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Jones, Brian C.
Condiciones del microclima dentro de las hojas enrolladas de Lepanthes helleri (Orchidaceae)
Microclimate conditions within the rolled leaves of Lepanthes helleri (Orchidaceae)
Epiphytic orchids of the cloud forest experience wet conditions from the high input of mist, but also drying from the potentially desiccating conditions of wind and light. The ability to minimize water loss while still maintaining photosynthetic levels is pivotal in an individuals survival. This study examined Lepanthes helleri and the role of leaf-curling as a mechanism to regulate microclimate conditions. Parameters measured included the size of the leaf and the width of its opened curl, relative humidity of the ambient environment, air and temperature inside and outside of the leaf. The relationships between the width of the leaf opening (aperture) and stomatal density, the percent of open stomata, and the presence of flowers, buds, or neither were tested. Air temperature and leaf height, when factored into a model testing significance, had the most impact on how open the leaf was (stepwise multiple regression; R2 adj = 0.18, P < 0.05, DF = 58 and n = 61). Specifically, as leaf height and aperture size increased, the inside temperature increased. There was a significant difference between the mean temperatures inside and outside the leaf (Paired t-test; t = 7.48, P < 0.0001, DF = 60, n = 61 for both) as well as a negative linear regression between aperture and stomatal density (F = 8.04, R2 adj = 0.12, P = 0.007, n = 52).
Las orqudeas epfitas del bosque nuboso viven en condiciones hmedas debido a la neblina y tambin a las condiciones desecadas por el sol y el viento. La habilidad de reducir la prdida de agua mientras que mantiene los niveles de fotosntesis es lo ms importante para la sobrevivencia de una planta. Esta investigacion examino a Lepanthes helleri y el papel de la hoja enrollada como un mecanismo de cambio en las condiciones del microclima.
Text in English.
Orchids--Costa Rica--Puntarenas--Monteverde Zone
Orqudeas--Costa Rica--Puntarenas--Zona de Monteverde
Tropical Ecology 2008
Ecologa Tropical 2008
t Monteverde Institute : Tropical Ecology
1 FIGURE 1. Lepanthes helleri with its characteristic curled leaves drawing: Luer, 1987. Microclimate conditions within the rolled leaves of Lepanthes helleri Orchidaceae Brian C. Jones Department of Biology, Fairfield University ABSTRACT Epiphytic orchids of the cloud forest experience wet conditions from the high input of mist, but al so drying from the potentially desiccating conditions of wind and light. The ability to minimize water loss while still maintaining photosynthetic levels is pivotal in an individualÂ€s survival. This study examined Lepanthes helleri and the role of leaf c urling as a mechanism to regulate microclimate conditions . Parameters measured included the size of the leaf and the width of its opened curl, relative humidity of the ambient environment, air and temperature inside and outside of the leaf . The relations hips between the width of the leaf opening ÂapertureÂ‚ and stomatal density, the percent of open stomata, and the presence of flowers, buds, or neither were tested. Air te mperature and leaf height, when factored into a model testing significance, had the most impact on how open the leaf was stepwise multiple regression; R 2 adj = 0.18, P < 0.05, DF = 58 and n = 61 . Specifically, as leaf height and aperture size increased, the inside temperature increased. There was a significant difference between the mean temperatures inside and outside the leaf Paired t test; t = 7.48, P < 0.0001, DF = 60, n = 61 for both as well as a negative linear regression between aperture and stomatal density F = 8.04, R 2 adj = 0.12, P = 0.007, n = 52 . RESUMEN Las orquÃ deas epifitas del bosque nuboso viven en condiciones hÃºmedas debido a la neblina y tambiÃ©n condiciones secas por el sol y el viento. La capacidad de reducir la perdida de agua y mantener los niveles de fotosÃntesis es lo mÃ¡s importante para la sobrevivenc ia de una planta. Esta investigaciÃ³n examinÃ³ Lepanthes helleri y el propÃ³sito de la curvatura de la hoja como un mecanismo de cambio en las condiciones del microclima. Los parÃ¡metros examinados incluyeron el tamaÃ±o de la hoja y el ancho de la curvatura a bierta, humedad relativa del medio ambiente, y la temperatura del aire dentro y afuera de la hoja. Las relaciones entre el ancho de la apertura y concentraciÃ³n de estomas, el porcentaje de estomas abiertas , y si hay flores, brotes, o nada se probaron. La temperatura del aire y el tamaÃ±o de la hoja, cuando se usa un modelo de significancia, el impacto mÃ¡s significante en el model es el ancho de la apertura de la hoja y su tamaÃ±o regresiÃ³n mÃºltiple por pasos; R 2 adj = 0.18, P < 0.05, DF = 58 y n = 61. Ta mbiÃ©n, cuando el tamaÃ±o de la hoja y la apertura crecen, la temperatura crece. Hay una diferencia tambiÃ©n de la temperatura a dentro y afuera de la hoja T pareada; t = 7.48, P < 0.0001, DF = 60, n = 61 para los dos y un relaciÃ³n lineal negativa entre la apertura de la hoja y la concentraciÃ³n de estomas F = 8.04, R 2 adj = 0.12, P = 0.007, n = 52. INTRODUCTION The genus Lepanthes Orchidaceae: Pleurothallidinae, has over 600 species, including Lepanthes helleri , a miniature epiphytic canopy orchid f rom Nicaragua to Panama Luer 1986. This species exhibits a unique growth form found only in the Monteverde region of Costa Rica and was once hypothesized to be endemic to Monteverde L. comet halleyi due to the way its leaves curl Atwood 2000. Unlik e other individuals found outside Monteverde, the leaf rolls so that the underside is on the inside of the curl Fig. 1; Luer . Like all plants, L. helleri has structures called stomata which are pores found on the surface of the leaf that regulate gas an d water
2 exchange. On each side of the pore is a guard cell that either swells to open the pore or shrinks to close it to permit or prevent exchange respectively Raven et al. 1986. When 90 to 95 percent of water lost by leaves is through a plantÂ€s stoma ta, t he careful regulation of these pores allows plants to reduce the potentially desiccating effects of transpiration Hopkins 1995. In all plants there also exists a boundary layer consisting of a thin layer of air which lies upon the surface of the le af and affects the rate of transpiration. Increased wind speed can decrease this boundary layerÂ€s thickness and reduce the path of diffusion of water vapor from stomata thus increasing rates of evaporation from the leaf Hopkins 1995. In environments wh ere there is lower moisture content and higher winds, plants such as epiphytic orchids experience increased risk of high levels of water loss due to such conditions . Cloud forest epiphytes depend heavily upon mist as a source of water necessary for ph otosynthesis and regular metabolic processes Fig. 2b; Nadkarni 1986. Because the canopy is raised above all other vegetation, organisms that reside in its branches receive high levels of mist throughout the day . However, individuals are also exposed mo re frequently to high levels of sun and wind which increase rates of transpiration and put the plant at risk of desiccation Fig 2a; Hopkins 1995. Lepanthes helleri grows in such a habitat and frequently oc curs on trees left standing in open pastures due to similarities in abiotic conditions. As a response to such conditions, it has evolved physiological and structural adaptations to help offset the consequences of high levels of wind and sun; succulent leaves better store water and smaller leaves reduce the area avai lable from which to lose water Dressler 1986. The majority of stomata for orchids are found on the underside of the leaf Goh et al. 1977; Kinziger 2000. In the case of L. helleri , stomata are also found on the underside of the leaf insi de the roll suggesting the hypothesis that such a growth form may serve as an adaptation for reducing water loss. The structure of a curled leaf may create a micro climate by trapping moisture and maintaining cooler air. This may allow for prolonged open ing of pores during the day by raising the relative humidity level inside the leaf and lowering temperature within the space created. Doing so would allow the plant to continue photosynthesizing while reducing the amount of water lost. In this study I i nvestigate whether there is a difference between the average temperature inside and outside of the curled leaf. . I will also determine whether the distance between the edges of the curled leaf the ÂapertureÂ‚ is related to outside air temperature, tempe rature differential, stomatal density, the percent of open stomata, the percent relative humidity outside the leaf, or leaf height. I predict that there is a microclimate present within the curl of the leaf, and that the smaller the aperture size the cool er the temperature, the greater the density of stomata, and the higher the percentage of open stomata. Additionally, I will explore whether the presence of flowers or buds relate to aperture size. Because species within this genus have been observed to b e pollinated by pseudocopulation with fungal gnats, I predict that a curled leaf will show a positive correlation between the presence of flowers and a smaller aperture creating a more stable environment in which the gnat can pollinate. FIGURE 2. Photo s of natural climate conditions of Monteverde. a mist essential to many epiphytic organisms; b dry, windy conditions present in pastures similar to that of the canopy. b a
3 MATERIALS AND M ETHODS STUDY SITE . Âƒ I took d ata on twenty seven L. helleri individuals between April 24, 2008 and May 3, 2007 in a pasture on the Guindon Family property in Monteverde, Costa Rica. All data were collected between early morning to mid afternoon when sunl ight and wind were moderate and consistent. I measured o nly individuals growing between approximately breast and head height on the trunk or low reaching braches of trees . Also, only leaves that were developmentally mature, noted by their larger size, d ramatically curled shape, and darker color, were included. This minimized variation that might arise due to age differences. TEMPERATURE . Âƒ For each individual leaf a digital, I used a digital, probe thermometer to determine the temperature inside and outside of the leaf. The thermometer was allowed to equilibrate in each setting for approximately two minutes or until t he reading no longer fluctuated . I also noted percent relative humidity of the external environment when first inserting the probe in to the curl . Before taking the data of another leaf , I allowed the thermometer to equilibrate again with the outside air temperature before taking the readings of the next individual . I used a paired t test to determine if there was a significant differe nce between the temperature inside and outside the curl. STOMATA DENSITY and PERCENT STOMATA OPEN . Âƒ For each individual , I painted a thin, even layer of clear chip resistant nail polish to the underside of the leaf and allowed it to dry before removing it with tweezers. Peels were examined under a compound microscope at 100x and all visible stomata were counted. In order to prov ide a better estimate, I took counts from two sites for each peel and used the average of the two sites in calculating stomata l density. If a peel was not able to be read or did not have sufficient area to provide two sites than it was excluded from statistical analyses concerning stomata. With respect to scoring open stomata , I used the same technique as described above. Indis tinguishable stomata were not scored and partially opened stomata were counted as open as long as they were able to be seen as clearly more than halfway open. The data from the two sites were also averaged to achieve a better estimate. Finally, I used a regression analysis to test the significance of stomatal density and percent open stomata related to aperture size. FLOWERS and BUDS . Âƒ I noted for each leaf whether there were flowers, buds, or no flowers at all present within the curl of the leaf typic al of L. helleri K.L. Masters 2008, pers. comm.. The height of each leaf was measured using micro calipers. I measured the height as the distance from the base of the leaf where it joins the stem to the tip of the leaf where the midvein ends. The ape rture distance was also recorded using micro calipers measuring where the edges of the widest part of the leafÂ€s margin were curled towards each other. All data taken with the micro calipers were recorded in centimeters. I tested the significance between the presence of flowers, buds, or nothing and aperture size using a Kruskal Wallis test. Finally, to test which of the previous measured parameters temperature differential, percent relative humidity, leaf height, and air temperature had the greatest i mpact on aperture size, I used a stepwise multiple regression analysis.
4 RESULTS The parameters of temperature differential partial R 2 = 0.008, percent relative humidity partial R 2 = 0.13, leaf height partial R 2 = 0.10, and air temperature partial R 2 = 0.20 were tested to see their individual effects on aperture size. When temperature differential was plotted as a function of aperture the trend was negative y = 0.2588x + 0.8725 . Also, when aperture was plotted as a function of percent relativ e humidity, percent relative humidity was not a determinant of aperture. When the parameters were all tested together using multiple regression analyses , the combined impact of leaf height and air temperature were significant stepwise multiple regression ; R 2 adj = 0.18, P < 0.05, df = 58, n = 61 for all. Leaf height showed a negative regression in relation to aperture Fig. 3 and ambient air temperature a positive regression Fig. 4. Temperature from inside the curl of the leaf was found to be signifi cantly greater than that of ambient air temperature Paired t test; t = 7.48, P < 0.0001, df = 60, n = 61 for both; Fig. 5. This showed that, on average, the inside of the curled leaf is hotter than the outside air temperature. The regression analysis f or stomatal density and aperture size was significant F = 8.04, R 2 adj = 0.12, P = 0.007, n = 52; Fig. 6. However, aperture size and the percent open stomata did not have a significant relationship although a trendline shows a positive regression F = 3 .20, R 2 adj = 0.041, P = 0.08, n = 52, y = 0.4399x + 0.1241. There was no significant relationship between the mean aperture size of the leaf and whether or not there were flowers, buds, or nothing within the curl Kruskal Wallis test; Â„ 2 = 5.16, P = 0.08, n flowers = 11, n buds = 11, n nothing = 39, SD flowers = Â± 0.15, SD buds = Â± 0.26, SD nothing = Â± 0.31. 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.30 1.50 1.70 1.90 2.10 Leaf Height cm Aperture cm FIGURE 3 . Aperture as a function of leaf height. The trendline illustrates a significant negative linear regression y = 0.4013x + 0.8593 .
5 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 18 20 22 24 26 Ambient Air Temperature Â€C Aperture cm FIGURE 4 . Aperture as a function of ambient air temperature. The trendline illustrates a significant p ositive linear regression y = 0.0646X Âƒ 1.1856 . 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 Mean Temperature Â€C Inside Outside FIGURE 5 . Temperature inside and outside the curl of the leaf . Error bars represent SD = Â± 1.71 for inside the leaf and SD = Â± 1.43 for outside the leaf. The mean value for outside the leaf is 21.96 and for inside is 22.77.
6 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 Stomatal Density stomata/mm^2 Aperture cm FIGURE 6 . Aperture versus stomatal density. The trendline illustrates a significant negative linear regression y = 0.0125x + 0.6827. DISCUSSION The results of this study did not s upport the prediction that conditions within the curled leaf were cooler and thus minimizing water loss. Instead temperature conditions were found to be higher inside the leaf than that of ambient conditions. Although this difference may aid L. helleri i n preventing loss of water due to open stomata, it is not clear as to how this affects its fitness and photosynthetic capacity. The temperature inside and outside the leaf are within the range at which transpiration is greatest for a plant Moore et al . 1 995 . When rates of transpiration increase 5 % for every 1 Â°C increase it is vital for L. helleri to close its stomata. Studies have shown that increases in temperature raise rates of cellular respiration and carbon diox ide concentrations within leaf tis sue causing pores to close Raven et al. 1986. For a plant that lives in such drying conditions , raising the temperature inside the curl causing closure of stomata could benefit it on days when open stomata would otherwise mean desiccation. The curl cou ld possibly achieve this by stabilizing warmer air thus magnifying the effect of conditions inside the curl and having stomata open as low as 22.7 % open on average. Results of this study also show that , when ambient conditions are hotter , aperture size in creases. The cooling effect of wind could aid the plant in its capacity to open its aperture more under such conditions. Increased levels of wind will disturb the boundary layer of the leaf thinning it, cooling the leaf, and opening stomata Hopkins 1995 . The trend in this study, although not significant, that percent open stomata increases as aperture size increases , supports the hypothesis that opening the leafÂ€ s aperture creates cooler conditions in the curl that allow stomata to open on warmer days. Paradoxically, despite higher rates of transpiration resulting from a disturbed boundary layer, it may be more of a benefit energetically to open stomata under such cooler conditions in order to photosynthesize . Another way that L. helleri seems to have adapted to hot and windy conditions is by reducing the number of stomata it has per leaf. The results of this study showed that as aperture size increased stomatal density decreased. As seen previously, when the aperture is wider
7 conditions are created t hat, although allow stomata to open, are also desiccating because they increase rates of transpiration. I ndividuals that are born with higher densities of stomata have significantly smaller apertures which could possibly be a method of compensating for hi gher possible rates of transpiration associated with greater stomatal densities. This could help the plant to offset the water most likely being lost from greater concentrations of open stomata that may otherwise be helping the plant to photosynthesize. The unique leaf shape of L. helleri could also be related to its pollinator and flower . Studies on other species within Lepanthes with very similar flower morphology have shown the pollinator to be a small fungus gnat. They were observed to always fly do wnwind, as if directed by a phero mone trail , when approaching the flower before engaging in pseudocopulation which results in pollination Dressler 1993: Blanco and Barboza 2005. In the case of L. helleri , a curled leaf could act as a funnel for concentr ating pheromones when windy conditions in the canopy or pasture would otherwise cause them to be diffusely scattered. Such a shape could also provide a stable environment in which the plantÂ€s pollinator could more easily engage in psuedocopulation without disturbance from high levels of wind present in such a habitat. Finally, with such a delicate flower structure, the curl could also better protect an individualÂ€s flower from damage due to factors such as harmful weather conditions . This study focused p rimarily on beginning to explore and quantify the m icroclimate conditions inside the rolled leaves of L. helleri . F urther studies examining humidity both inside and out of the curl and how stomat a respond would also help to better define the microclimate conditions. Taking into consideration varying weather conditions within pastures and the canopy, sto matal response to wind speed and percent relative humidity changes should be examined in order to further understand how L. helleri adjusts its microclimat e to daily fluctuating in weather . Studies have also shown evidence that other species of Lepanthes have potential to be classified as Crassulacean acid metabolism CAM in which stomata open more at night allowing metabolic processes to occur with greatl y reduced risk of water loss Hopkins 1995 ; Becklund 2000. It is important to note that even if this particular species of Lepanthes is a CAM species it still may also utilize typical metabolic and photosynthetic pathways during the day in order to meet energetic demands with available sunlight . Ho w stomatal openness, leaf morphology, and alteration of its microclimate relate to L. helleri meeting its energetic demands in this way would be s ignificant in understanding if there are implications of a curle d leaf on metabolic processes . Finally, studies must be performed to determine if there is a n increased reproductive fitness with a cur l ed leaf in relation to the organismÂ€s pollinator . With these accompanying investigations we will be better able to ass ess how such a fascinating organism responds to the daily challenges its habitat puts forth. ACKNOWLEDG MENTS Thank you to la EstaciÃ³n BiolÃ³gica de Monteverde for providing a space in which to carry out the laboratory portions of this study. Thank you to Lucky Guindon for the generous use of her farm and house and to Martha Campbell for initially offering her farm as a site. Thank you profusely to the continual patience , support, and advice from Karen Masters throughout the forming and execution of this project. Finally, thanks to Alan Masters for helping with initial brainstorming sessions, Taegan McMahon and Nikol Biermaier for assistance with statistical analyses and formatting difficulties, and CIEE and everyone that made this experience as memorable as it was.
8 LITERATU R E CITED Atwood, J. T. 2000. Orchids of Monteverde . In, Monteverde: Ecology and Conservation of a Tropical Cloud Forest, Nadkarmi, N.M., and Wheelwright, N.T., ed. Oxford University, New York, NY, pp. 524. Becklund, K. 2006. Sto matal density and aperture in four species of pleurothallid orchids Orchidaceae. In, CIEE Spring 2006 Tropical Ecology and Conservation. Blanco, M. and G. Barboza. 2005. Psuedocopulatory pollination in Lepanthes Orchidaceae: Pleurothallidinae by Fun gus Gnats. Annals of Botany. 955: 763 772. Dressler, R. L. 1986. The Orchids : Natural History and Classification. Harvard University Press. pp. 28. ÂƒÂƒÂƒÂƒÂƒÂƒ . 1995. Field Guide to the Orchids of Costa Rica and Panama. Cornell University Press. Goh, C ., P. Avadhani, C. Loh, C. Hanegraaf, and J. Arditti. 1977. Diurnal stomatal and acidity rhythms in orchid leaves. New Phytology. 78:365 372. Hopkins, W. 1995. Introduction to Plant Physiology. John Wiley and Sons, Inc. United States of America. pp . 43, 45 46. Kinziger, A. 2000. Effects of microhabitat on fitness and physiological strategies of Lepanthes helleri comet halleyi Orchidaceae. In, CIEE Spring 2000 Tropical Ecology and Conservation. Luer, C. A. 1987. New Lepanthes species from Costa Rica and Panama. Lindleyana; Scientific Journal of the American Orchid Society 24: 185 217. ÂƒÂƒÂƒÂƒÂƒ . 1986. Icones Pleurothallidinarum, I. Systematics of the Pleurothallidinae Orchidaceae. Monographs in systematics botany , Missouri Botanical Gardens. 1 5: 1 81. In: Tremblay, R. L. Distribution and dispersion patterns of individuals in nine species of Lepanthes Orchidaceae. Biotropica 29: 38 45. Moore, R., W. D. Clark, K. R. Stern, D. Vodopich. 1995. Botany. Wm. C. Brown Communications, Inc. pp. 4 98. Raven, P., R. Evert, S. Eichhorn. 1986. Biology of Plants. Worth Publishers, Inc. New York, NY. pp. 548, 550.