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Relaciones pteridofitas tropicales con los hongos micorrcicos
Tropical pteridophyte relationships with mycorrhizal fungi
It is known that pteridophytes have a mutualistic association with mycorrhizal fungi, which play a critical role in the capture of nutrients from the soil, to help with the acquisition of phosphorus and nitrogen, perhaps the most limiting factors for plant growth (Brundett 1983). Few studies have been conducted in the tropics; although there is much descriptive evidence from elsewhere that suggests that mycorrhizal relationships are important (Wagner et al.1983). This study was conducted in the San Luis premontane forest and Monteverde lower montane wet forests in Costa Rica. Twenty-four epiphytic individuals and 20 terrestrial individuals were collected to investigate the frequency of infection. The samples were dyed and examined for the presence or absence of mycorrhizae. Thirty-nine of the 44 individuals were infected. The terrestrial individuals exhibited a greater bias towards vesicles and the epiphytic individuals more prone to hyphal infection. All seven families were infected with mycorrhizae with all ten species having at least two individuals infected. The five individuals without mycorrhizae suggest that pteridophytes are capable of developing and sustaining plant growth without the mutualistic interaction.
Se sabe que las pteridofitas tienen una asociacin mutualista con los hongos micorrcicos, que desempean un papel crtico en la captura de alimentos nutritivos de la tierra, para ayudar con la adquisicin del fsforo y el nitrgeno, quizs los factores ms limitantes para el crecimiento de la planta (Brundett 1983). Pocos estudios han sido realizados en los trpicos; sin embargo hay mucha evidencia descriptiva de otras partes que sugiere que las relaciones micorrizas son importantes (Wagner et al. 1983). Este estudio se llev a cabo en el bosque premontano de San Luis y Monteverde, en los bosques hmedos bajos de montaa en Costa Rica. Veinticuatro individuos de epfitas y 20 individuos terrestres fueron colectados para investigar la frecuencia de la infeccin.
Text in English.
Tropical Ecology 2006
Ecologa Tropical 2006
t Monteverde Institute : Tropical Ecology
1 Tropical pteridophyte relationships with mycorrhizal fungi Katie Heard Department of Ecology and Evolutionary Biology, University of Colorado at Boulder ABSTRACT It is known that pteridophytes have a mutualistic association with mycorrhizal fungi, whi ch play a critical role in the capture of nutrients from the soil, to help with the acquisition of phosphorus and nitrogen, perhaps the most limiting factors for plant growth Brundett 1983. Few studies have been conducted in the tropics; although there i s much descriptive evidence from elsewhere that suggests that mycorrhizal relationships are important Wagner et al.1983. This study was conducted in the San Luis premontane forest and Monteverde lower montane wet forests in Costa Rica. Twenty four epiphy tic individuals and 20 terrestrial individuals were collected to investigate the frequency of infection. The samples were dyed and examined for the presence or absence of mycorrhizae. Thirty nine of the 44 individuals were infected. The terrestrial individ uals exhibited a greater bias towards vesicles and the epiphytic individuals more prone to hyphal infection. All seven families were infected with mycorrhizae with all ten species having at least two individuals infected. The five individuals without mycor rhizae suggest that pteridophytes are capable of developing and sustaining plant growth with out the mutualistic interaction . RESUMEN Se sabe que pteridofitas tienen una asociaciÃ³n mutua con los hongos micorricicos, los cuales tienen un papel crÃtico en l a captura de aliment os nutritivos de la tierra, ayu dando con la adquisiciÃ³n del fÃ³sforo y el nitrÃ³geno, quizÃ¡s los factores mÃ¡s restrictivos para el crecimiento de la planta Brundett 1983. Pocos estudios han sido realizados en los trÃ³picos; constante mente hay mucha evidencia descriptiva de otras partes que sugiere que estas relaciones de los micorricicos son importantes Wagner et al.1983. Este estudio fue realizado en el bosque de San Luis y el bosque de Monteverde recogidos en Costa Rica. Veinticuatro indi viduos de epi fitas y 20 individuos terrestres fueron reunidos para investigar la frecuencia de la infecciÃ³n. Las muestras fueron teÃ±idas y examinadas para la presencia o la ausencia de los hongos micorricicos. Treinta y nueve de los 44 individuos fuer on infectados. Los individuos terrestres exhibieron una tendencia mÃ¡s grande hacia vesÃcul as y los individuos de epi fitas fueron mÃ¡s pronos a la infecciÃ³n de hypha. Todas las siete familias fueron infectadas con los hongos micorricicos y todas las diez es pecies tuvieron por lo menos dos individuos infectados. Los cinco individuos sin los hongos micorricicos sugieren que pteridofitas son capaces de desarrollar y crecer sin la interacciÃ³n mutua. INTRODUCTION The roots of most plants are colonized by mychor rhizal fungi, which play a critical role in the capture of nutrients from the soil, and therefore in plant nutrition Smith et al. 1997. Such an association provides the fungus with a renewable source of food through access to fixed carbon from the plant. In return, the plant takes advantage of the increased nutrient uptake ability of the fungal hyphae. This association is initiated as young roots secrete exudates that attract the fungi. Fungus plant interaction s are especially important in the tropics, wh ere a deficit in nutrients is caused by year round leaching and
2 weathering Jordan 1985. Mycorrhizae have been shown to improve productivity in soils of low fertility and are important in increasing the uptake of difficult to obtain nutrients such as phos phorus, zinc and nitrogen. Additionally, mycorrhizae increase their plant symbionts tolerance to adverse soil conditions, influence their response to severe climatic conditions, and are essential in natural ecosystems Quilambo 2003. There are two type s of mycorrhizae: ecto and endomycorrhizae. Ectomycorrhizal fungi are identified by growth of hyphae around the root cells . Endomychorrhizae are characterized by having both inter and intracellular hyphal fungal growth in the root cortex, forming structur es referred to as vesicles and arbuscles. Vesicles are spherical structures formed within the root cortex and are used for storage , whereas arbuscles are short lived spherical structures believed to play an essential role in exchange with the host plant. In a mycorrhizal relationship, the first structures to appear are hyphae, immediately followed by arbuscles and later vesicles Brundett 2002. The latter two features give endomycorrhizae the common name of vescular arbuscular mycorrhizae. VAM. Ves cular arbuscular mycorrhizae exists in 80 % of all terrestrial plant species and are found in a wide range of habitats usually in the roots of angiosperms, gymnosperms, and pteridophytes Quilambo, 2003. Extant VAM fungi are placed in the taxonomic order Glomales, which currently consists of six genera. Glomales comprises the most common underground symbiont, present in 95% of the world's extant species of vascular plants Quilambo 2003. Evidence is abundant that Glomalean fungi were present in pteridop hyte archetypal interactions. Early evolutionary data suggest colonization by VAM fungi , as structures reminiscent of the vesicles seen in modern associations were seen and photographed in underground axes of chert fossils from Devonian times Read et al. 2000. These anatomically preserved chert fossils are part of the rhynie material; in which detailed information is visible in polished specimens of early land plants. Rhynie material suggests a presence of similar structures to arbuscles, indicating a con tinuous presence of these structures during land development. Threads assumed to be fungal hyphae are seen entering plant material, possibly acting as mycorrhiza. Perhaps the most interesting suggestion of the ancestral relationship between terrestrial pla nts and Glomales fungi comes from the analysis of substitutions in nucleic base pairs. This analysis reveals that Glomales emerged around the same period of evolutionary history as land plants. Interestingly enough, pteridophytes were amongst the first vas cular plants to develop during this time period, suggesting that fungal associations may have assisted the development of ferns. These data suggest that the fern mycorrhizal fungal relationship is one of ancestral origin, which was thought to have develope d with the colonization of land Wang 2005. Archetypal data, fossil evidence, and fungus pteridophyte studies suggest that VAM fungi of the order Glomales would be the most probable symbiont of tropical ferns Iqbal et al. 1980. In Monteverde, there ar e currently 350 known species of ferns. The high community richness may be due to the moist, shaded environments of the rainforest. Very little research has been conducted on fungus pteridophyte relationships in Costa Rica, although there is much descripti ve evidence from studies elsewhere that mycorrhizal relationships are important Wagner et al. 1983. In order to fill the gap on Costa Rica fern relationships, I conducted a broad survey of the incidence of mycorrhiza l
3 infections in epiphytic and terrestr ial ferns in pre montane and lower montane wet forests in Monteverde, Costa Rica. Similar researc h conducted on Hawaiian pteridophytes in tropical habitats showed that 83% of the terrestrial species and 55% of the epiphytic species exhibited mycorrhizal ass ocations Gemma et al. 1992. Based on nutrient limitation in tropical soils, I expected to find ferns exhibiting mycorrhizal associations in the Monteverde area . METHODS Sixteen individual ferns were collected within five hundred meters of the Biologica l Station in Monteverde. Twenty eight individuals were taken from the fifteen hectare wooded area surrounding the Eco lodge in San Luis, making a total count of 44 ferns. These 44 individuals included ten species of ferns found in eight genera and seven fam ilies , with four to six individuals per species for the 10 species analyzed. The genera were Ctenitis sp. A Tectariaceae , Thelypteris sp. A Thelypteriaceae, Thelypteris sp. B Thelypteriaceae, Pteris sp. A Pteridaceae , Chystopteris sp. A Woodsiace ae , Pleopeltis sp. A Polypodiaceae, Pleopeltis sp. B Polypodiaceae, Asplenium sp. A Aspleniaceae , Polytaenium sp. A Vittariaceae , and Scoliosorus sp. A Vittariaceae . Of the species collected, 24 ferns were of epiphytic origin located between one and two meters above the ground in a sample of three Acnistus arborescens trees at the Ecolodge in San Luis. The remaining were terrestrial ferns from the wooded areas of San Luis n = 4 and Monteverde n=16 . All pteridophytes collected were young indiv iduals, with frond length no greater than 12 cm. Two 2 cm sections were cut from the root tip of each pteridophyte and soaked in a 10 % KOH solution for 24 hours. The root tips were then soaked in 1 % HCL for one hour. The root section was stained using a 4:2:1 solution of 50 % glycerol, 1% HCL, and 0.05 % Trypan blue dye for one hour. Cross section s of each of the two root samples per fern were taken and examined under the microscope at 40x for the presence of vescular arbuscular mycorrhizae, specifically the genus Glomus . Because Glomus has been shown to be present in mycorrhizal associations with bromeliads Rowe 2001, bromeliad root samples were examined. Numerous studies have cited Glomus also being present in pteridophytes, and thus the root samples from both pteridophyte and Vreisea individuals were compared to identify a Glomales association Read et al. 2000. Three young individuals of epiphytic Vreisea bromeliads were collected within 500 ms of the Biological Station in Monteverde. All were coll ected at breast height on one tree . Root samples from these bromeliads were stained using the same procedure with one slight di fference : the three cleared root samples were stained in Trypan blue dye for only twenty minutes before using light microscopy R owe 2001. A chi squared goodness of fit test was used to determine whether the frequency of mycorrhizal association s in epiphytic and terrestrial pteridophytes can by explained by chance . RESULTS Of the 44 individuals of pteridophytes examined, 39 were infected with some type of vescular arbuscular mycorrhizae, with evidence of mycorrhizae in all genera and families Table 2 . There were eight terrestrial individuals that only exhibited infected vesicles, compared to zero epiphytic. Infected hyphae had a greater proportion in epiphytic species
4 with 13 individuals compared to a terrestrial three. Only five individuals were completely devoid of any type of infection. Of the eight genera with infected hyphae, only one lacked infected filaments; only one spec ies with vesicles lacked infected hyphae Table 1 . Examination of bromeliad root samples under light microscopy displayed presence of vescular mycorrhizal fungi. Characteristic features of Glomus including dark staining and oval vesicles were present in the slides examined. The Chi square goodness of fit test showed that the incidence of mycorrhizal association within epiphytic and terrestrial pteridophytes is non random Ã°c 2 =15. 884 , df = 3, p Ã°< 0.05. Table 1 Species Family n Location and Habit Presence of fungal structure Asplenium sp. A Aspleniaceae 4 SL, epi hyphae Chystopteris sp. A Woodsiaceae 4 MV, terr hyphae, vesicles Ctenitis sp. A Tectariaceae 4 MV, terr hyphae, vesic les Pleopeltis sp. A Polypodiaceae 6 SL, epi hyphae, vesicles Pleopeltis sp. B Polypodiaceae 4 SL, epi hyphae, vesicles Polytaenium sp. A Vittariaceae 4 SL, epi hyphae, vesicles Pteris sp. A Pteridaceae 4 MV, terr hyphae, vesicles Scoliosorus sp. A Vittariaceae 6 SL, epi hyphae, vesicles Thelypteris sp. A Thelypteriaceae 4 SL, terr hyphae, vesicles Thelypteris sp. B Thelypteriaceae 4 MV, terr vesicles Table 2 Location and Habit Hyphae only Vesicles Only Both Neither Total MV, terr 2 8 5 1 16 SL, terr 1 0 3 0 4 SL, epi 13 0 7 4 24 DISCUSSION The purpose of this study was to examine the frequency of mycorrhizal infection in pteridophytes in the Monteverde San Luis area. These data are important because they represent the first broa d survey conducted in Monteverde. The data have demonstrated that there is a widespread relationship of this nature with mycorrhizae in tropical pteridophytes. All families, all genera, and all species examined were infected with mycorrhizae , supporting pr evious studies that have displayed similar results Figure 2 Iqbal 1980 . The results showed that among the terrestrial pteridophytes, there was a higher incidence of vesicles whereas epiphytic species were more prone to being infected
5 by hyphae Figure 1. Through comparisons with root samples known to contain Glomus , the order Glomales has been identified in this experiment as the main symbiont with ferns. Samples from both Vreisea and the pteridophyte species, when examined with light microscopy, demo nstrated dark staining with arbuscles being dense and compact , a pattern shown to be present in Glomus colonization Brundett 2002. Oval vesicles, characteristic of this order, were present in between root cortex cells in both Vreisea and the pteridophyte s . Moreover, numerous studies have cited fossil evidence and genetic explanations for Glomales associations with vascular plants in the Devonian period. Visual evidence, comparative photographs, and fossil evidence support Glomales existence in tropical pt eridophytes Read et al. 2000. In addition to concluding that most pteridophyte genera show evidence of an association with Glomalean fungi, this study has presented thought invoking data suggesting potentially facultative mycorrhizal relationships. It is interesting that five individuals did not have any evidence of mycorrhizal fungi Figure 2. In the individuals lacking this association, 4 families with 4 different genera were represented. This trend is worth investigating to confirm the absence of my corrhizae and to understand its assocation with pteridophytes of those genera. Potential reasons for this data include the possibility that the pteridophytes examined were too young to develop a relationship with mycorrhizae. Other studies have presented d ata where young vascular plants lacked a mycorrhizal relationship. A study conducted in Connecticut freshwater transition zone revealed a colonization of all mature plants by fungi but only facultative associations in the younger individuals Cooke et al. 1998. The second hypothesis for an absence of mycorrhizae relies on the possibility that microscopic spores may not be present in the microhabitats of the pteridophytes. A third possible conclusion to be drawn is that the development of pteridophytes is n ot contingent upon the nutrient uptake ability provided by the mycorrhizal fungus. Another interesting pattern arises when considering the type of VAM structures present in differing microhabitats. For example, vesicles are more common in Monteverde ferns , all of which are terrestrial whereas hyphal infection is predominant in in the San Luis epiphytic individuals. T his trend may be explained by age differences in epiphytic and terrestrial individuals. The epiphytic ferns collected were visually smaller than the terrestrial ferns, with four out of five species having a frond length less than nine centimeters whereas fronds of terrestrial ferns were all approximately 12 cm long . In young individuals, hyphae should and did have a greater presence because these structures exist in early developmental stages Brundett 2002. Hyphae would benefit epiphytic individuals greatly as nutrient uptake in non terrestrial species is more important because of lower levels of nutrient availability. The conditions for th e epiphytic individuals in San Luis are not favorable to phosphorus or water. The San Luis Valley experiences relatively small amounts of wind driven precipitation and minimal cloud immersion during the transition period and dry seasons Nadkarni et al. 20 00. It would make sense that more epiphytic individuals would display hyphae to adapt to the nutrient poor lifestyle. Along the same lines, it is also possible that terrestrial individuals would benefit by having a greater abundance of vesicles as opposed to hyphae. It is likely that storage vesicles are essential in tropical soils with limited nutrient availability so as to best retain and utilize the minerals obtained by the hyphae.
6 Another reason for the overwhelming presence of vesicles in Monteverde terrestrial individuals could be that an alternative kind of mycorrhizal fungi exists in Monteverde. It is possible that this different species produces more vesicles than those present in San Luis. The last hypothesis for this pattern is that all pterido phyte individuals collected were growing on a single species of Acnistus arborescens , and the results may differ if a wide variety of host plants were used. It is clear that much more research needs to be conducted on the intricacies of VAM associations within tropical pteridophyte habitats . Data needs to be collected on the exact nutrient levels in both terrestrial and epiphytic habitats in order to accurately compare nutrient uptake. A valuable research project would entail a study conducted on epiphyt ic individuals whose host tree did not exhibit the spongy bark capable of water uptake as Acnistus arborescens can. Yet, based on this research, it is clear that t here is a mycorrhizal association with tropical pteridophytes in Monteverde, Costa Rica. ACK NOWLEDGEMENTS I thank Karen Masters for her assistance with research and statistics , and her persistence in speaking to the Eco Lodge about facility use. Thanks to Willow Zuchowski for help in identifying the ferns collected. I would like to thank Alan Ma sters for supporting a research project that has been little explored in the tropics. Many thanks to both Cameryn and Tom for their tolerance and help with identifying mycorrhizal fungi. Most of all, I would like to thank my parents for giving me the oppor tunity to be entrenched in a place of wondrous beauty and to conduct a project in Costa Rica. LITERATURE CITED Brundett, M.C. 1996. Ectomycorrhizas. Mycorrhiza 6 :509 Brundett, M. C. 2002. Coevolution of roots and mycorrhizas of land plants. New Phytologist 154 : 275 304 Cooke, J. C., Lefor, M.W. 1998. The Mycorrhizal Status of Selected Plant Species from Connecticut Wetlands and Transition Zones. Restoration Ecology 6 2 :214 Gemma, J.N.,Koske, R.E., Flynn, T.1992. Mycorrhizae in Hawaiian Pte ridophytes: Occurence and Evolutionary Significance. American Journal of Botany 79 8 : 843 852 Iqbal, S.H., Yousaf, M., Younus, M. 1980. A Field Survey of Mycorrhizal Associations in Ferns of Pakistan. New Phytol: 87 : 69 79 Jordan, C. 198 5. Nutrient Cycling in Tropical Forest Ecosystems. Pg. 24. John Wiley and Sons, Great Britian. Lellinger, D.B. 1985. Ferns and Fern Allies. Pg. 15 33. Washington D.C. Smithsonian Institution Press.
7 Mills, T. J. 2006. Presence of endomycorhizae on roo ts of epiphytic orchids. Pg. 1 5. Tropical and Conservation Ecology Summer. Moran, R.C. 2004. A natural history of ferns. Pg. 218 233. Portland, OR. Timber Press Inc. Clark, K., Lawton, R.O., Butler, P. R. 2000. The physical environment. In N. Nadk arni and N. Wheelwright Eds.. In Monteverde Ecology and Conservation of a tropical cloud forest. Pg. 19. Oxford University Press, New York Quilambo, O. A. 2003. The vesicular arbuscular mycorrhizal symbiosis. African Journal of Biotechnology: 2 1 2 : 539 546 Read, D.J., Duckett, J.G., Francis, R., Ligrone, R., Russell, A. 2000. Symbiotic fungal associations in "lower" land plants. The Royal Society: 355 : 815 831 Rowe, A. 2001. Mycorrhizae in bromeliads. Pg. 731. UCEAP Fall. Smith, S.E., Rea d, D.J. 1997. Mycorrhizal symbiosis. Pg. 312 313. San Diego, CA. Academic Press Inc. Taylor, T.N., Kerp, H., Hass,H. 2005. The rynie chert and its flora. Mycologia: 97 : 269 285 Wang, Y.L. 2006. Phylogenetic distribution and evolution of mycorrhizas in land plants. Mycorrhiza: 16 : 299 363 Wagner, W., Gomez, L.D. 1983. Pteridophytes. In D.H. Janzen Ed.. Costa Rican Natural History, pg. 311 316. The University of Chicago Press, Chicago, Illinois. Witbacht, M. 2000. The occurrence of vesicular ar buscular mycorrhizae in terrestrial and epiphytic ferns. Pg. 754. UCEAP Fall.
8 FIGURE 1. The number of epiphytic or terrestrial individuals identified by incidence of infection in vesicles and hyphae. The incidence of mycorrhizal structures seen in ter restrial and epiphytic species is non random Ã°c 2 = 15.884, df = 3, p Ã°< 0.05. Terrestrial species have a greater incidence of vesicles and epiphytic species are more prone to hyphal infection. 0 2 4 6 8 10 12 14 Both vesicles and hyphae Vesicles Hyphae None infected Infection Type Number of Individuals Terrestrial Epiphytic
9 FIGURE 2. Number of individual s with infected vesicles or hyphae in each of the ten species.. The incidence of mycorrhizal structures seen in terrestrial and epiphytic species is non random Ã°c 2 = 15.884, df = 3, p Ã°< 0.05. 0 1 2 3 4 5 6 7 Aspenium sp. A SL, epi Chystopteris sp. A MV, terr Ctenitis sp. A MV, terr Pleopeltis sp. A SL, epi Pleopeltis sp. B SL, epi Polytaenium sp. A SL, epi Pteris sp. A MV, terr Scoliosorus sp .A SL, epi Thelypteris sp. A SL, terr Thelypteris sp. B MV, terr Species Number of Individuals Hyphae Vesicles