Pâmela Lavor 1,2, Claudia M. Jacobi 3, Flávio F. Carmo 3, Leonardo M. Versieux 1,2,4

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1 CONFERENCE SCIENCE CORNER Notes on the floral biology, seed morphology and post-seminal development of Vriesea minarum L.B.Sm., an endangered Bromeliaceae of Southeastern Brazil Pâmela Lavor 1,2, Claudia M. Jacobi 3, Flávio F. Carmo 3, Leonardo M. Versieux 1,2,4 Abstract Vriesea minarum is a bromeliad endemic to the Iron Quadrangle region in Minas Gerais state, Brazil and is currently listed as an endangered species. The aim of our study was to investigate the species floral biology, breeding system, and post-seminal development. Observations and experiments were conducted in the Rola-Moça State Park. Flowers have morphological characteristics typical for the genus but we discuss the position of the ovary, oversimplified in some papers as superior. Seed set indicates that V. minarum is predominantly outcrossing, but self-compatible as well. The floral biology indicates flower protandry, and suggests a mixed pollination syndrome, as was observed recently in other bromeliads. Due to the survival risk facing this species, we suggest that more studies investigating the potential to preserve germplasm should be conducted, since its seeds appear to have a limited viability. Key words - endemic species, Iron Quadrangle, Rola-Moça State Park, Tillandsioideae Introduction Vriesea minarum L.B. Sm. is an endemic species from the Iron Quadrangle (IQ), a region in the southern portion of the Espinhaço mountain range in Minas Gerais state, Brazil (Versieux 2005, 2011, Versieux & Wendt 2006, Jacobi et al. 2007, Versieux et al. 2008). This rupicolous bromeliad has at least 12 populations within the Iron Quadrangle (Lavor et al. 2014). Due to severe habitat loss by mining, it is currently included as an endangered species in the Red List of Brazilian Endangered Plant Species (CNCFlora 2013) and it is officially protected only in three full protection conservation units: Serra de Gandarela National Park, Monumento Natural da Serra da Moeda and Rola-Moça State Park (Versieux 2011, Lavor et al. 2014). Considering this threatening scenario, it is desirable to inquire about its floral biology, since the behavior of flowers and their pollinators will affect plant breeding systems and fertility, which will be fundamental factors when assessing endangered 1 Programa de Pós-Graduação em Sistemática e Evolução, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil. 2 Laboratório de Botânica Sistemática, Departamento de Botânica e Zoologia, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil. 3 Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil. 4 Corresponding author: Leonardo Versieux (lversieux@yahoo.com.br) 87

2 species population viability and choosing appropriate conservation measures (Paggi et al. 2007, 2013). The relationships between plant, pollinator and disperser are also important for understanding the population structure, as they may influence the spatial distribution of the genetic diversity through gene flow (Yamamoto et al. 2007). Several studies are available in the literature about the reproductive biology of bromeliad species (e.g. Martinelli 1994, 1997, Varassin & Sazima 2000, Wendt et al. 2001, 2002, Matallana et al. 2010) or on the strategies used by the species of the subfamily Tillandsioideae to disperse propagules (e.g. Paggi et al. 2010). Although the Espinhaço range may be considered a Bromeliaceae hotspot few reproductive data are available for its unique bromeliads. Thus, the aim of this study was to provide some detailed information on V. minarum reproductive patterns, including floral biology, reproductive systems, seed morphology and post-germination development which may help conserve this threatened taxon. Material and methods Study area - The study was conducted between February and March 2012 in Rola-Moça State Park (RMSP) ( S W), Minas Gerais, Brazil. The species occurs in an area of open rocky field, more specifically ironstone outcrops (locally known as canga ) (Jacobi et al. 2007, Lavor et al. 2014). Floral biology We studied the morphology of V. minarum flowers through observation and photographic documentation in situ and ex situ, followed by dissection under stereomicroscope of the components of the floral whorls (calyx, corolla, androecium and gynoecium and septal nectaries), completing the description made by Versieux (2011). We conducted daily visits to observe the floral biology of eight genets separated by a minimum distance of 5 meters from March 6 to March 27, We observed the time of anthesis and floral senescence, flower duration (in days), period of pollen availability, and number of open flowers per day per genet. We tested the stigmatic receptivity three times a day (8:00 am, 12:00 and 5:00 pm) with hydrogen peroxide, using a 10x magnification hand lens to observe the formation of bubbles (Dafni et al. 2005). We measured and monitored nectar volume production in flowers (N = 3) at three different periods (morning, noon and late afternoon) by removing all nectar accumulated at the base of the ovary with a graded microsyringe (Hamilton) and analyzed sugar concentration with the aid of a hand-held refractometer 0-50% Brix (Kearns & Inouye 1993). To qualitatively investigate the floral odor we conducted a nose bioassay (Kearns & Inouye 1993). Flowers (N=4) and flower buds (N=4) were enclosed in a clean vial for 24 hours. After that, we questioned four volunteers if the flowers or buds had odor, and if so, how they would describe it. We conducted visual observations during the day, for one week, totaling 12 hours of direct observation, noting the time and number of floral visitors (and collection of possible visitors) in eight flowering individuals. 88

3 Reproductive biology, seed morphology and post-seminal development To obtain seeds, we performed a preliminary test of reproductive biology. We used four treatments to check if fruit and seeds would be produced and, after that, used the seeds obtained to analyze morphology and post-seminal characteristics. The treatments were performed on 8 inflorescences from 8 different genets, separated by ca. 5 m from each other, along the top of one mountain. With the aid of pollinator and pollen exclusion nets, a total of 59 flowers were treated. Treatments were: (1) manual self-pollination: to test for self-compatibility, unopened flowers were bagged and, on the next day, after anthesis, were pollinated with their own pollen and bagged again (N=12); (2) manual cross-pollination: to test for cross-fertilization, bagged flowers were pollinated with pollen from other flowers (preferably from plants at least 5 m apart to avoid collecting pollen from relatives) and re-bagged (N=12); (3) spontaneous self-pollination: to test the need for pollinator vectors, buds were bagged and no artificial pollination was performed (N=16); (4) to test the effective contribution of pollinators in the system, senescent flowers were bagged, serving as a control treatment (N=19). All the seeds obtained in all treatments were counted (total=4,400), put to germinate in Petri plates with filter paper soaked in distilled water, and placed under natural light at room temperature for 30 days. The start of germination was measured by the appearance of the radicle. Daily observations were made to verify the emergence and growth of the radicle and other vegetative organs of the seedling, which were documented by photos, using a portable digital microscope (Celestron 10x - 150x digital Microscope). Due to results obtained for germination indicating low values of viability (data not shown) we will only briefly discuss fruit and seed set, and not germination rates. Results Observations and floral biology - The reproductive period (measured from the beginning to the end of the inflorescence production) of V. minarum is from January to March. During January (the first month) there was the emergence of the inflorescence peduncle and, in the second month, flower buds were already visible in an immature inflorescence. In March, the inflorescences were mature and had two overlapping phenophases (flower buds and open flowers) (Fig. 1). Inflorescence maturation occurs from base toward the apex, with the opening of a single flower per day (Fig. 1a). Flowers last for two days and have diurnal anthesis (after 8:00 am), remaining open during the first night. Although observations were not carried out overnight, we deduced that flowers remain opened during the night since from our last observation at 6:00 pm until our first observations the next morning at 6:00 am, the flowers were still open. The first sign of anthesis is the spreading of the petal apexes (Fig. 1b). After that, the stigma emerges, but at this point it is not receptive yet (Fig. 1c), followed by the appearance of the stamens, which nearly 3 hours later will be more exposed and with the anthers completely exserted (Fig. 1d). 89

4 Figure 1. Specimen of Vriesea minarum growing on typical ironstone habitat. (a) blooming individual (b) flower bud (c) stigma protruding (d) stamens with closed anthers (e) fully open flower (f) senescent flower. Bars: (a) = 10 cm, (b f) = 1 cm. Photos by Pâmela Lavor. The anthers appeared close to the petal apex a few hours after anthesis, starting to release pollen after 9:00 am, and continuing throughout the day until the early hours of the following morning. The stigma was receptive from the end of the first day (around 4:00 pm) (Fig. 1e) until the morning of the second day. Flower senescence starts on the morning of the second day. Senescence in V. minarum flowers is characterized by the loss of the yellow color of the sepals and darkening of the petals (Fig. 1f). Nectar accumulated at the base of the corolla, and its production started around 8:00 am on the first day (mean = ml ± 0.02 SD). There was a general trend of decrease (Fig. 2), with the end of the production around 2:00 pm on the second day. Similarly, the mean sugar concentration in the nectar was higher on the first day (mean = 13% ± 9.5 SD). On the second day the average sugar concentration was 9%. As with production, there was a general trend of decreasing sugar concentration throughout the day (Fig. 2). When nectar was removed, the flower restored the same average amount of nectar throughout the day and its output coincides with thecal dehiscence and the release of pollen. Flowers have no perceivable odor after being kept in sealed vials. Unfortunately, no floral visitors or pollinators were observed, although many different species of visitors (hummingbirds, butterflies, bees and beetles) were noticed 90

5 Figure 2. Nectar production (ml) and nectar sugar concentration (%) along a period of 2 days in Vriesea minarum flowers. Figure by Pâmela Lavor. around other plant species that were flowering in the same area. In one of the observed V. minarum plants, an unidentified ant circulated along the axis of the inflorescence, and got into the flowers sporadically without touching stamen or stigma or causing damage to the floral parts. The flowers of V. minarum have a trimerous perianth (three sepals and three petals) (Fig. 3a), with two basal petal appendages per petal (Fig. 3c). The ovary is nearly superior (Figs. 3a and 3g), and is 3-locular (Figs. 3b and 3d), with a fraction of each locule base going below the point of petal attachment. Nectaries are infralocular, with septal nectaries well developed (Fig. 3a). The anthers are dorsifixed and have longitudinal dehiscence (Figs. 3e and 3f). The stigma type (Fig. 3h) is classified as convolute blade in the Brown and Gilmartin (1984, 1989) system. Reproductive biology All treatments produced fruits and seeds, indicating that the species can produce seed under either self- or cross-pollination (see Table 1). The number of fruit was proportionally higher in the control, untouched and unbagged, in which 18 out 19 (94.7%) flowers treated set fruit, followed by manual self-pollination (75%), spontaneous self-pollination (68.8%), and by the manual cross-pollination (41.7%). The proportion of seeds per fruit (seed set divided by the fruit set), however, was higher in manual cross-pollination (263.6), followed by manual self-pollination (199.6), spontaneous self-pollination (93.2) and by the control (14.4). In all the treatments, we observed aborted seeds that were not counted. 91

6 Figure 3. Flower morphology of Vriesea minarum. (a) longitudinal section of V. minarum showing the well-developed infra-locular septal nectaries (*), the locules ( ), chamber delimited between the ovary and the petal appendages (γ), and appendages (»); (b) transversal section of the ovary showing the locules and the terminal portions of the nectaries opening (arrow head); (c) dissected basal petal appendages; (d) ovary sectioned along the mid portion, showing placentation and ovules; (e) anther in front view; (f) anther in dorsal view; (g) conical shape of the mostly superior portion of the ovary; (h) convolute blade stigma. Bars = 1 mm. Photos by Pâmela Lavor. Seed morphology and postseminal development - Vriesea minarum has seeds nearly 6 mm long (± 0.06 SD; N = 5), which are filiform, brown, bearing whitish feathery appendages along the base (Fig. 4a). The germination was initiated at days after imbibition (Fig. 4b). The radicle arises close to the cotyledonary sheath. A post-seminal characteristic in V. minarum is the presence of the undeveloped hypocotyl. The first leaf (eophyll) appears five days after radicle (primary root) emergence (Fig. 4c), and it takes five more days before the second leaf becomes visible (Fig. 4d). After the appearance of the third leaf, leaves (eophylls) develop so intertwined that the shape of a miniature rosette, typical of Bromeliaceae, is already apparent (Fig. 4e). Discussion Observations and floral biology - The reproductive phase observed here for V. minarum in RMSP coincides with the rainy season, corroborating Versieux s data (2011). Nevertheless, the flowers only started to open in March. In other areas of the IQ the blooming of V. minarum appears to be longer, from December to March based on herbarium records (Versieux & Wendt 2006, Versieux 2011). The lack of floral visitors observed may be due to the size of the population analyzed (8 blooming individuals, a small number if compared to the massive bloom seen in other rocky outcrops, L.M. Versieux pers. obs.) and by the low number of flowers on individual plants and in the whole population. A fire in the RMSP preceeding the study was responsible for these low numbers. The opening 92

7 Table 1 Results of the pollination treatments for Vriesea minarum in Rola-Moça State Park, Minas Gerais. Manual selfpollinaion Manual Crosspollintion Spontanous self-pollination (untouched and bagged) Control (untouched and unbagged) Flowers treated FRUIT SET % Fruit set per treatment SEED SET of a single flower a day per inflorescence in V. minarum, could be less attractive if compared to several options of flowers in other shrubs and herbs nearby. Moreover, as the observations were made only during the day and there are indications that its flowers remain open during the night, perhaps the floral visitors were more active in this time. A similar fact is reported by Van Sluys & Stotz (1995) with other species of Vriesea that, by presenting a limited offer of flowers per inflorescence during the flowering season, received fewer floral visitors, while groups of flowering plants with a larger number of open flowers received more visits by hummingbirds. The basic characteristics of the flowers of V. minarum suggest a mixed pollination syndrome. Mixed pollination systems are found in several Mesoamerican Bromeliaceae species, with some species of Tillandsia presenting flowers that last for almost 48 hours, a strategy to allow access of pollinators that are active during the night (Benzing 2000b). The flower of V. minarum has typical ornithophily features, such as exposed flowers and inflorescence, tubular and odorless flowers, diurnal anthesis, conspicuous or contrasting colors among floral parts, lower concentration of nectar sugar, stigma and anthers away from the nectaries and positioned to optimize the contact with birds, and absence of a landing platform (Machado & Semir 2006). Marques & Lemos-Filho (2008) observed birds visiting V. minarum (at that time identified as V. citrina) in Serra da Piedade, 41 km northeast of RMSP. These authors also noted that above 1,500 m.a.s.l. in the area of Serra da Piedade ornithophily was most common among the Vriesea species. Other morphologically similar species were also indicated as ornithophilous, as is the case of V. marceloi Versieux & Machado, from Serra do 93

8 Figure 4. (a) Mature seed of V. minarum. (b-e) Post seminal development of Vriesea minarum. ; (b) onset of germination with the emergence of the radicle (primary root); (c) the first leaf (eophyll) appearance; (d) formation of the second leaf; (e) appearance of the third leaf. Bars = 2 mm (Legends: Co = Coma; Cs = Cotyledonary sheath; Eo = Eophyll; Pr = Primary root; Se = Seed). Line drawings by Rhudson Cruz. Caraça (Versieux & Machado 2012). Versieux (2011) also made personal observations of hummingbird visits to V. minarum. On the other hand, flowers of V. minarum have a duration of two days, do not close during the night, and the stigma becomes receptive in the late afternoon, suggesting visits by nocturnal pollinators, maybe bats. Currently, 36 species of bats have been inventoried in the caves in the ironstone outcrops, four nectarivorous species and 12 frugivorous (Gomes et al. 2015). Bats are the main pollinators in several bromeliad genera, including Vriesea Section Xiphion (e.g. Vogel 1969, Sazima et al. 1989, 1995, Martinelli 1997, Kessler & Kromer 2000, Benzing 2000c), although most bat-pollinated Vriesea species have wide-mouthed flowers and stamens positioned on one side of the flower (Sazima et al. 1995). Bats can be opportunistic and sometimes visit flowers that do not conform to the classic bat pollination syndrome ; the color of bat flowers ranges from white, brown and green to pink, fuchsia and yellow. Bat flowers/inflorescences can be roughly divided into three categories based on their shape: (1) shaving-brush or 94

9 stamen ball with many projecting stamens; (2) bell-shaped with the corolla forming a tube; and (3) cupshaped with an open corolla (Fleming et al. 2009). Recent works indicate interesting results for bromeliads that have flowers remaining open during day and night, and this strategy may be similar in V. minarum (Marques et al. 2015, Aguilar-Rodrıguez et al. 2014, 2015, Queiroz et al. 2016). Pollination by bats may explain recent results of population genetics for the species. Lavor et al. (2014) found a low genetic population structure attributed either to efficient seed or pollen dispersal among separated mountains. Bats can travel great distances to feeding sites (up to 38 km), whereas many species of hummingbirds are territorial, thus restricting cross-pollination distances (Proctor et al. 1996). Nevertheless, the low levels of gene flow obtained with the similarly rupicolous and closely related genus Alcantarea lead Barbará et al. (2007) to suggest that bats do not contribute so much to crosspollination between inselberg (isolated granitic rock outcrops) bromeliad populations. Considering the floral morphology, our dissections indicate that the ovary in this species has a positioning similar to what is seen in Alcantarea (Versieux & Wanderley 2015). The basal 1/8 of locules is below the point of attachment of the petals (Fig. 3a). This would classify the ovary as cryptically half-inferior or nearly superior (as described by Mez 1934, Smith & Till 1998). In contrast, some descriptions of Vriesea, suggest that a fully superior ovary is the only type found in the entire genus. Reproductive biology - There are many characteristics that may influence the proportions of selfing and outcrossing breeding systems in Bromeliaceae. Protandry is the main means of promoting outcrossing in self-compatible species (Benzing 2000b) and this is an attribute observed in V. minarum. This strategy was recorded by Martinelli (1994) in 17 Vriesea species pollinated by hummingbirds and bats in southeastern Brazil Atlantic rainforest. The mean number of seeds produced by both manual and spontaneous self-pollination (Table 1) indicates that the species is self-compatible and capable of reproducing in case of pollinator shortage, despite the higher value of seed set seen for manual cross-pollination. For V. minarum the reproductive pattern is like those observed in the bromeliad species studied by Martinelli (1994) and Matallana et al. (2010) that presented characteristics of an outcrossing breeding system (an advantage when pollinator availability is high). However, inferences about the reproductive system of this species can not be fully understood now, and are still speculative in the present work and will demand additional experiments, including overnight observations (to make sure bats are visiting the inflorescences) and seed viability tests. Because the flowering season was atypical that year and only 8 rosettes were blooming for this experiment, only a limited number of inflorescences and flowers were available for treatments. This limited the number of flowers in each treatment category. We hope to expand the study in the future with a larger numbers of flowers and to avoid any loss of seed viability due to delays in transportating and counting. Germination rates 95

10 could then be used to determine which of the treatments would, in fact, guarantee more efficient reproduction. Morphology of seeds and post-seminal development - The seeds of V. minarum are morphologically similar to those from other Tillandsioideae species. This subfamily is characterized mainly by the coma, the flight apparatus allowing anemochory. The appendages increase the surface/volume ratio, reducing speed of the fall during dispersal by air streams or winds in the dry season (Paula & Silva 2004). Seed germination time is considered intermediate according to the Pereira et al. (2008) classification and similar to what have been described for other Tillandsioideae, as well as their postseminal characteristics, such as the presence of the undeveloped hypocotyl, and straight cotyledonary sheath and reduced primary root (Tillich 2007). Conservation - In terms of ecological importance, the family Bromeliaceae is remarkable, but the amount of scientific literature available about the floral biology, phenology and reproductive systems is still limited compared to the richness and dominance of Bromeliaceae taxa in many habitats, although an increase in the last decade is noticed (e.g. Martinelli 1997, Siqueira-Filho & Machado 2001, Nara & Webber 2002, Machado & Semir 2006, Pereira & Quirino 2008, Rios et al. 2010, Marques et al. 2015, Aguilar-Rodríguez et al. 2014, 2015, Queiroz et al. 2016). This knowledge is fundamental, since the bromeliads are biodiversity promoters and help in the establishment and maintenance of other organisms, such as other plants, invertebrates, and vertebrates (Benzing 2000b). Although no floral visitors were seen in the present study, data presented here on nectar production in V. minarum show the plant may be considered an important source of nectar for diurnal and nocturnal fauna in the ferruginous fields. As proposed by Lavor et al. (2014) we suggest that more conservation units be created within the entire IQ to protect micro-endemic taxa such as V. minarum, and that more studies investigating the potential to preserve germplasm of this taxon (including techniques to prolong seed viability) should be conducted. Acknowledgements We thank CAPES for the first author s M.Sc. CMJ and LMV thank the Brazilian National Council for Scientific and Technological Development (CNPq) for their Research Productivity Scholarships and research grants (304778/ and /2014-8). The Rola-Moça State Park and the State Forest Institute (IEF-MG) are gratefully acknowledged for providing personnel to assist with collection and observation of specimens in the field. We also thank Profs. Fabio de Almeida Vieira and Vânia Cristina Rennó Azevedo for their constructive suggestions to an earlier version of this manuscript, as well as the anonymous reviewers and editors. Interns A.S. Medeiros and E.C. Tomaz are acknowledged for their patience and help while counting seeds. 96

11 Literature cited Aguilar-Rodríguez, P.A., Krömer, T., García-Franco, J.G., Mac Swiney, G.M.C From dusk till dawn: nocturnal and diurnal pollination in the epiphyte Tillandsia heterophylla (Bromeliaceae). Plant Biology 18: Aguilar-Rodríguez, P.A., MacSwiney, G.M.C., Krömer, T., García-Franco, J.G., Knauer, A., Kessler, M First record of bat-pollination in the species-rich genus Tillandsia (Bromeliaceae). Annals of Botany 113: Barbará, T., Martinelli, G., Fay, M.F., Mayo, S.J., Lexer, C Population differentiation and species cohesion in two closely related plants adapted to neotropical high-altitude inselbergs, Alcantarea imperialis and Alcantarea geniculata (Bromeliaceae). Molecular Ecology 16: Benzing, D.H. 2000a. Pollination. In: D.H. Benzing (ed.). Bromeliaceae: profile of an adaptive radiation. Cambridge University Press, Cambridge. Benzing, D.H. 2000b. Relationship with the fauna. In: D.H. Benzing (ed.). Bromeliaceae: profile of an adaptive radiation. Cambridge University Press, Cambridge. Benzing, D.H. 2000c. Reproductive structure. In: D.H. Benzing (ed.). Bromeliaceae: profile of an adaptive radiation. Cambridge University Press, Cambridge. Brown, G.K & Gilmartin, A.J Stigma structure and variation in Bromeliaceae neglected taxonomic characters. Brittonia 36: Brown, G.K. & Gilmartin, A.J Stigma types in Bromeliaceae a systematic survey. Systematic Botany 14: CNCFlora - Centro Nacional de Conservação da Flora Lista Vermelha de Espécies. Available from (accessed in 20 Feb 2016). Dafni, A., Kevan, P.G., Husband, B.C Practical Pollination Biology. Enviroquest, Ltd. Cambridge, Ontario, Canada. Dutra, V.F., Vieira, M.F., Garcia, F.C.P., Lima, H.C Fenologia reprodutiva, síndromes de polinização e dispersão em espécies de Leguminosae dos campos rupestres do Parque Estadual do Itacolomi, Minas Gerais, Brasil. Rodriguésia 60: Fleming, T.H., Geiselman, C., Kress, W.J The evolution of bat pollination: a phylogenetic perspective. Annals of Botany 104: Gomes, A.M., Silva, L.H.D., Tavares, V.C Morcegos associados a Ecossistemas Ferruginosos de Minas Gerais. In: F.F. Carmo, Kamino, L.H.Y. (Org.). Geossistemas Ferruginosos do Brasil: áreas prioritárias para a conservação da diversidade geológica e biológica, patrimônio cultural e serviços ambientais. 3i Editora Ltda. Belo Horizonte. Jacobi, C.M., Carmo, F.F., Vincent, R.C., Stehmann, J.R Plant communities on ironstone outcrops: a diverse and endangered Brazilian ecosystem. 97

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13 Paggi, G.M., Sampaio, J.A.T., Bruxel, M., Zanella, C.M., Goetze, M., Buttow, M.V., Palma-Silva, C., Bered, F Seed dispersal and population structure in Vriesea gigantea, a bromeliad from the Brazilian Atlantic Rainforest. Botanical Journal of the Linnean Society 164: Paggi, G.M., Silveira, L.C.T., Zanella, C.M., Bruxel, M., Bered, F., Kaltchuk- Santos, E., Palma-Silva, C Reproductive system and fitness of Vriesea friburgensis: a self-sterile bromeliad species. Plant Species Biology 28: Paula, C.C. & Silva, H.M.P Cultivo prático de bromélias. Viçosa, Universidade Federal de Viçosa. Pereira, F.R.L. & Quirino, Z.G.M Fenologia e biologia floral de Neoglaziovia variegata (Bromeliaceae) na caatinga paraibana. Rodriguésia 59: Pereira, A.R., Pereira, T.S., Rodrigues, A.S., Andrade, A.C.S Morfologia de sementes e do desenvolvimento pós-seminal de espécies de Bromeliaceae. Acta Botanica Brasilica 22: Proctor, M., Yeo, P., Lack, A The Natural History of Pollination. Timber Press, Portland, Oregon. Queiroz, J.A., Quirino, Z.G.M., Lopes, A.V., Machado, I.C Vertebrate mixed pollination system in Encholirium spectabile: A bromeliad pollinated by bats, opossum and hummingbirds in a tropical dry forest. Journal of Arid Environments 125: Rios, P.A.F., Silva, J.B., Moura, F.B.P Visitantes florais de Aechmea constantinii (Mez) L. B. Sm. (Bromeliaceae) em um remanescente da Mata Atlântica do Nordeste Oriental. Biotemas 23: Sazima, I., Vogel, S., Sazima, M Bat pollination of Encholirium glaziovii, a terrestrial bromeliad. Plant Systematics and Evolution 168: Sazima, M., Buzato, S., Sazima, I., Polinização de Vriesea por morcegos no sudeste brasileiro. Bromélia 2: Siqueira Filho, J.A., Machado, I.C.S Biologia reprodutiva de Canistrum aurantiacum E. Morren (Bromeliaceae) em remanescente da Floresta Atlântica, Nordeste do Brasil. Acta Botanica Brasilica 15: Smith, L.B. & Till, W Bromeliaceae. In: K. Kubitzki (ed.). The families and genera of vascular plants, IV flowering plants, Monocotyledons Alismatanae and Commelinanae (except Gramineaee). Springer Verlag, Berlin, pp Tillich, H.J. Seedling diversity and the homologies of seedling organs in the order Poales (Monocotyledons). Annals of Botany 100: Van Sluys, M. & Stotz, D.F Patterns of hummingbird visitation to Vriesea neoglutinosa in Espírito Santo, Southeastern Brazil. Bromélia 2: Varassin, I.G. & Sazima, M Recursos de Bromeliaceae utilizados por beija- -flores e borboletas em mata atlântica no sudeste do Brasil. Boletim do Museu de Biologia Mello Leitão 11/12: Versieux, L.M Bromeliáceas de Minas Gerais: catálogo, distribuição geográ- 99

14 fica e conservação das espécies. Universidade Federal do Rio de Janeiro, Rio de Janeiro. Versieux, L.M Brazilian plants urgently needing conservation: the case of Vriesea minarum (Bromeliaceae). Phytotaxa 28: Versieux, L.M Bromeliaceae. In: M.C. Jacobi & Carmo, F.F. (Orgs.). Diversidade florística nas cangas do Quadrilátero Ferrífero. IDM Editora, Belo Horizonte, Minas Gerais. Versieux, L.M. & Machado, T.M A new ornithophilous yellow-flowered Vriesea (Bromeliaceae) from Serra do Caraça, Minas Gerais, Brazil. Phtotaxa 71: Versieux, L. M. & Wanderley, M.G.L Bromélias-gigantes do Brasil. Capim Macio & Offset, Natal, pp 200. Versieux, L.M. & Wendt, T Checklist of Bromeliaceae of Minas Gerais, Brazil, with notes on taxonomy and endemism. Selbyana 27: Versieux, L.M. & Wendt, T Bromeliaceae diversity and conservation in Minas Gerais, Brazil. Biodiversity and Conservation 16: Versieux, L.M., Wendt, T., Louzada, R.B., Wanderley, M.G.L Bromeliaceae da Cadeia do Espinhaço. Megadiversidade 4: Vogel, S Chripoterophilie in der neotropischen Flora. Neue Mitteilugen III. Flora 148: Wendt, T., Canela, M.B.F., de Faria, A.P.G., Rios, R.I., Reproductive biology and natural hybridization between two endemic species of Pitcairnia (Bromeliaceae). American Journal of Botany 88: Wendt, T., Canela, M.B.F., Klein, D.E., Rios, R.I Selfing facilitates reproductive isolation among three sympatric species of Pitcairnia (Bromeliaceae). Plant Systematics and Evolution 232: Yamamoto, L.F., Kinoshita, L.S., Martins, F.R Síndromes de polinização e de dispersão em fragmentos da Floresta Estacional Semidecídua Montana, SP, Brasil. Acta Botanica Brasilica 21: [Editor s notes: In a floral biology study, the aim is to determine in detail the way a plant is able to produce seeds. Do the flowers need cross-pollination, or can they self-fertilize? If pollinated, how do they interact with pollinators? We are interested in these characteristics for endangered species because the details may provide clues to the forces limiting the population growth of the species. For instance, pollinators having a long history with the species - and presumably quite efficient in carrying out pollination - may suddenly become unavailable due to changes in rainfall patterns, temperature or the arrival of new competitors and/or predators. If pollination is now carried out by a less efficient pollinator, the potential seed set could well be limited. Of course, such details may be intriguing for non-endangered as well as endan- 100

15 gered species. The plants in your own collection provide you with an opportunity to make similar observations. You can start by determining when the flowers open and when they close. Most of the cultivated bromeliads have seasonal blooming cycles. In southern Florida, there are plants, such as Billbergia species and the members of several different species groups in Aechmea that bloom during the Northern Hemisphere Fall and Winter. Larger Neoregelia species tend to flower primarily in Spring. A large variety of species bloom preferentially during the Summer. It is also possible, and interesting, to keep track of when the flowers open and close during the day. Most bromeliad flowers last only a single day or part of a day, but - as indicated in this article - there are exceptions. Usually, a fully open flower can be recognized by outward bending of the petal tips. In Neoregelia, and several other genera, these tips are easily seen due to their large size and the way they bend sharply away from the tube formed by the lower portion of the petals, often ending up at right angles to the tube. In the case of Billbergia, it is common for the petal tips to arch away from each other gradually with the two lower petals ultimately recurving. In contrast, many species of Vriesea have only a small proportion of the petal (less than 1/8 of an inch or 2-3 mm) bending out. Also, there are some species where the petals expand, but the tips remain curved inward, and the only opening is a hole where the tips of the petals pull slightly away from each other. In either case, once you can recognize an open flower, you start observing your blooming plant at progressively earlier times. With some plants, you may have to be out watching the flowers before the sun comes up. With a night-blooming plant, you might have to start your observations in the evening to catch the hour of opening. It is also of interest to know how long the flower stays open, so you want to record what hour the flower closes. Flower closing is usually marked by a change in the appearance of the petals. Sometimes the petals will darken, sometimes they will start to turn a light brown. Often, this color change will be visible before the petals show any sign of changing physically, but Neoregelia provides a major exception. In Neoregelia species, the spreading lobes of the petals typically retain color while the lobes unbend over a rather long period of time until they are erect and touching each other. It is probable the biological closure of the flower occurs sometime within this period of petal straightening, but we can define the hour of flower closure as the time the petal lobes touch again. Another note regarding Neoregelia: the time of flower closing is strongly influenced by the cultural conditions of the plant. In general, the flower opens (the petal lobes spread outward) early in the morning. For plants grown under high light conditions, the petal lobes can start to unbend before noon. When plants are grown in more shade - or are observed on a cloudy day - the fully spread petals persist for a longer time.] 101