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Williams, Nicole M.
Los hongos Micorrizas en las epfitas y en Oerstedella exasperata (Orchidaceae)
Mycorrhizal fungi in epiphytic and terrestrial Oerstedella exasperata (Orchidaceae)
All orchids have a relationship with mycorrhizae during germination. Nutrient availability in soils often determines whether these relationships are maintained into adulthood. The aim of this study was to investigate the frequency of mycorrhizae infection in epiphytic and terrestrial Oerstedella exasperata. Since orchids that are rooted in the ground may have greater access to, or more consistent supplies of, nutrients I predicted that mycorrhizae would be found less often in terrestrial individuals. Furthermore, since plant fitness may be affected by the presence or absence of mycorrhizae or the availability of nutrients, fitness of epiphytic and terrestrial plants, with and without mycorrhizae were compared. I found a difference in the frequency of infection between the two substrate types with terrestrial individuals showing a higher frequency than expected by chance and in frequencies that exceeded those for the epiphytic individuals. Significant differences were not found in fitness parameters for orchids between the two substrates but trends were found that showed individuals occurring on both substrate types did better with the presence of mycorrhizae.
Todas las orqudeas tienen una relacin con las micorrizas durante su germinacin. La disponibilidad de nutrientes en los suelos a veces determina si estas relaciones se mantienen hasta la edad adulta. El objetivo de este estudio fue investigar la frecuencia de la infeccin por micorrizas en las epfitas y en Oerstedella exasperata.
Text in English.
Tropical Ecology 2007
Ecologa Tropical 2007
t Monteverde Institute : Tropical Ecology
1 Mycorrhizal Fungi in Epiphytic and Terrestrial Oerstedella exasperata Orchidaceae Nicole M. Williams Division of Biological Sciences, University of Missouri Columbia ABSTRACT: All orchids have a relationship with mycorrhizae during germination. Nutri ent availability in soils often determines whether these relationships are maintained into adulthood. The aim of this study was to investigate the frequency of mycorrhizae infection in epiphytic and terrestrial Oerstedella exasperata . Since orchids that ar e rooted in the ground may have greater access to, or more consistent supplies of, nutrients I predicted that mycorrhizae would be found less often in terrestrial individuals. Furthermore, since plant fitness may be affected by the presence or absence of m ycorrhizae or the availability of nutrients, fitness of epiphytic and terrestrial plants, with and without mycorrhizae were compared. I found a difference in the frequency of infection between the two substrate types with terrestrial individuals showing a higher frequency than expected by chance and in frequencies that exceeded those for the epiphytic individuals. Significant differences were not found in fitness parameters for orchids between the two substrates but trends were found that showed individuals occurring on both substrate types did better with the presence of mycorrhizae. RESUMEN: Todas las orquÃdeas tienen alguna relaciÃ³n con micorrizas durante su germinaciÃ³n. La disponibilidad de nutrientes en los suelos a veces determina si estas relacione s se mantienen. El objetivo de este estudio fue investigar la frecuencia de infecciones micorrizicas en Oerstedella exasperata epifiticas y terrestres. Ya que las orquÃdeas con raÃces pueden tener mayor acceso a nutrientes, yo predije que las micorrizas se rian encontradas con menos frecuencia en los individuos terrestres. AdemÃ¡s, como el valor adaptativo de la planta se puede ver afectado por la presencia o ausencia de micorrizas o la disponibilidad de nutrientes, se comparo el valor adaptativo de plantas e pifiticas y terrestres, con o sin micorrizas. Los individuos terrestres mostraron una mayor frecuencia de infecciÃ³n. No se encontraron diferencias en cuanto a los parÃ¡metros de valor adaptativo de las orquÃdeas entre sustratos, pero existe una tendencia ha cia mejores condiciones con micorrizas. INTRODUCTION: Mycorrhiza refers to the symbiotic relationship between a fungus and the roots of a vascular plant Dressler 1990, and mycorrhizal infection is a near universal feature of individuals in the famil y Orchidaceae Hadley & Williamson 1972. The relationship of
2 mycorrhizae with orchids is one of particular interest since all orchids pass through a seedling stage during which they are unable to photosynthesize and depend on a supply of carbohydrate, pro vided in nature, by mycorrhizal fungi Smith & Read 1997. Orchid mycorrhizal fungi are assumed to supply carbohydrates to the orchid seedlings with which they are associated and in adult orchids the mycorrhiza may be important for mineral acquisition, by penetrating the root cells and facilitating the uptake of nitrogen and phosphorus Smith 1966; Smith & Read 1997; Brundrett 2002, in Gebauer & Meyer 2003. While many orchids maintain relationships with the fungus throughout their life, some orchids are ab le to reject the fungus. Reasons for the rejection of mycorrhizae are still unclear although it is suggested that the rejection is related to the costs of the fungi relative to their benefits. The result of a previous study using Piperaceae suggests that many plant species that are commonly mycorrhizal when they grow terrestrially are not consistently mycorrhizal when they grow epiphytically Maffia et al. 1993. This could be due to the fact that the substrate type changes the amount of nutrients availab le. Since some tropical epiphytic orchids are non mycorrhizal when adult, while many terrestrial orchids remain mycorrhizal Burgeff 1932, in Gebauer & Meyer 2003; Smith & Read 1997, this study examines the frequency of infection between epiphytic and ter restrial Oerstedella exasperata orchids. The species O. exasperata was chosen because individuals can be found growing in abundant quantities, epiphytically and terrestrially, near the Monteverde Cloud Forest from 900 2500 m Hammel et al. 2003. There m ay be a relationship between the fitness of individual plants and whether they have mycorrhizae present. Results from Alexander and Hadley 1984 show mycorrhizal fungus enhances nutrient uptake and thus allows greater growth rates in the orchid Goodyera r epens . This would suggest that orchids with mycorrhizae should exhibit greater fitness than those without. The purpose of this study is to explore the hypotheses that nutrient availability determines whether or not a plant possesses mycorrhizae, and that increased nutrient supplies enhance the health of orchid individuals. To investigate these hypotheses, I will compare the frequency of mycorrhizae in O. exasperata orchids with terrestrial roots which should have greater access to nutrients to those of epiphytic individuals which should have less access to nutrients. Further, I will compare the size and reproductive status of terrestrial and epiphytic individuals, with and without mycorrhizae. METHODS: Orchid root samples were collected from October 22 to November 13, 2007 in Monteverde, Costa Rica from both epiphytic and terrestrial O. exasperata plants. Individuals were found growing on embankments and substrates above the embankments along Cerro Amigos trail between 1700 1800 meters. Samples fro m 40 individuals were collected, including twenty individuals from each substrate type the soil or on logs or trees. Each sample consisted of three 2 cm long segments cut from the tips of fresh, green roots. Root segments were first located by digging di rt and leaf litter out from around the exposed roots of terrestrial plants and clearing debris from the substrate for epiphytic plants, and then collected in labeled, plastic bags. No roots of terrestrial plants were pulled out of the soil. For each indivi dual
3 the following fitness parameters were recorded: a length of longest stem, in cm; b the number of inflorescences, and c the number of stems. Following collection, root samples were placed in vials containing 10% KOH for 24 48 hours to clear the roots. Root segments were then placed in H 2 O 2 for one hour to remove pigments and then transferred into 1% HCl for 20 minutes. Finally, the root segments were put into a 4:2:1 solution of 50% glycerol: 1% HCl: 0.05% trypan blue dye for one and a half hour s to stain. Cross sections were cut from the tip of each root and places on slides. Prepared samples were viewed under a compound microscope at 40X and 100X and the presence or absence of mycorrhizal fungi was recorded. A contingency table was constructe d to find how many individuals from each substrate type were infected or not infected with mycorrhizal fungi. A Spearman rank correlation was done to test correlations between the following fitness parameters: a the length of longest stem and the number of inflorescences and b the number of stems and the number of inflorescences. A Wilcoxon test was also done to evaluate if average fitness parameters varied between the two different substrate types. RESULTS: Of the 40 O. exasperata examined, 19 terr estrial and 14 epiphytic individuals showed presence of mycorrhizae. Terrestrial individuals showed a higher frequency than the epiphytic individuals of infection with mycorrhizae, and more than expected by chance Ã°l 2 = 4.34; df = 1; p < 0.05; likewise, t here were more epiphytic plants without mycorrhizae than terrestrial, and more than expected by chance Table 1. TABLE 1. Contingency table showing frequency of infection of mycorrhizae in Oerstedella exasperata individuals found growing terrestrially a nd epiphytically in Monteverde, Puntarenas. For an individual plant to score as having mycorrhizae, at least one of three root samples examined showed a mycorrhizal infection. Infection Status: Number of plants With mycorrhi zae Without mycorrhizae Terrestrial 19 1 Epiphytic 14 6 Figure 1 and 2 compare the rank number of inflorescences to the rank length of the longest stem and the rank number of inflorescences to the rank number of stems, respectively. Infected epiphytic orchids showed no significant correlations between the number of inflorescences and length of longest stem rho = 0.51; p > 0.05; n = 14 or number of inflorescences and number of stems rho = 0.18; p > 0.05; n = 14. Infe cted terrestrial orchids also did not show a correlation between number of inflorescences and length of longest stem rho = 0.34; p > 0.05; n = 19 but did show a significant positive correlation between number of inflorescences and number of stems rho = 0.52; p < 0.05; n = 19.
4 0 2 4 6 8 10 12 14 16 18 20 0 5 10 15 20 rank of length of longest stem rank of number of inflorescences EM ENM TM TNM FIGURE 1. Relationships between the number of inflorescences per plant and the lengths of the longest stem in Oerstedella exasperata orchids collected in Monteverde, Puntarenas that are epiphytic or terrest rial and that have or lack mycorrhizae. Number of individuals that are epiphytic with mycorrhizae, epiphytic without mycorrhizae, terrestrial with mycorrhizae, terrestrial without mycorrhizae = 14, 6, 19, 1, respectively. EM = epiphytic with mycorrhizae present; ENM = epiphytic without mycorrhizae present; TM = terrestrial with mycorrhizae present; TNM = terrestrial without mycorrhizae present.
5 0 2 4 6 8 10 12 14 16 18 20 0 5 10 15 20 rank of number of stems rank of number of inflorescences EM ENM TM TNM FIGURE 2. Relationships between the number of inflorescences per plant and the number of stems in Oe rstedella exasperata orchids collected in Monteverde, Puntarenas that are epiphytic or terrestrial and that have or lack mycorrhizae. Number of individuals that are epiphytic with mycorrhizae, epiphytic without mycorrhizae, terrestrial with mycorrhizae, t errestrial without mycorrhizae = 14, 6, 19, 1, respectively. Legend as in Figure 1. The number of stems varied in quantities from 1 to 5 in epiphytic O. exasperata and from 1 to 11 in terrestrial O. exasperata . The lengths of the longest ste m ranged from 40.4 cm to 136.6 cm in epiphytic orchids and 43.4 cm to 235.1 cm in terrestrial orchids. The number of inflorescences varied from zero to 11 in epiphytic orchids and from zero to 63 in terrestrial orchids. Between the two substrates, signific ant differences were not found in the number of stems z = 0.95; n 1 = 14, n 2 = 19; p = 0.34, the lengths of longest stem z = 1.3; n 1 = 14, n 2 = 19; p = 0.18 or the number of inflorescences z = 0.02; n 1 = 14, n 2 = 19; p = 0.98 . DISCUSSION: The purpos e of this study was to explore the hypotheses that nutrient availability determines whether or not a plant possesses mycorrhizae, and that increased nutrient supplies enhance the health of orchid individuals. The size and reproductive status of terrestrial and epiphytic individuals, with and without mycorrhizae was evaluated and the frequency of mycorrhizae in O. exasperata orchids with terrestrial roots was compared to O. exasperata individuals with epiphytic roots. The results showed that there was a si gnificant difference in the frequency of infection with mycorrhizae between epiphytic and terrestrial individuals. Terrestrial
6 orchids were infected at a higher frequency than were epiphytic orchids, and occurred at frequencies higher than expected by chan ce. Likewise, there were more epiphytic individuals that were uninfected than terrestrial individuals or than expected by chance. This was not predicted, since terrestrial roots should not need mycorrhizae as much as do epiphytic roots. The results did not show an overall difference in fitness parameters between the two substrate types although an obvious trend of increased fitness in individuals infected with mycorrhizae on both substrate types was found Figure 1 and 2. This was also not predicted since plants in the soil should have better access to minerals and nutrients. This leads to two questions: Why were more terrestrial orchids found with the presence of mycorrhizae than epiphytic orchids; and how does the cost benefit relationship between mycorrh izae and O. exasperata facilitate increased plant fitness? Since mineral nutrients are usually in short supply for epiphytes Benzing 1973, in Dressler 1990 mycorrhizal relationships are especially important for them. Nutrient availability is typically h igher in soils and more easily obtained, thus making the presence of mycorrhizae less necessary in terrestrial individuals than epiphytic ones. It is interesting, then, that more terrestrial individuals showed presence of mycorrhizae than did epiphytic ind ividuals. One hypothesis to explain this is that the clay soil in which O. exasperata is most often found is actually more nutrient poor than the substrates on which the epiphytic type grows and thus mycorrhizae is more necessary for terrestrial growth. Another hypothesis presented by Maffia et al. 1993, suggests that relatively high atmospheric inputs of dissolved inorganic nutrients that alleviate the requirement for mycorrhizae may explain the absence of mycorrhizae from epiphytic Piperaceae. Simila rities between all epiphytes make this hypothesis a likely explanation for the absence of mycorrhizae in the epiphytic orchids of this study. Maintenance of a mycorrhizal relationship comes at a cost to the plant so plants must be selective when evaluat ing whether or not the benefits of the relationship will outweigh the costs to maintain it. Since only one terrestrial individual failed to show presence of mycorrhizae, it suggests that the fungi are vitally important to helping the plants survive in the n utrient poor soils of Monteverde and that the distribution of mycorrhizal fungi must be influenced, to a significant extent, by soil conditions Hadley 1970. Studies show that nutrient availability plays a role in controlling the relationship between orch id and fungi Rasmussen 2000. The fact that a large fraction of plants on both substrate types studied here possessed mycorrhizae suggests that they are both extremely nutrient poor. Since mycorrhizal plants show a greater fitness due to increased nutrie nt uptake Smith & Read 1997, plants should establish a relationship with mycorrhizae whenever possible. However, my results showed a large number of epiphytic individuals that were uninfected with mycorrhizae. This was unexpected as fitness was actually shown to decrease in epiphytic individuals without mycorrhizae. The best hypothesis to explain this is that there simply are no mycorrhizae spores available for the roots to obtain, as presence of these spores is more limited in the air than on the ground. This is the best explanation because clearly, whether growing epiphytically or terrestrially, plants benefit more from having mycorrhizae. Therefore, if available, it is assumed that plants would adopt relationships with the fungi.
7 The results here showe d that the best option for an individual of O. exasperata is to be a terrestrial individual with mycorrhizae. These two strategies combined give an individual a greater chance of reaching maximum fitness levels although the mean fitness does not differ for the substrates. However, simply possessing mycorrhizae increases fitness on both substrates, so it is better to have mycorrhizae than not. Further studies could be done using a larger sample size to examine if the trends seen in this study can be solidif ied with statistical significance. Also, this study showed that the presence of mycorrhizae increased plant fitness in orchids growing both as epiphytes and as terrestrial plants but I did not analyze the amount of mycorrhizae present in root samples. A pr evious study by Lee 2006 studied the relationship between the amount of mycorrhizae and plant fitness in canopy orchids but found no significance. However, future studies could focus on this relationship in O. exasperata , which grow in very exposed areas . This would pose the question of why and how some individuals, growing on the same substrate, with presumably the same nutrient resources available, have more mycorrhizae than others. ACKNOWLEDGMENTS: I would like to thank Dr. Karen Masters and Dr. Ala n Masters for their guidance throughout this project, for assistance with data and statistical analyses, and Karen for her help with the yellow triangle. I would also like to thank Mary Snayd for being a great companion as we walked the clay road everyday to collect d ata. Muchas gracias to the S eidy Torres family for listening patiently as I tried to explain my project to them in Spanish and for opening their home to me. I would also like to thank my own family whose love and support made this Costa Rican a dventure possible. LITERATURE CITED: Alexander, C. and G. Hadley. 1984. The Effect of Mycorrhizal infection of Goodyera repens and Its Control by Fungicide. New Phytologist 97: 391 400. Benzing, D. H., 1973. Mineral Nutrition and Related Phenomen a in Bromeliaceae and Orchidaceae. Quarterly Review of Biology 48: 277 290. Brundrett, M. C. 2002. Coevolution of Roots and Mycorrhizas of Land Plants. New Phytologist 154: 275 304. Burgeff H. 1932. Saprophytismus und Symbiose. Jena, Germany: Gustav Fis cher Verlag. Dressler, R. L. 1990. The Orchids: Natural History and Classification. Harvard University Press, Cambridge, Massachusetts. Hadley, G. 1970. Non Specificity of Symbiotic Infection Orchid Mycorrhiza. New Phytologist 69: 1015 1023. Hadley, G. and B. Williamson. 1972. Features of Mycorrhizal Infection in some Malayan Orchids. New Phytologist 71: 1111 1118. Hammel, B. E., M. H. Grayum, C. Herrera, and N. Zamora Eds.. 2003. Manual de Plantas de Costa Rica, Volume 3. Missouri Botanical Garden Press, St. Louis, Missouri. Lee, Y. Y. C. 2006. Mycorrhizae Concentration and Fitness of Canopy Orchids in the Cloud Forest of Monteverde, Costa Rica. CIEE Tropical Ecology and Conservation: Fall.
8 Maffia, B., N. M. Nadkarni, and D. P. Janos. 1993. Ves icular arbuscular Mycorrhizae of Epiphytic and Terrestrial Piperaceae Under Field and Greenhouse Conditions. Mycorrhiza 4: 5 9. Rasmussen, H. M. 2002. Recent Developments in the Study of Orchid Mycorrhiza. Plant and Soil 244: 149 163. Smith, S. E. 1966. Physiology and Ecology of Orchid Mycorrhizal Fungi with Reference to Seedling Nutrition. New Phytologist 65: 488 499. Smith, S. E., and D. J. Read. 1997. Mycorrhizal Symbiosis, 2nd edition. Academic Press, San Diego, California.