Stomatal density and aperture in four species of pleurothallid orchids Orchidaceae Kristen Becklund Department of Biology, University of Wisconsin Â€ Madison ABSTRACT This experiment explored stomata density and aperture in four different species of epiphytic orchids in the sub tribe Pleurothallidinae. Pleurothallids are likely to exhibit stomatal adaptations that reduce transpirational water loss because they are subject to the xeric conditions of the canopy environment and lack water storing pse udobulbs. I calculated daytime and nighttime frequencies of open stomata on leaves of Masdevallia chasei, M. striatella, Lepanthes monteverdensis, and L. ciliisepala in order to test their ability to respond to environmental fluctuations in the short term . In order to see if pleurothallids can respond adaptively to water stress over longer time scales, I calculated stomatal densities on young and old leaves of each species to test for an effect of seasonality. L. ciliisepala and L. monteverdensis showed significantly higher daytime and nighttime frequencies of open stomata than M. chasei and M. striatella 1 way ANOVA, F = 12.23, p < 0.01 and F = 4.84, p < 0.01, respectively. Leaves from the two species of Masdevallia showed higher frequencies of open s tomata at night than during the day, suggesting the potential for Crassulacean Acid Metabolism CAM in these species. Masdevallia striatella had significantly higher densities of stomata on young leaves as compared to old leaves, which may be a result o f a peak in plant growth during the dry season 1 way ANOVA, F = 5.25, p < 0.01. Understanding the environmental cues that influence stomata density, opening, and closure and identifying how water conserving stomatal adaptations vary between species will allow scientists to make predictions about how different epiphytic orchids will fare in the face of human induced climate change. RESUMEN Este estudio investigÃ³ la densidad y la apertura de los estomas en cuatro especies diferentes de orquÃdeas epÃfita s en la subtribu Pleurothallidinae. Es probable que las pleurotÃ¡lidas tengan adaptaciones que disminuyan la pÃ©rdida del agua por transpiraciÃ³n, ya que viven bajo condiciones secas en el ambiente del dosel y carecen de seudobulbos. Se calcularon las frecue ncias de estomas abiertos en hojas of Masdevallia chasei, M. striatella, Lepanthes monteverdensis, y L. ciliisepala durante el dÃa y la noche para examinar su habilidad para reacionar a las fluctuaciones ambientales a corto plazo. Para determinar si las pleurotÃ¡lidas pueden reacionar adaptivamente a la carencia de agua a largo plazo se calcularon las densidades estomatales de hojas jÃ³venes y viejas de cada especie para averiguar si habÃa un efecto de estaciÃ³n. Lepanthes ciliisepala y L. monteverdensi s mostraron frecuencias significativamente mÃ¡s altas que M. Chasei y M. Striatella ANOVA, F = 12.23, p < 0.01 y F = 4.39, p < 0.01, respectivamente. Las hojas de las dos especies de Masdevallia mostraron frecuencias mayores de estomas abiertos durante la noche que durante el dÃa, sugiriendo la capacidad potencial para el metabolismo crasulÃ¡ceo Ã¡cido CAM en estas especies. Masdevallia striatella tuvo densidades significativamente mÃ¡s altas de estomas en hojas jÃ³venes que en hojas viejas; esto puede ser el resultado de un punto crÃtico en el crecimiento de las plantas durante la Ã©poca seca ANOVA, F = 5.25, p < 0.01 . Entender las seÃ±ales ambientales que influencian la densidad, apertura y cierre de los estomas y el identificar cÃ³mo las adaptaciones de la conservaciÃ³n del agua varÃan entre especies permitirÃ¡n a los cientÃficos predecir cÃ³mo las diversas orquÃdeas epifÃticas reacionarÃ¡n a cambios en el clima causados por los seres humanos.
INTRODUCTION Carbon dioxide uptake drives photosynthesis and therefore requires an interface between intracellular plant air spaces and the surrounding atmosphere. Guard cells control gas exchange by mediating photosynthesis and transpiration through the widening and constriction of stomata, the openings that faci litate carbon dioxide entry and water vapor exit in plants Hopkins 1995. Therefore, although photosynthesis is an essential plant function, it is always coupled with the potential for water loss. Thus any study of the efficacy of gas exchange in plants must include a cost benefit analysis of water loss through transpiration and metabolic gain through photosynthesis. Transpiration is responsible for a large portion of the water lost from plants, thus elucidating the triggers that govern stomatal opening and closure is critical for predicting how, and how well, different plant species will respond to Earth s changing climate. This is of particular importance for species that are already subject to water stress, species that are especially sensitive to sm all scale changes in microclimate, and species that are dependent upon the periodicity or constancy of certain environmental variables. Based on these parameters, epiphytes are ideal study organisms. When epiphytes were evolutionarily cut off from their ties to the ground, they were thrust into a world where water acquisition and conservation is of utmost importance. Many epiphytes were forced to rely almost entirely on atmospheric sources for nutrient and water absorption. The nature of the epiphytic g rowth form and the seasonality of cloud forest mist and precipitation probably created strong selection pressure for water conservation adaptations. Far removed from sources of soil moisture and ground water, epiphytes developed a variety of morphological and physiological traits to deal with the often xeric conditions of the canopy environment. Conspicuous morphological characteristics such as pseudobulbs, velamen, trichomes, and succulence can indirectly enhance water conservation, but the cellular proce sses of plants may actively reduce water loss by responding to changing moisture conditions across multiple temporal and spatial scales. Plants may regulate the opening and closing of their stomata to reduce transpirational water loss caused by daily env ironmental fluctuations. Changes in stomatal aperture is probably modulated by several factors, including temperature, light availability, carbon dioxide, and humidity Waggoner and Zelitch 1965. While high light and carbon dioxide levels favor photosyn thesis, warm temperatures and low humidity may promote transpiration as water vapor flows down its concentration gradient from high levels between plant cells to low levels in the outside air Thut 1939. Crassulacean acid metabolism CAM has evolved in some plants because it is a photosynthetic pathway that circumvents this trade off. CAM plants open their stomata at night and close them during the day when transpiration rates are highest, using carbon dioxide stored from the night before to perform pho tosynthesis. Few and Hue 1982 studied CAM in several species of epiphytic orchids in Singapore, and although the specific metabolic behavior of many Neotropical species of orchids remains undocumented, one might expect a significant number of them to re ly on nighttime stores of carbon dioxide for at least a portion of their photosynthetic needs. Plants might also reduce transpirational water loss by adjusting stomatal density. Although stomata density may be somewhat species specific Carroll 1937, i t may also
be related more broadly to species functional type. For example, one might expect orchids subject to chronic water stress to produce relatively few stomata or exert tight control over stomatal movements. In fact, one study has demonstrated tha t orchids with pseudobulbs exhibit significantly higher stomatal densities than their non pseudobulb counterparts Beebe 2003, suggesting that species already endowed with sufficient water storage adaptations need not place such a high premium on reducing transpiration. Pleurothallids comprise a physiologically unique group of orchids of particular interest to the study of stomatal regulation because they lack pseudobulbs. This distinct characteristic has several potential consequences for pleurothallid fitness, especially in light of global warming. First of all, pleurothallids may exhibit other adaptations that allow them to cope with water stress effectively, and this flexibility may allow them to combat climate change at a proximate level. However, it is also possible that pleurothallids may be sensitive to even small departures from normal environmental ranges of temperature, humidity, or precipitation, and may therefore show an increased susceptibility to climate aberrations resulting from global warming. A paucity of data on the physiology and ecology of many pleurothallid species makes it difficult to predict how individual species will fare in the face of anthropogenic changes to their abiotic environment. Epiphytic orchids are often very wel l adapted to specific microhabitats within tree crowns, but these evolved physiological features and regulatory mechanisms may or may not be adaptive as climate conditions change. Declining mist frequency has been recorded in Monteverde in recent years Po unds et al. 1999, thereby compounding the already desiccating dry season conditions and possibly pushing certain orchid species past their thresholds for water stress tolerance. In an ongoing study in Monteverde, Costa Rica, pleurothallid orchid species in the genera Lepanthes and Masdevallia are being exposed to ambient and experimentally increased mist frequencies in order to detect the effects of local climate change. After only 64 weeks of observation, Lepanthes spp. are already responding significan tly differently to the two treatments K.L. Masters 2006, pers. comm.. In this study, I examined the frequency and aperture of the stomata of four species of pleurothallids in response to environmental variables across two time scales. To assess the s tomatal responses of species in the short term, I calculated the frequency of open stomata on the same leaves during the day and night, and predicted higher frequencies at night possibly due to CAM metabolism in these species. Recent studies have shown th at certain environmental cues can trigger the adjustment of stomata density on leaves produced at different times in a plant s lifetime Webb and Baker 2002. Perhaps some species are able to respond to the distinct yet predictable conditions that charact erize different times of the year, and produce different numbers of stomata on leaves manufactured in different seasons. To investigate the possibility that species adaptively adjust stomata on a slightly longer, seasonal time scale, I calculated the dens ity of stomata on old and young leaves of the same individual, invoking leaf age as a proxy for the season in which it was produced. I predicted that young leaves, which I assumed were produced during the dry season, would have fewer stomata in order to r educe transpiration during the time of year when water is most limiting.
METHODS STUDY SITE. The study was conducted in an experimental orchid garden near the Monteverde Biological Station in Costa Rica. Data were collected at the end of the dry season, from April 25 th May 7 th , 2006. STUDY ORGANISMS. I observed stomata frequencies and patterns of opening and closure for four species of orchids in the sub tribe Pleurothallidinae. Pleurothallids are very small, often miniature, orchids that can grow either epiphytically or terrestrially Luer 2003. There are over 4000 species currently described, and cloud forests like those of Monteverde harbor the greatest species richness Dressler 1981; Pridgeon 1982. The four species I studied were M asdevallia chasei, Masdevallia striatella, Lepanthes monteverdensis, and Lepanthes ciliisepala. Lepanthes monteverdensis is endemic to the lower montane rain forest of Monteverde, and all but M. striatella are endemic to Costa Rica Luer 2003. Species i n the genus Lepanthes are generally smaller and more delicate than the Masdevallia species, and they tend to have restricted distributions. The two genera also differ in their habitat specificity; Masdevallia tend to occur on exposed branch tips whereas L epanthes are generally shade loving and occur in more protected areas within the tree crown K.L. Masters 2006, pers. comm.. All four species are currently included in a long term experiment documenting the effects of declining mist frequencies on orchid fitness, and trends so far show notably different responses between the two genera. Fourteen to 20 individuals of each species were selected for study within the orchid garden. The only requirement for selection was that each individual have at least o ne old and one young, photosynthetically active leaf. I marked selected leaves so that samples could be paired throughout the study. No orchids were removed from the garden as stomata imprints were captured on leaves in the field. STOMATA DENSITY. S tomata density was calculated for young and old leaves occurring on the same individual. Developmentally mature leaves had previously been marked during an ongoing study by Karen Masters, thus providing a reliable method for the discrimination between old and young leaves. I assumed that marked leaves were most likely produced during the wet season and unmarked leaves were most likely produced during the dry season. An even coat of clear nail polish was applied to the underside of each selected leaf, and, once sufficiently dry, peels were removed with tweezers. Peels were taken from leaf undersides because that is where the majority of stomata tend to be found on orchids Goh et al. 1977. I used a compound microscope to examine each leaf peel. I counte d all visible stomata in the field of view at 100x magnification for three sites per leaf. I usually selected sites near the leaf midvein, but the quality of the nail polish peel often constrained site selection. The average density of stomata per leaf was calculated as the average of the three replicate sites. STOMATA REGULATION I N DAY VS. NIGHT. The marked, old leaves selected for calculating stomata density were also used to calculate daytime and nighttime frequencies of open stomata. Due to the intensive time required, day and night leaf peels from all four species were not collected within the same 24 hour period. However, the
maximum variation in ambient humidity allowed between different collection times was set at 10%, and ranged from 58 68 %. I examined each leaf peel at 400x magnification and scored the first 40 stomata encountered as either open or closed. Partially open or indistinguishable stomata were not scored. RESULTS STOMATA DENSITY. Stomata densities varied significantly a cross the old leaves of all four species 1 way ANOVA, F = 5.25, df = 3, p < 0.01 and between the old and young leaves of one species Figure 1. The old leaves of M . chasei had significantly higher stomata densities than young leaves of M. striatella, L . ciliisepala, and L. monteverdensis Fisher s PLSD; p = 0.010, p < 0.01, p < 0.01, respectively. The young leaves of M. striatella had significantly higher stomata densities than the young leaves of L. ciliisepala and L. monteverdensis 1 way ANOVA; F = 2.06, p = 0.04, p = 0.05 respectively. Additionally, the young leaves of M. striatella had significantly higher stomata densities than old leaves on the same individual Paired t Test, t = 2.68, p = 0.015. Figure 1. Average number of stomata for four species of pleurothallids Orchidaceae. Averages were taken from stomata counts from three replicate sites per leaf at 100x magnification N = 20 individuals of M. striatella , N = 20 for M. chasei, N = 18 for L. monteverdensis, N = 14 for L. ciliis epala; one old and one young leaf were examined for each individual. Stomata density ranged from 66 to 270 stomata per 3.14 mm 2 of leaf area e.g. one field of view at 100x. Young leaves of M. striatella had significantly higher stomata densities than the young leaves of the other three species. Variation in stomata density of young leaves was not significant. Masdevallia striatella was the only species to show a significant difference between the stomata densities of its young and old leaves. Old le aves were likely produced during the wet season and young leaves were likely produced during the dry season, thus variation in stomata density on leaves of different ages may reflect plant adjustments to seasonality. Error bars represent + one standard er ror. 0 20 40 60 80 100 120 140 160 Lepanthes ciliisepala Lepanthes monteverdensis Masdevallia chasei Masdevallia straitella Species old leaves new leaves # 0f stomata
STOMATA REGULATION I N DAY VS. NIGHT. Daytime leaf peels for all species were taken on Wednesday, April 26 th ambient humidity = 58%. Night leaf peels for M. chasei and M. ciliisepala were taken on Tuesday, May 2 nd ambient humidity = 68%, and ni ght leaf peels for L. monteverdensis and L. ciliisepala were taken on Sunday, May 7 th ambient humidity = 61%. Overall, the frequency of open stomata was relatively low in both the night and day for all species, ranging between 0 and 34% Figure 2. How ever, significant trends between daytime and nighttime stomata regulation were found at the genus level Figure 3. Both species of Lepanthes had significantly higher averages of open stomata during the day than the two Masdevallia species 1 way ANOVA, d f = 3, F= 12.23, p < 0.01. Similar although slightly less significant patterns were found between the two genera at night 1 way ANOVA, df = 3, F= 4.84, p < 0.01; L. ciliisepala leaves had significantly higher frequencies of open stomata than those of M . striatella Fisher s PLSD , p = 0.05 and L. monteverdensis leaves had significantly higher frequencies of open stomata than those of M. striatella and M. chasei Fisher s PLSD, p < 0.01 and p < 0.01, respectively. Intraspecific comparisons of day and n ighttime open stomata frequencies show that both Lepanthes species open more stomata during the day and Masdevallia species open more stomata at night, although these trends are only significant in the case of L. ciliisepala Paired t Test, p < 0.05. Figure 2. Frequency of open vs. closed stomata on Pleurothallid leaves in the day and night. All four species exhibited relatively low frequencies of open stomata 0 34% regardless of time of day. Lepanthes ciliisepala and L. monteverdensis exhibited h igher frequencies of open stomata than M. chasei and M. striatella . At night, the frequency of open stomata decreased for both Lepanthes species but increased slightly for both Masdevallia species. 0 20 40 60 80 100 L. ciliisepala day L. ciliisepala night L. monteverdensis day L. monteverdensis night M. chasei day M. chasei night M. striatella -day M. striatella night closed stomata open stomata
Figure 3. Daytime and nighttime averages of number of open stomata on old leaves of four species of pleurothallid orchids. One leaf peel was taken from each individual, and forty stomata were scored at 400x magnification for each leaf N = 19 for M. chasei and M. striatella , N = 18 for L. monteverdensis, and N = 15 for L. ciliisepala . The two species of Lepanthes showed significantly higher frequencies of open stomata than the Masdevallia species during both the day and night. Error bars represent + one standard error. DISCUSSION The maximum freque ncy of open stomata observed in any species was 34%, a surprisingly low value considering the paramount importance of photosynthesis to plant fitness. The consistently low frequencies of open stomata may, in fact, reflect an underlying adaptation for wate r conservation in all four species. By only opening a small proportion of stomata at any given time, transpirational water loss is necessarily reduced while still permitting ongoing, low levels of photosynthesis. Perhaps for epiphytic orchids, the costs incurred due to high numbers of open stomata outweigh the potential benefits of optimal levels of photosynthetic activity. Despite the fact that all species showed relatively low frequencies of open stomata overall, two interesting trends were revealed. The first pertains to the relative frequencies of open stomata between species, and the second relates to the relative day and nighttime frequencies of open stomata within species. First , the two species of Lepanthes exhibited between two and six times m ore open stomata per unit of leaf area in both the day and night, relative to the two species of Masdevallia . Additionally, leaf peels taken from Lepanthes individuals at night show lower frequencies of open stomata than peels taken from the same leaf dur ing the day. Although this is only significant in the case of L. ciliisepala, the trend is evident for L. monteverdensis as well. The opposite was true for the two species of Masdevallia, which showed increases in the frequency of open stomata between da y and night sampling. Although these results are not sufficient to identify M. chasei and M. striatella as strict -1 1 3 5 7 9 11 13 15 L. ciliisepala L. monteverdensis M. chasei M. straitella Species night mean day Average number o f open stomata per leaf open
CAM plants, they do demonstrate that proportionately more of their photosynthetic activity is dependent on nighttime carbon dioxide stores, t hereby reducing the amount of water lost to transpiration through open stomata during the day. These findings could indicate that L. monteverdensis and L. ciliisepala are less adaptively equipped than M. striatella and M. chasei in terms of their control over stomatal aperture. The mechanisms regulating stomatal opening closure could be less sensitive in Lepanthes species, or individuals could simply be less responsive due to a broader tolerance for environmental fluctuations. The difference in the ada ptive abilities of Lepanthes and Masdevallia may be a consequence of their microhabitat uses within tree crowns. The strength of selection pressure to reduce transpirational water loss probably varies significantly with height from the ground and distanc e from the trunk, as these factors determine epiphyte exposure to increasingly desiccating conditions. Masdevallia species tend to inhabit the upper crown and outer branch tips, in conditions where water conservation is most critical and adaptations that reduce water loss are most advantageous. Lepanthes, on the other hand, is often found on darker, more sheltered locations within the crown or on the trunk, and may not derive as much benefit from adaptations that regulate stomatal opening and closure. T he results of the stomatal density counts were unexpected and intriguing. Contrary to prediction, more stomata were located on young leaves than on old leaves of the same individual. This trend applied to three out of the four pleurothallid species and w as significant for M. striatella. This result is corroborated by a study on turf grasses in which greater stomatal densities were found under dry, exposed conditions Carroll and Welton 1937. This suggests that there may be a selective advantage to load ing leaves produced in the dry season with high densities of stomata. Although the declining mist frequency in recent years has created severely stressful conditions for some orchids, historically the dry season may have been the most favorable season for growth. In contrast to the constant downpours and dark skies that diminish photosynthetic potential during the wet season, the dry season is typically characterized by a high availability of light and sufficient sources of moisture in the form of atmosph eric mist, which may lead to peaks in leaf production and plant growth during this time of year. If this is the case, epiphytic orchids would be expected to produce high stomatal densities and exhibit high frequencies of open stomata during the dry season in order to take advantage of this opportunity for unhampered photosynthesis. Perhaps M. striatella evolved under conditions that made this growth strategy especially beneficial, and the resulting high stomatal densities were retained, even after conditi ons changed. Therefore, patterns of stomatal density in pleurothallids may be vestiges of past responses to seasonality, but the matter of whether or not they can be altered on intermediate, contemporary timescales remains unresolved. The fact that signif icant results and noteworthy trends were found at both the species and genus level provides insight into the types of factors that may influence variability in stomatal regulation. For example, the young and old leaves of M. striatella showed a significan t difference in density, whereas those of M. chasei did not, and the frequencies of open stomata in L. ciliisepala showed significant differences between night and day, whereas those of L. monteverdensis did not. In each case, the species within each genu s showed similar tendencies but different degrees of significance.
Members of the same genus share similar morphology and physiological motifs, but species specific factors such as phylogenetic constraints, natural history, and genetic variability within populations may give individuals of one species an edge under certain conditions. This study focused on those factors that act over short and intermediate time scales, but observations of the differential responses of these four species to the ongoing mist enhancement experiment can help to resolve the uncertainty in which of these factors can operate over the long term. Lepanthes species under ambient conditions are experiencing significantly increased mortality and reduced fitness as compared to tho se subject to the experimental treatment of daily mist applications. These observations provide a fundamental framework for an informed interpretation of the results of this study, and also provide a preliminary context within which subtle trends can be e xtrapolated and predictions made. All orchids are not created equal. In fact, it seems that the smaller and more delicate Lepanthes may be more susceptible to climate change in the long term, although the extent to which individual factors such as stomat al density and aperture contribute to this vulnerability remains unknown. Alternatively, species in the genus Masdevallia seem to possess the ability to induce appropriate responses in their stomata under water stress in the short term. However, whether or not their potential stomatal adaptability will translate into actual and effective adjustments over longer time scales is a question that merits urgency in light of the impending threats of global climate change. ACKNOWLEDGMENTS Profuse thanks to Ka ren for her constant guidance and unending patience as I changed projects three times and consistently overestimated the amount of work I could complete in the time provided. I cannot thank her enough for entrusting her babies to me for this experiment. I would also like to thank Ollie for the hours he spent rigging trees on the ridge for my first project attempt, despite the fact that I never got to climb them. Thanks to Maria for her assistance in answering my plethora of statistics and formatting rela ted questions, and thanks to Alan and Javier for never failing to cheer me when things looked grim. Finally, I would like to thank everyone who endured the incessant clicking sound for days on end as I counted stomata in the nerdery. LITERATURE CITED Baker, A. and A. Webb. 2002. Stomatal biology: new techniques, new challenges. New Phytologist. 153:365 370. Beebe, C. 2003. Stomata densities of pleurothallids Orchidaceae in different microhabitats of Monteverde. In, CIEE Fall 2003 Tropical E cology and Conservation. Carroll, J. and F. Welton. 1937. Daily periodicity of stomata in certain species of turf grasses. Botanical Gazette. 992:420 423. Dressler, R. 1981. The Orchids: Natural History and Classification. Harvard 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.
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