank the Pró-Reitoria de Pesquisa of the University of São Paulo for the help with the cost of the publishing. PCG and JMRBVA thank the grant provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The authors thank the staff of Instituto de Botânica for providing assistance with cultivated material of P. edulis and Joel Caitano (Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto) for providing material of C. pepo. PCG also thank Wilton José da Rocha Lima (Central Analítica – Instituto de Química, Universidade de São Paulo) for technical assistance with confocal microscopy. JMRBVA thanks Laura de Castro Lamonica for helping with practical work. FB acknowledges the research productivity grant received from the Brazilian National Council for Scientific and Technological Development (CNPq). We thank the two anonymous referees for their valuable comments to the manuscript and their constructive suggestions.The authors thank the PróReitoria de Pesquisa of the University of São Paulo for the help with the cost of the publishing. PCG and JMRBVA thank the grant provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The authors thank the staff of Instituto de Botânica for providing assistance with cultivated material of P. edulis and Joel Caitano (Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto) for providing material of C. pepo. PCG also thank Wilton José da Rocha Lima (Central Analítica – Instituto de Química, Universidade de São Paulo) for technical assistance with confocal microscopy. JMRBVA thanks Laura de Castro Lamonica for helping with practical work. FB acknowledges the research productivity grant received from the Brazilian National Council for Scientific and Technological Development (CNPq). We thank the two anonymous referees for their valuable comments to the manuscript and their constructive suggestions

Reproductive biology of Echinodorus longipetalus (Alismataceae): Sexual morphs, breeding system and pollinators

The floral biology, pollinators and breeding system of Echinodorus longipetalus Micheli were studied in a marshy area of the district of Taquaritinga (State of Sao Paulo), southeastern Brazil. E. longipetalus is gynodioecious and as far as is known, this is the first record of unisexual flowers, besides perfect flowers, in Echinodorus. Proportion of female individuals in the studied population is 50% and produces 31% more flowers than hermaphrodites. Perfect and pistillate flowers of E. longipetalus are similar in appearance and are pollinated by several species of Hymenoptera (mainly by Xylocopa (Neoxylocopa) suspecta Moure & Camargo). Perfect flowers offer pollen as a reward. Pistillate flowers attract floral visitors by deceit with their staminodes that resemble the stamens of the perfect flowers. Visits to pistillate flowers are quick (1-2 s), while visits to perfect flowers last up to 120 s. The perfect flowers are self-compatible and produce fruits through spontaneous self-pollination (control flowers), whereas the pistillate ones only set fruits through cross-pollinations. Perfect and pistillate flowers set more fruits under natural conditions than in manual treatments, respectively. Although the pistillate and perfect flowers bear a strong similarity, the selective pollinator behavior seems to be responsible for the increase of fruit set in perfect flowers. (C) 2008 Elsevier B.V. All rights reserved

Biologia reprodutiva e morfologia floral de especies de Orchidaceae em diferentes ambientes no Estado de São Paulo

Orientador: Maria do Carmo Estanislau do AmaralDissertação (mestrado) - Universidade Estadual de Campinas. Instituto de BiologiaResumo: Foram estudadas a biologia reprodutiva e morfologia floral de quatro espécies de Orchidaceae: Cirrhaea dependens Rchb. f, Epidendrum secundum Jacq., Polystachya estrellensis Rchb. f e Psilochilus modestus Barb. Rodr. que ocorrem tanto em matas de altitude do interior do Estado de São Paulo (Serra do Japi, Jundiaí e Pedra Grande, Atibaia) quanto em matas superúmidas da planície litorânea (picinguaba, Ubatuba). Na região de Picinguaba foi investigada ainda a biologia reprodutiva de Polystachya concreta (Jacq.) Garay & H.R. Sweet, e a especificidade dos agentes polinizadores de mais duas espécies do gênero Cirrhaea, C. saccata Lindl. e C. loddigesii Lindl. Com a presença de uma espécie distinta de Stanhopea para cada ambiente, sendo que Stanhopea lietzeii (Regel) Schltr. ocorre na Serra do Japi e S. insignis Frost ex Hook. em Picinguaba, foram também realizados estudos comparativos entre as duas espécies. As espécies que oferecem pólen (P. modestus), pseudopólen (P. estrellensis) e néctar (E. secundum e P. modestus) são mais generalistas em relação aos agentes polinizadores. Das espécies que oferecem óleos voláteis como recompensa, ambas as espécies de Stanhopea apresentam alta especificidade,por outro lado, C. dependens e C. saccata apresentam baixo grau de especificidade. O grupo de polinizadores das espécies investigadas tende a ser o mesmo em ambos os ambientes. Entretanto, diferenças marcantes existem quando comparamos os microhábitats onde as plantas ocorrem. Exceto para C. dependens,. todas as espécies são autocompatíveis, e algumas exibem mecanismos prevenindo autopolinização. Os tratamentos indicam que, em condições experimentais, as espécies produzem mais frutos em relação às populações controle. Desse modo, a abundância e freqüência dos polinizadores nas flores é fator limitante no sucesso reprodutivo das espécies, exceto para P. estrellensis, na qual a maioria das flores são cleistógamas. A fragrância das flores é muito importante na especificidade dos agentes polinizadoresdas Stanhopeinae. Para as demais espécies, a fragrância atua como um atrativo secundário no processo de polinização. Apesar de terem sido observadas diferenças na morfologia das flores de ambos os ambientes, estas diferenças não são relevantes para a polinização das espécies. A ocorrência de E. secundum na região de Picinguaba está relacionada à ação antrópica, tomado-a simpátrica com E. fulgens Brongn. Nesta área, é observada a presença de diversas formas híbridas entre as duas espéciesAbstract: The floral morphology and pollination biology of four orchid species: Cirrhaea dependens Rchb. f, Epidendrum secundum Jacq., Polystachya estrellensis Rchb. f and Psilochilus modestus Barb. Rodr., were studied. These species occur either in montane mesophytic forests (Serra do Japi, Jundiaí and Pedra Grande, Atibaia) or in Atlantic RainForests sensu stricto (picinguaba, Ubatuba) as well. In Picinguaba, the floral and reproductive biology (inc1uding polIinator specificity) ofother three orchid species: Polystachya concreta (Jacq.) Garay & H.R. Sweet, Cirrhaea saccata Lind1. and Cirrhaea loddigesii Lind1., was also studied. Comparative studies between two Stanhopea species were also performed. Stanhopea lietzeii (Regel) Schltr. occurring in Serra do Japi and S. insignis Frost ex Hook., in Picinguaba. Orchid species offeringpollen (P. modestus), pseudopollen (P. estrellensis) or nectar (E. secundum and P. modestus) as rewards for pollinators, havequite generalistic pollen vectors. Among the orchid species which offer aromatic compounds as a reward, the two Stanhopea spp. showed quite specific pollination systems. Incomparison,both Cirrhaea dependens and C. saccata showed much lower pollinator specificity. The groups of pollinators, as a whole, tend to remain the same in both principal study areas. With the exception of C. dependens, alI the species studied are self-compatible, some exhibiting mechanisms to. avoid non automatic self;.pollination. Plants of all species studied,except P. estrellensis, showed an increased ftuit set than those at control, ffee-livingpopulations. Therefore, the abundance andffequency of pollinators is the limiting-factor for reproductive success, except for P.estrellensis. Most flowers of the last-named species are c1eistogamous. Whereas, flower ffagrances are very important for the pollinator-specificity in the Stanhopeinae, they play a secondary role in the other species studied. Flower morphological variations between populations are not relevant in the context of pollination biology. The antropogenic habitat perturbation seems to be responsible for the occurrence of E. secundum in Picinguaba, where it is sympatric with E. fulgens Brongn. Various hybrid forms between these species were recorded.MestradoMestre em Biologia Vegeta

A família Orchidaceae na Serra do Japi, São Paulo, Brasil

RESUMO Este estudo apresenta o inventário das espécies de orquídeas ocorrentes na Serra do Japi, no estado de São Paulo. A região é composta por áreas de floresta mesófila estacional semidecídua baixo montana e de altitude e afloramentos rochosos. A família está representada por 125 espécies, distribuídas em 61 gêneros. O gênero mais representativo é Epidendrum (10 spp.), seguido de Oncidium e Habenaria (9 spp. cada). A maioria das espécies (79 spp., 63,2%) é epífita, sendo que 40 espécies (32%) são terrícolas, 31 espécies. (24,8%) são rupícolas, duas são hemiepífitas e apenas uma é saprofítica. A floresta mesófila estacional semidecídua é o tipo de vegetação que abriga o maior número de espécies (88 spp., 70,9%). A maioria das espécies floresce no verão, entre dezembro e março. Duas espécies, Habenaria sp. e Acianthera sp. provavelmente são novas para a ciência. A Serra do Japi, por estar em uma zona de transição entre a as florestas ombrófilas (Serra do Mar) e as florestas estacionais semideciduais do planalto paulista, abriga espécies de ambas as formações. Apesar da grande diversidade de Orchidaceae na Serra do Japi, a região sofre com a intervenção humana. A preservação e o estudo integrado da Serra do Japi é uma urgente necessidade científica, com reflexos sociais, econômicos e preservacionistas

Facultative autogamy in Cyrtopodium polyphyllum (Orchidaceae) through a rain-assisted pollination mechanism

Cyrtopodium includes similar to 42 species, among which is Cyrtopodium polyphyllum (Vell.) Pabst ex F. Barros that occurs in a rainforest in south-eastern Brazil. Its non-rewarding flowers, which attract Centridini bees by deceit, are rain-assisted self-pollinated, a phenomenon rarely found in orchids and other plant families. In addition, self-pollination has never been reported in Cyrtopodiinae and data on the pollination of South American orchids are scarce. Flowers were observed at different times of the day, on both sunny and rainy days, to record floral morphology, visitors and the effects of rainfall on flowers. On rainy days, water accumulates on the stigma and dissolves the adhesive substance of the stigmatic surface. A viscous drop thus forms, which contacts the pollinarium. When evaporation makes the viscous drop shrink, the drop moves the pollinarium with the anther onto the stigmatic surface and promotes self-pollination. Fruit set in natural habitat was low, with 2.4% at one study site, where a similar value (2.2%) was recorded in flowers self-pollinated by rain. In C. polyphyllum, facultative self-pollination assisted by rain is thus an important strategy that guarantees fruit set when pollinator's visits are scarce, which is common in species pollinated by deceit564363367CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP43.703/200403/12595–7; 04/12531–

Facultative autogamy in Cyrtopodium polyphyllum (Orchidaceae) through a rain-assisted pollination mechanism

Cyrtopodium includes similar to 42 species, among which is Cyrtopodium polyphyllum (Vell.) Pabst ex F. Barros that occurs in a rainforest in south-eastern Brazil. Its non-rewarding flowers, which attract Centridini bees by deceit, are rain-assisted self-pollinated, a phenomenon rarely found in orchids and other plant families. In addition, self-pollination has never been reported in Cyrtopodiinae and data on the pollination of South American orchids are scarce. Flowers were observed at different times of the day, on both sunny and rainy days, to record floral morphology, visitors and the effects of rainfall on flowers. On rainy days, water accumulates on the stigma and dissolves the adhesive substance of the stigmatic surface. A viscous drop thus forms, which contacts the pollinarium. When evaporation makes the viscous drop shrink, the drop moves the pollinarium with the anther onto the stigmatic surface and promotes self-pollination. Fruit set in natural habitat was low, with 2.4% at one study site, where a similar value (2.2%) was recorded in flowers self-pollinated by rain. In C. polyphyllum, facultative self-pollination assisted by rain is thus an important strategy that guarantees fruit set when pollinator`s visits are scarce, which is common in species pollinated by deceit