Stomata Densities of Pleurothallids (Orchidaceae ) in Different Microhabitats of Monteverde Chelsea Beebe Department of EPOBiology, University of Colorado Boulder ___________________________________________________________________ ABSTRACT The purpose of this study was to test whether there is a difference in the number of open and closed stomata on the northeast and southwest facing sides of trees among seven species of Pleurothallids and one pseudobulb species. Stomata impressions were tak en from the leaves using clear finger nail polish and they were then examined under a microscope. The results showed that there was a significant difference between the number of open and closed stomata for every species, always with a higher frequency of closed stomata. Significant affects of species type and position on the tree on stomata density was also found with branch tip orchids having a lower stomata density then non branch tip species. A possible explanation for the low frequency of open stomata is that they might only open in the rain, when water loss is at a minimum. RESUMEN El propÃ³ sito de este estudio debÃa proba r si hay una diferencia en el nÃº mero de abierto y cerrÃ³ stomata en el noreste y el suroeste frente a lados de Ã¡ rboles entre siete especies de Pleurothallids y una especie de pseudobulb. Las impresiones de Stomata se tomaron de las hojas que utilizan el esmalte para las uÃ± as claro de dedo y ellos entonces fueron examinados abajo un microscopio. Los resultados mostraron que habÃa una d iferencia significativa entre el nÃºmero de abierto y cerrÃ³ stomata para cada especie, siempre con una frecuencia m Ã¡s alta de cerrÃ³ stomata. Los afectos significativos de tipo de especie y la posiciÃ³n en el Ã¡ rbol en la densidad de stomata se encontrÃ³ tambiÃ© n con orquÃ deas de punta de rama, teniendo una densidad mÃ¡ s baja de stomata entonces no la especie de punta de rama. Una explicaciÃ³ n posible para la frecuencia baja de stomata abierto es que ellos pueden sol o abi erto en la lluvia, cuando la pe rdida de agu a estÃ¡ en un mÃ nimo. Introduction The family Orchidaceae is a large and very diverse group among the Angiosperms with between 20,000 and 25,0 00 species in 725 genera (Dressl er 1981). With approximately 1,200 species in Costa Rica, it is the largest group of flowering plants (Hartshorn 1983; Walter 1986). The majority of orchid species are epiphytic (Walter 1986). Epiphytes are more exposed to harsh conditions and thus require specific abiotic and biotic factors for successful growth. One necessary abiotic, factor is a high amount of continuous moisture. With 2500 3500 mm of rainfall annually, as well as a frequent misty period s , the cloud forest of Monteverde, Costa Rica, is a very hospitable habitat for epiphytic orchids (Haber
2000). Orchid diversity is especially high in this type of ecosystem (Dressl er 1993). The Monteverde trade winds blow from northeast to southwest most pr ominently from November through January and more moderately from February to April (Clark et al. 2000). This wind has both positive and negative effects on epiphytic growth, bringing with it either high amounts of mist or drying winds. In desiccating envir onments, plants have evolved many adaptations to protect themselves from dehydration. One such adaptation is Crassulacean acid metabolism (CAM) photosynthesis. In this process, plants open their stomata, the structures that regulate gas exchange and water up take, at night instead of during the day (Raven and Johnson 1986). This method of carbon fixation accumulates C 4 compounds at night, allowing the plants to enter the Calvin cycle during the day and thus minimize water lost to harsh daytime environmental factors (Raven and Johnson 1986). Stomata densities are not uniform throughout a species. Different leaves of the same species can have different stomata densities depending on their ages as well as the conditions of the microhabitat into which the leaf w as born (Chicurel 2000). Since the loss of water through the s tomata is proportional to the stomata density, a leaf born in a drier environment will likely have lower stomata density in order to limit the loss of water to such factors as wind, intense UV light, and increased daytime temperatures (Fitter and Hay 1987). The sub tribe Pleurothallidinae occurs in cloud forests in great diversity (Dressl er 1993). This group is often epiphytic and can be distinguished by their fleshy leaves, thick cuticle, oft en, sunken stomata, lack of pseudobulbs, and limited storage tissue for water (Walter 1986; Dressl er 1993). They are most common in mid to high elevation areas and are thought to be a very vulnerable group to drought and other microhabitat variation (Walte r 1986; Frohlich 2002). Due to their lack of pseudobulbs and limited storage tissue, these orchids rely heavily on the rains and misty conditions of the cloud forest for survival. In a study done on mist variation and epiphytic orchid abundance, it was fou nd that Pleurothallids were most abundant where the daily mist frequencies were the highest and most frequent (Frohlich 2002). This study tested the whether there was a higher frequency of stomata open at night versus the day on the northeast facing side o f the tree or the southwest facing side of the tree for seven species of the sub tribe Pleurothallidinae, and one pseudobulb species. On the postulation that Pleurothallids utilize CAM photosynthesis due to their lack of water storage structures, a higher frequency of open stomata at night was anticipated. Likewise there was a predicted higher frequency of open stomata in the day for the pseudobulb species because they are not limited by water availability, due to their morphological adaptations. Within these results, there was also anticipated a higher number of open stomata on the southwest side of the tree due to their protected location from the northeast trade winds. Materials and Methods This study was conduct ed in a field of secondary growth and open space behind the EstaciÃ³n BiolÃ³ gica in Monteverde, Puntarenas, Costa Rica. T he elevation is approximately 1580 m above sea level. Eight species of epiphytic orchids, seven from the family Pleurothallidinae includin g Pleu rothallis sanchoi, Lepanthis pyg meae,
Speklinia aristata, Pleurothallis tuerckheimii, Lepanthes monteverdensis, Lepanteis sp., and Masdevallia nidifica and one species of pseudobulb orchid were chosen for the study. The samples were taken from the northeast and southwest facing sides of the lower two meters of the trees. Twenty leaves from each direction were taken in the day between the hours of 9:00a.m. and 2:00 p.m., and at night between the hours of 8:00p.m. and 1 1:00 p.m. The six trees from which the samples were taken were chosen based on a mist study previously performed in the same field by Hanna Meisner Bagdahn in 2003. Her study measured the effects of mist frequency on Pleurothallid abundance and survival. Ba sed on these data, I chose trees from that study that all had more misty days on the north facing side of the tree as compared with the south facing side. These trees are located along the western and northwestern sides of the field . Impressions of stomata were taken using clear finger nail polish and applying a small amount to the abaxial side of each orchid leaf. After sufficient drying time, the finger nail polish was removed with tweezers and placed in an envelope for transport. Each sample was then placed under a compound microscope at 400x magnification and viewed in three randomly selected locations. At each location the number of open and closed stomata was counted and recorded. A 2 way ANOVA test was performed on the data to measure the effects of species type and position on the densities of stomata. The data from each species from the northeast and southwest and day and night were then summed and analyzed using a 2x4 Contingency test. Results After running a 2 way ANOVA (ind ependent variables = species and position; dependent variables = number of stomata), I found a significant effect (P = 0.0307) of species on number of stomata as well as a significant interaction effect (Table 1 and Fig. 1). I also ran a Fisher's PLSD post hoc test and found many species pairs to be significantly different (Table 2). I then ran a 2x4 contingency test on the summed data of open and closed stomata from each species and found a significant difference between the north and the south sides for every species (Table 3 and Fig. 2). Discussion The results (Table 1 and Fig. 1) showed that the species of orchid has a significant effect on the stomata density of each leaf. There are also a number of significant differences between pairs of species. The re is a significant difference between P. sanchoi and M. nidifica and all other Pleurothallids. This can be explained in a number of ways. All the other Pleurothallids here, except for P. tuerckheimii, are considered branch tip species of orchids. This means that, in the canopy where orchids are more common, these species are usually found on the very tips of the host branches (K.L Masters, 2003, pers comm.). Here, the orchids are exposed to more wind and sunlight than in other areas where non branch tip orchids such as P. sanchoi and M. nidifica generally grow. Branch tip micro habitats, which are potentially be more desiccating, would be likely to have orchids with lower stomata density than the stomata densi ty of orchids found else where o n trees.
There was a significant difference between the total number of stomata on the pseudobulb species and the other Pleurothallids. Pseudobulb orchids can store water for the plant in their enlarged stems. This constant source of water a vailability protects the plant from desiccation and thus allows it to have more stomata. There was also a higher frequency of open stomata on the southwest side of the host tree. The se data suggest that the pseudob ulbs species could possibly be more suscep tible to the trade winds and thus has a higher frequency of stomata on the more protective southwestern side of the tree. The comparis on of the frequency of open and closed sto mata in the night and day and on the northeast and southwest side of the tree yielded a nonrandom pattern. Specifically, more stomata were closed then was expected by chance, for both the night and day and the northeast and southwest aspect. These results were true for both the Ple urothallids and well as the pseudobulb species. This means that the stomata of both groups generally keep their stomata closed regardless of their aspect on the tree or the time of day. This may suggest the importance of water conservation to all orchids, since the high frequency of closed stomata was dramatic. Reasons for the abundant closure of stomata may be explained by a great need to avoid water loss during evapotranspiration. Perhaps they only open their stomata when there is abundant moisture in the air, i. e. when it is raining or heavily misting. Since it is difficult to take good samples in the rain, all night samples were taken in dry weather thus excluding any raining nights. Also, according to Dressl er, the CAM system is very flexible, allowing stomata to open in the day in times of moisture abundance, i.e. the wet season in Monteverde. This might have been missed due to infrequent sampling. Further studies need to be preformed to answer this puzzling question . Acknowledgements I would like to thank Karen Masters for all of her help and expertise during this project, especially for Gasner and Andrew Rodstrom, and Carmen Rojas for all their help in proof reading and material acquisition and a special thanks to Matt for the late night stomata sampling. Additional thanks to Amish Patel for all the help on the figures. I would also like to thank the staff of the EstaciÃ³n BiolÃ³gica and all the CIEE fall 2003 nut bags for all the good times and laughter. _________________________________________________________________________________________________ Literature Cited Chicurel, M.R. 2000. Water conservation strategies in six species of epiphytic orchids at the EstaciÃ³n BiolÃ³gica Monteverde, Puntarenas, Costa Rica. CIEE Summer Monteverde, Costa Rica. Clark, K., R. Lawton, and P. Butler. 2000. The physical environment . In: Nadkarni, N. and N. Wheelwright, eds. 2000. Monteverde. Oxford Uni versity Press, Inc, pp. 15 38 Dressler, R.L. 1981. The Orchids: Natural history and classification. Harvard University Press, London England. Dressler, R.L. 1993. Field Guide to the Orchids of Costa Rica and Panama. Cornell University Press, New York, pp. 7 25.
Fitter, A.H. and R. Hay. 1987. Environmental Physiology of Plants, 2 nd Edition. Academic Press Limited, London, page 140. Frolich, C. 2002. Mist variation and epiphytic orchid abundance in a Monteverde Cloud Forest. CIEE Spring Monteverde, Costa Rica . Haber, W. 2000. Plants and vegetation . In: Nadkarni, N. and N. Wheelwright, eds. 2000. Monteverde. Oxford University Press, Inc., pp. 39 41. Hartshorn, G.S. 1983. Introduction. In: Costa Rican Natural History, D.H. Janzen, ed. The University of Chicago P ress, Chicago IL, page 148. Labay, B.J. 2000. Effects of microhabitat on morphological adaptations of Pleurothallis sanchoi (Orchidaceae). CIEE fall Monteverde, Costa Rica. Meisner Bagdahn, H. 2003. Climate Change and Microclimatic Variation: Effects on the Fitness and Abundancy of Cloud Forest Orchids. ACM Spring, Monteverde, Costa Rica. Raven, P.H. and G.B. Johnson. 1986. Biology. Times Mirrory/Mosby College Publishing, St. Louis, MO., page 644. St arr, C. and R. Taggart. 1981. Biology: The unity and diversity of life, 2 nd edition. Wadsworth Publishing Company, Belmont, CA. page 374. Walter, K.S. Orchidaceae . In: Costa Rican Natural History, D.H. Janzen, ed. The University of Chicago Press, Chicago, IL, pp. 282 289.
Table 1 . ANOVA table showing effects of orchid species and position (northeast and southwest) on number of stomata on orchid leaves. DF F Value P Value Species 7 39.162 <.0001 Position 1 2.903 0.0895 Species by position 7 2.244 0.0307 Residual 304 Table 2. Pairs of significantly different species in terms of stomata density. Species 1 2 3 4 5 6 7 Pseudobulbs P. sanchoi * * * * * X * L. pygmeae * * * X X * * S. aristata * * X X X * * P. tuerckheimii * * X X * * * L. monteverdensis * X X X X * * Lepanthes sp. * X X * X * * M. nidifica X * * * * * * Pseudobulb * * * * * * * a * = significantly different pair b X = not significant pair Table 3. Comparisons of the difference in the number of open and closed stomata on eight species of orchids. Species XÂ² DF P Value P. sanchoi 26.922 3 <.0001 L. pygmeae 10.230 3 <.0001 P. tuerckheimii 18.621 3 <.0001 S. aristata 10.230 3 <.0001 L. monteverdensis 46.853 3 <.0001 Lepanthes sp. 52.992 3 <.0001 M. nidifica 64.643 3 <.0001 Pseudobulb 49.763 3 <.0001
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Stomata Densities of Pleurothallids (Orchidaceae) in Different Microhabitats of Monteverde
Digitized by MVI
The purpose of this study was to test whether there is a difference in the number of open
and closed stomata on the northeast and southwest facing sides of trees among seven
species of Pleurothallids and one pseudobulb species. Stomata impressions were taken
from the leaves using clear finger nail polish and they were then examined under a
microscope. The results showed that there was a significant difference between the
number of open and closed stomata for every species, always with a higher frequency of
closed stomata. Significant affects of species type and position on the tree on stomata
density was also found with branch tip orchids having a lower stomata density then nonbranch
tip species. A possible explanation for the low frequency of open stomata is that
they might only open in the rain, when water loss is at a minimum.
Text in English.
Costa Rica--Puntarenas--Zona de Monteverde--Monteverde
Costa Rica--Puntarenas--Zona de Monteverde--Monteverde
Tropical Ecology Fall 2003
Ecologa Tropical Otoo 2003
t Monteverde Institute : Tropical Ecology