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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 relati onship with mycorrhizae during germination. Nutrient availability in soils often determines whether these relationships are maintained into adulthood. The aim of this study was to i nvestigate the freque ncy of mycorrhizae infection in epiphyt ic and terrestrial Oerstedella exasperata Since orchids that are rooted in the ground may have greater access to, or more consistent s upplies of, nutrients I predicted that mycorrhizae w ould 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, f itness of epiphytic and te rrestrial 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 tr ends were found that showed individuals occurring on both substrate types did bette r with the presence of mycorrhizae. RESUMEN: Todas las orqudeas tienen alguna relacin con micorrizas durante su germinacin. La disponibilidad de nutrientes en los suelos a veces determina si estas relaciones se mantienen. El objetivo de este estudio fue investigar la frecuencia de infecciones micorrizicas en Oerstedella exasperata epifiticas y terrestres. Ya que las orqudeas con races pueden tener mayor acceso a nutrientes, yo predije que las micorrizas serian encontradas con menos frecuencia en los indi viduos terrestres. Adems, como el valor adaptativo de la planta se puede ver afectado por la presencia o ausencia de micorrizas o la disponibilidad de nutrientes, se compar o el valor adaptativo de plantas epifiticas y terrestres, con o sin micorrizas. Los individuos terrestres mostraron una mayor frecuencia de infeccin. No se encontraron diferencias en cuanto a los parmetros de valor adaptativo de las orqudeas entre sustratos, pero existe una tendencia hacia mejores condiciones con micorrizas. INTRODUCTION: Mycorrhiza refers to the symbiotic relati onship between a fungus and the roots of a 1
vascular plant (Dressler 1990), and mycorrhizal infection is a near-universal feature of individuals in the family Orchidaceae (H adley & Williamson 1972). The relationship of mycorrhizae with orchids is one of particular interest since all orchids pass through a seedling stage during which they are unabl e to photosynthesize and depend on a supply of carbohydrate, provided in nature, by mycorrhizal fungi (Smith & Read 1997). Orchid mycorrhizal fungi are assumed to supply car bohydrates to the orchid seedlings with which they are associated and in adult or chids the mycorrhiza may be important for mineral acquisition, by penetrati ng the root cells and facilita ting the uptake of nitrogen and phosphorus (Smith 1966; Smith & Read 1997; Brundrett 2002, in Gebauer & Meyer 2003). While many orchids maintain relationshi ps with the fungus throughout their life, some orchids are able to reject the fungus. Reasons for the rejection of mycorrhizae are still unclear although it is suggest ed that the rejection is rela ted 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 (Ma ffia et al. 1993). This could be due to the fact that the substrate type changes the amount of nutrients availabl e. Since some tropical epiphytic orchids are non-mycorrhizal when a dult, while many terrestrial orchids remain mycorrhizal (Burgeff 1932, in Gebauer & Me yer 2003; Smith & Read 1997), this study examines the frequency of infecti on between epiphytic and terrestrial Oerstedella exasperata orchids. The species O. exasperata was chosen because individuals can be found growing in abundant quantities, epiphytically a nd terrestrially, near the Monteverde Cloud Forest from 900-2500 m (Hammel et al. 2003). There may 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 repens This would suggest that or chids 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 indivi duals. To investigate these hypotheses, I will compare the frequency of mycorrhizae in O. exasperata orchids with terrestrial roots (which should have greater ac cess to nutrients) to those of epiphytic individuals (which should have less access to nutrients). Furt her, 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 embankment s and substrates above the embankments along Cerro Amigos trail between 1700-1800 meters. Samples from 40 individuals were colle cted, including twenty individuals from each substrate type (the soil or on logs or tr ees). Each sample consisted of three 2-cm long segments cut from the tips of fresh, green roots. Root segments were first located by digging dirt and leaf litter out from around the exposed root s of terrestrial plants and 2
clearing debris from the substrate for epiphyt ic plants, and then collected in labeled, plastic bags. No roots of terrestrial plants we re pulled out of the soil. For each individual 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 c ontaining 10% KOH for 24-48 hours to clear the roots. Root segments were then placed in H2O2 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 hours to stain. Cross secti ons 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 constructed to find how many individuals from each substrate type were infected or not infect ed with mycorrhizal fungi. A Spearman rank correlation was done to test corr elations between the following fitness parameters: (a) the length of longest stem and the number of in florescences 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 th e two different substrate types. RESULTS: Of the 40 O. exasperata examined, 19 terrestrial and 14 epiphytic individuals showed presence of mycorrhizae. Terrestrial indivi duals showed a higher frequency than the epiphytic individuals of infection with myco rrhizae, and more than expected by chance ( 2 = 4.34; df = 1; p < 0.05); likewise, there were more epiphytic plants without mycorrhizae than terrestrial, and more than expected by chance (Table 1). TABLE 1. Contingency table showing fre quency of infection of mycorrhizae in Oerstedella exasperata individuals found growing terres trially and epiphytically in Monteverde, Puntarenas. For an individual plan t to score as having mycorrhizae, at least one of three root samples examined showed a mycorrhizal infection. Infection Status: Numb er of plants With mycorrhizae 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 s howed no significant correlations between the number of inflorescences and length of l ongest 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). Infected 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). 3
0 2 4 6 8 10 12 14 16 18 20 05101520rank of length of longest s t EM ENM TM TNM FIGURE 1. Relationships between the number of inflorescences per pl ant and the lengths of the longest stem in Oerstedella exasperata orchids collected in Monteverde, Puntarenas that are epiphytic or terrestrial and that have or lack mycorrhizae. (Number of individuals that are epiphytic with my corrhizae, epiphytic without mycorrhizae, terrestrial with mycorrhizae, terrestrial without mycorrhizae = 14, 6, 19, 1, respectively.) EM = epiphytic with mycorrhizae presen t; ENM = epiphytic without mycorrhizae present; TM = terrestrial with mycorr hizae present; TNM = terrestrial without mycorrhizae present. 4
0 2 4 6 8 10 12 14 16 18 20 0 5 10 15 20rank of number of stemsEM ENM TM TNM FIGURE 2. Relationships between the number of inflorescences per plant and the number of stems in Oerstedella exasperata orchids collected in Monteverde, Puntarenas that are epiphytic or terrestrial and that have or lack mycorrhizae. (N umber of individuals that are epiphytic with mycorrhizae, epiphytic without mycorrhizae, terrestrial with mycorrhizae, terrestrial 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 stem 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 tw o substrates, significant differences were not found in the number of stems (z = 0.95; n1 = 14, n2 = 19; p = 0.34), the lengths of longest stem (z = 1.3; n1 = 14, n2 = 19; p = 0.18) or the number of inflorescences (z = 0.02; n1 = 14, n2 = 19; p = 0.98) DISCUSSION: The purpose of this study was to explore the hypotheses that nut rient availability determines whether or not a plant possesses mycorrhizae, and that increased nutrient supplies enhance the health of orchid indi viduals. 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 significant difference in the frequency of infection with mycorrhizae between epiphytic and terrestrial indi viduals. Terrestrial 5
orchids were infected at a higher frequency th an were epiphytic orchids, and occurred at frequencies higher than expected by chance. Likewise, there were more epiphytic individuals that were uninfected than terrestr ial individuals or than expected by chance. This was not predicted, since te rrestrial 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 t ypes although an obvious trend of increased fitness in individuals infected with my corrhizae 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: W hy were more terrestrial orchids found with the presence of mycorrhizae than epiphytic orchids; and How does the cost-benefit relationship between mycorrhizae 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 es pecially important for them. Nutrient availability is typically higher in soils and more easily obtained, thus making the presence of mycorrhizae less necessary in terrestria l individuals than epiphytic ones. It is interesting, then, that more terrestrial indi viduals showed presen ce of mycorrhizae than did epiphytic individuals. One hypothesis to ex plain 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 my corrhizae is more necessary for terrestrial growth. Another hypothesis presented by Maffia et al. (1993), suggests that relatively high atmospheric inputs of dissolved inorganic nut rients that alleviate the requirement for mycorrhizae may explain th e absence of mycorrhizae fr om epiphytic Piperaceae. Similarities between all epiphytes make th is hypothesis a likely explanation for the absence of mycorrhizae in the epip hytic orchids of this study. Maintenance of a mycorrhizal relationship comes at a cost to the plant so plants must be selective when evaluating whether or not the benefits of the relationship will outweigh the costs to maintain it. Since onl y one terrestrial individual failed to show presence of mycorrhizae, it sugge sts that the fungi are vitally important to helping the plant survive in the nutrient 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 orchid and fungi (Rasmussen 2000). The fact that a large fr action of plants on both substrate types studied here possessed mycorrhizae s uggests that they are both extremely nutrient poor. Since mycorrhizal plants show a greater fitness due to increased nutrient 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 unexp ected 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. 6
The results here showed that th e best option for an individual of O. exasperata is to be a terrestrial individual with mycorrhi zae. These two strategies combined give an individual a greater chance of reaching maximu m 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 solidified with st atistical significance. Also, this study showed that the presence of mycorrh izae increased plant fitness in orchids growing both as epiphytes and as terrestrial pl ants but I did not analyze th e amount of mycorrhizae present in root samples. A previous study by Lee (2006) studied the relationship between the amount of mycorrhizae and pl ant 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 que stion of why and how some individuals, growing on the same substrate, with presumab ly the same nutrient resources available, have more mycorrhizae than others. ACKNOWLEDGMENTS: I would like to thank Dr. Karen Masters and Dr. Alan 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 be ing a great companion as we walked the clay road everyday to collect data. Muchas gracias to the Sadie Torres family for lis tening 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 Ri can adventure possible. LITERATURE CITED: Alexander, C. and G. Hadley. 1984. The Effect of Mycorrhi zal infection of Goodyera repens and Its Control by Fungicide. Ne w Phytologist 97: 391-400. Benzing, D. H., 1973. Mineral Nutrition and Related Phenomena in Bromeliaceae and Orchidaceae. Quarterly Review of Biology 48: 277-290. Brundrett, M. C. 2002. Coevolution of Root s and Mycorrhizas of Land Plants. New Phytologist 154: 275. Burgeff H. 1932. Saprophytismus und Sy mbiose. Jena, Germany: Gustav Fischer Verlag. Dressler, R. L. 1990. The Orchids: Natu ral History and Classification. Harvard University Press, Cambridge, Massachusetts. Hadley, G. 1970. Non-Specificity of Symbio tic 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. Herre ra, and N. Zamora (Eds.). 2003. Manual de Plantas de Costa Rica, Volume 3. Missour i Botanical Garden Press, St. Louis, Missouri. Lee, Y. Y. C. 2006. Mycorrhizae Concentra tion and Fitness of Canopy Orchids in the Cloud Forest of Monteverde, Costa Rica. CIEE Tropical Ecology and Conservation: Fall. 7
8 Maffia, B., N. M. Nadkarni, and D. P. Ja nos. 1993. Vesicular-arbuscu lar 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 Or chid Mycorrhizal Fungi with Reference to Seedling Nutrition. Ne w Phytologist 65: 488-499. Smith, S. E., and D. J. Read. 1997. Mycorrhi zal Symbiosis, 2nd edition. Academic Press, San Diego, California.
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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