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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 we t conditions from the high input of mist, but also drying from the potentially desiccating conditions of wind and ligh t. 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 microclimat e conditions. Parameters measured included the siz e of the leaf and the width of its opened curl, relative humidity of the ambient environment, air and temperature in side and outside of the leaf. The relationships between the width of the leaf opening (aperture) and stomata l density, the percent of open stomata, and the presence of flower s, buds, or neither were tested. Air temperature a nd leaf height, when factored into a model testing significance, ha d the most impact on how open the leaf was (stepwis e multiple regression; R2 adj = 0.18, P < 0.05, DF = 58 and n = 61). Specifically, as lea f height and aperture size increased, the inside temperature increased. There was a signific ant 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 n egative linear regression between aperture and stomatal density (F = 8.04, R2 adj = 0.12, P = 0.007, n = 52). RESUMEN Las orqudeas epifitas del bosque nuboso viven en c ondiciones hmedas debido a la neblina y tambin co ndiciones secas por el sol y el viento. La capacidad de redu cir la perdida de agua y mantener los niveles de fo tosntesis es lo ms importante para la sobrevivencia de una planta. Esta investigacin examin Lepanthes helleri y el propsito de la curvatura de la hoja como un mecanismo de cambio en las condiciones del microclima. Los parmetros examinados incluyeron el tamao de la hoja y el anc ho de la curvatura abierta, humedad relativa del me dio ambiente, y la temperatura del aire dentro y afuera de la hoja. Las relaciones entre el ancho de la a pertura y concentracin de estomas, el porcentaje de estomas abiertas, y si hay flores, brotes, o nada se probar on. La temperatura del aire y el tamao de la hoja, cuando se usa un modelo de significancia, el impacto ms significante en el model es el ancho de la apertura de la hoja y su tamao (regresin mltiple por pasos; R2 adj = 0.18, P < 0.05, DF = 58 y n = 61). Tambin, cuando el tamao de la hoja y la apertura crecen, la temperatura crece. Hay una diferencia tambin de la temperatura a dentro y afu era de la hoja (T pareada; t = 7.48, P < 0.0001, DF = 60, n = 61 para los dos) y un relacin lineal negativa entre l a apertura de la hoja y la concentracin de estomas (F = 8.04, R2 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 from Nicaragua to Panama (L uer 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). Unli ke other individuals found outside Monteverde, the leaf roll s so that the underside is on the inside of the curl (Fig. 1; Lue r). Like all plants, L. helleri has structures called stomata which are pores found on the surface of the leaf that regulate gas and 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 respectiv ely (Raven et al. 1986). When 90 to 95 percent of water lost by leaves is through a plants stomat a, the careful regulation of these pores allows plants to reduce the potentially desiccating effect s 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 leaf and affects the rate of transpiration. In creased wind speed can decrease this boundary layers thickness and reduce the path of diffusion of water vapor from stomata thus increasing rates of evaporation from the leaf (Hopkins 1995). In environments where there is lower moisture content and higher winds, plants such as e piphytic 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 photosynthesis and regular m etabolic processes (Fig. 2b; Nadkarni 1986). Because the ca nopy is raised above all other vegetation, organisms that reside i n its branches receive high levels of mist throughout the day. Ho wever, individuals are also exposed more frequently to hig h 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 occurs on trees le ft standing in open pastures due to similarities in abiotic conditions. As a response to such conditions, it has evolved physiological and s tructural adaptations to help offset the consequences of high levels of wind and sun; succulent leaves better store water and sm aller leaves reduce the area available from which to lose water (Dressler 1986). The majority of stomata for orchids are found on th e 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 inside the roll suggesting the hypothesis that such a growth form m ay 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 opening o f pores during the day by raising the relative humidity level insi de the leaf and lowering temperature within the space created. Doi ng so would allow the plant to continue photosynthesizing while reducing the amount of wate r lost. In this study I investigate whether there is a diff erence between the average temperature inside and outside of the curled leaf. I will al so determine whether the distance between the edges of the curled leaf (the aperture) is relate d to outside air temperature, temperature 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 cooler t he temperature, the greater the density of stomata, and the higher the percentage of open stom ata. Additionally, I will explore whether the presence of flowers or buds relate to aperture size Because species within this genus have been observed to be 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 polli nate. FIGURE 2. Photos 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 METHODS STUDY SITE. I took data on twenty-seven L. helleri individuals between April 24, 2008 and May 3, 2007 in a pasture on the Guindon Family property in Monteverd e, Costa Rica. All data were collected between early morning to mid afternoon when sunligh t and wind were moderate and consistent. I measured only individuals growing between approxi mately breast and head height on the trunk or low reaching braches of trees. Also, only leave s that were developmentally mature, (noted by their larger size, dramatically curled shape, and d arker color), were included. This minimized variation that might arise due to age differences. TEMPERATURE. For each individual leaf a digital, I used a digita l, probe thermometer to determine the temperature inside and outside of the leaf. The th ermometer was allowed to equilibrate in each setting for approximately two minutes or until the reading no longer fluctuated. I also noted percent relative humidity of the external environme nt when first inserting the probe into the curl. Before taking the data of another leaf, I allowed t he 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 difference bet ween the temperature inside and outside the curl. STOMATA DENSITY and PERCENT STOMATA OPEN. For each individual, I painted a thin, even layer o f clear chip-resistant nail polish to the underside of the leaf and allowed it to dry before removing i t with tweezers. Peels were examined under a compound microscope at 100x and all visible stomata were counted. In order to provide a better estimate, I took counts from two sites for each pee l and used the average of the two sites in calculating stomatal density. If a peel was not ab le to be read or did not have sufficient area to provide two sites than it was excluded from statist ical analyses concerning stomata. With respect to scoring open stomata, I used the sa me technique as described above. Indistinguishable stomata were not scored and parti ally opened stomata were counted as open as long as they were able to be seen as clearly more t han halfway open. The data from the two sites were also averaged to achieve a better estimate. F inally, I used a regression analysis to test the significance of stomatal density and percent open s tomata related to aperture size. FLOWERS and BUDS. I noted for each leaf whether there were flowers, b uds, or no flowers at all present within the curl of the leaf (typical of L. helleri ) (K.L. Masters 2008, pers. comm.). The height of each leaf was measured using micro calipers. I measured the heig ht as the distance from the base of the leaf where it joins the stem to the tip of the leaf wher e the midvein ends. The aperture distance was also recorded using micro calipers measuring where the edges of the widest part of the leafs margin were curled towards each other. All data ta ken 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 par ameters (temperature differential, percent relative humidity, leaf height, and air tem perature) had the greatest impact on aperture size, I used a stepwise multiple regression analysi s.
4 RESULTS The parameters of temperature differential (partial R2 = 0.008), percent relative humidity (partial R2 = 0.13), leaf height (partial R2 = 0.10), and air temperature (partial R2 = 0.20) were tested to see their individual effects on aperture size. Whe n 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 relative 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 ai r temperature were significant (stepwise multiple regression; R2 adj = 0.18, P < 0.05, df = 58, n = 61 for all). Leaf height sho wed 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 fo und to be significantly 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 th e curled leaf is hotter than the outside air temperature. The regression analysis for stomatal density and a perture size was significant (F = 8.04, R2 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, R2 adj = 0.041, P = 0.08, n = 52, y = 0.4399x + 0.1241). There was no significant relationship be tween the mean aperture size of the leaf and whether or not there were flowers, buds, or nothing within the curl (Kruskal-Wallis test; 2 = 5.16, P = 0.08, nflowers = 11, nbuds = 11, nnothing = 39, SDflowers = 0.15, SDbuds = 0.26, SDnothing = 0.31). 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.101.301.501.701.902.10Leaf 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 1820222426 Ambient Air Temperature (C)Aperture (cm) FIGURE 4. Aperture as a function of ambient air te mperature. The trendline illustrates a significant positive linear regression (y = 0.0646X 1.1856). 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0Mean Temperature (C)Inside Outside FIGURE 5. Temperature inside and outside the curl o f the leaf Error bars represent SD = 1.71 for inside the leaf and SD = 1.43 for outsi de 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 1.0015.0020.0025.0030.0035.0040.0045.0050.0055.00Stomatal Density (stomata/mm^2)Aperture (cm) FIGURE 6. Aperture versus stomatal density. The t rendline illustrates a significant negative linear regression (y = -0.0125x + 0.6827). DISCUSSION The results of this study did not support the predi ction that conditions within the curled leaf were cooler and thus minimizing water loss. Instead tem perature conditions were found to be higher inside the leaf than that of ambient conditions. A lthough this difference may aid L. helleri in 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 an d outside the leaf are within the range at which transpiration is greatest for a plant (Moore et al. 1995). When rates of transpiration increase 5 % for every 1 C increase it is vital fo r L. helleri to close its stomata. Studies have shown that increases in temperature raise rates of cellular respiration and carbon dioxide concentrations within leaf tissue 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 ot herwise mean desiccation. The curl could possibly achieve this by stabilizing warmer air thu s 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 increases. The cooling effect of wind could aid th e plant in its capacity to open its aperture more under such conditions. Increased levels of wind wi ll 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 leafs aperture cre ates cooler conditions in the curl that allow stomata to open on warmer days. Paradoxically, des pite 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. Th e 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 that, although allow stomata to open, are also desiccating because they increase rates of transpiration. Individuals that are born with higher densities of stomata have significantly smaller apertures which could possibl y be a method of compensating for higher possible rates of transpiration associated with gre ater stomatal densities. This could help the plant to offset the water most likely being lost fr om greater concentrations of open stomata that may otherwise be helping the plant to photosynthesi ze. 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 ob served to always fly downwind, as if directed by a pheromone trail, when approaching the flower b efore engaging in pseudocopulation which results in pollination (Dressler 1993: Blanco and B arboza 2005). In the case of L. helleri a curled leaf could act as a funnel for concentrating 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 plants p ollinator could more easily engage in psuedocopulation without disturbance from high leve ls of wind present in such a habitat. Finally, with such a delicate flower structure, the curl could also better protect an individuals flower from damage due to factors such as harmful w eather conditions. This study focused primarily on beginning to explo re and quantify the microclimate conditions inside the rolled leaves of L. helleri Further studies examining humidity both inside and out of the curl and how stomata respond would a lso help to better define the microclimate conditions. Taking into consideration varying weat her conditions within pastures and the canopy, stomatal response to wind speed and percent relative humidity changes should be examined in order to further understand how L. helleri adjusts its microclimate to daily fluctuating in weather. Studies have also shown ev idence that other species of Lepanthes have potential to be classified as Crassulacean acid met abolism (CAM) in which stomata open more at night allowing metabolic processes to occur with gr eatly 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 met abolic and photosynthetic pathways during the day in order to meet energetic demands with availab le sunlight. How stomatal openness, leaf morphology, and alteration of its microclimate rela te to L. helleri meeting its energetic demands in this way would be significant in understanding i f there are implications of a curled leaf on metabolic processes. Finally, studies must be perf ormed to determine if there is an increased reproductive fitness with a curled leaf in relation to the organisms pollinator. With these accompanying investigations we will be better able to assess how such a fascinating organism responds to the daily challenges its habitat puts f orth. ACKNOWLEDGMENTS Thank you to la Estacin Biolgica de Monteverde fo r providing a space in which to carry out the labor atory 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, su pport, and advice from Karen Masters throughout the forming and execu tion of this project. Finally, thanks to Alan Mast ers for helping with initial brainstorming sessions, Taegan McMahon and Nikol Biermaier for assistance with st atistical analyses and formatting difficulties, and CIEE and everyone that made this experience as memorable as it was.
8 LITERATURE CITED Atwood, J. T. 2000. Orchids of Monteverde In, Monteverde: Ecology and Conservation of a Tropical Cloud Forest, Nadkarmi, N.M., and Wheelwr ight, N.T., ed. Oxford University, New York, NY, pp. 524. Becklund, K. 2006. Stomatal density and aperture in four species of pleurothallid orchids (Orchidaceae). In, CIEE Spring 2006 Tropical Ecol ogy and Conservation. Blanco, M. and G. Barboza. 2005. Psuedocopulatory pollination in Lepanthes (Orchidaceae: Pleurothallidinae) by Fungus Gnats. Annals of Bot any. 95(5): 763-772. Dressler, R. L. 1986. The Orchids: Natural History and Classification. Harvard University Press. pp. 28. . 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. 78:365372. Hopkins, W. 1995. Introduction to Plant Physiolog y. John Wiley and Sons, Inc. United States of America. pp. 43, 45-46. Kinziger, A. 2000. Effects of microhabitat on fitne ss and physiological strategies of Lepanthes helleri ( comet-halleyi ) (Orchidaceae). In, CIEE Spring 2000 Tropical Eco logy and Conservation. Luer, C. A. 1987. New Lepanthes species from Costa Rica and Panama. Lindleyana; S cientific Journal of the American Orchid Society 2(4): 185-2 17. . 1986. Icones Pleurothallidinarum, I. Systema tics of the Pleurothallidinae (Orchidaceae). Monographs in systematics botany Missouri Botanical Gardens. 15: 1-81. In: Trembl ay, R. L. Distribution and dispersion patterns of indi viduals 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. 498. Raven, P., R. Evert, S. Eichhorn. 1986. Biology o f Plants. Worth Publishers, Inc. New York, NY. pp. 548, 550.
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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