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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
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 asso ciation with mycorrhizal f ungi, 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 eviden ce 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 th e frequency of infection. The samples were dyed and examined for the presence or absence of mycorrhizae. Th irty-nine of the 44 indivi duals 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 w ith mycorrhizae with all ten species having at least two individuals infected. The fi ve individuals without mycorrhizae sugges t that pteridophytes are capable of developing and sustaining plant growth without the mutualistic interaction. RESUMEN Se sabe que pteridofitas tienen una asociacin mutua con los hongos micorricicos, los quales tienen un papel crtico en la captura de alimentos nutritivos de la tierra, ayundando con la acquesision de el fsforo y el nitrgeno, quizs los f actores ms restrictivos para el crecimiento de la planta (Brundett 1983). Pocos estudios han sido realizados en los trpicos; nonostante hay mucha evidencia descriptiva de otras partes que sugiere que estas relaciones de lo s micorricicos son importantes (Wagne r et al.1983). Este estudio fue realizado en el bosque de San Luis y el bosque de Monteverde recogidos en Costa Rica. Veinticuatro individuos de epiphytic y 20 individuos terrestres fueron reunidos para investigar la frecuencia de la infeccin. Las muestras fueron tei das y examinadas para la presencia o la ausencia de los hongos micorricicos. Treinta y nueve de los 44 individuos fueron infectados. Los individuos terrestres exhibieron una tendencia ms grande hacia vesculas y los individu os de epiphytic fueron ms pronos a la infeccin de hypha. Todos las siete familias fu eron infectadas con los hongos micorricicos y todas las diez especies tuvieron por lo menos dos individuos infectados. Los cinco individuos sin los hongos micorricicos sugieren que pteridofitas son capaces de desarro llar y crecer sin la interaccin mutua. INTRODUCTION The roots of most plants are co lonized by mychorrhizal fungi, wh ich play a critical role in the capture of nutrients from th e soil, and therefore in plant nutrition (Smith et al. 1997). Such an association provides the fungus w ith a renewable source of food through access to fixed carbon from the plant. In return, the plant takes advant age of the increased nutrient uptake ability of the fungal hyphae. This associati on is initiated as young roots secrete exudates that attract the fungi. Fungus-plant interactions are especially important in the tropics, where a deficit in nutrients is caused by year-round leaching and 1
weathering (Jordan 1985). Mycorrhizae have be en shown to improve productivity in soils of low fertility and are important in increa sing the uptake of difficult to obtain nutrients such as phosphorus, zinc and nitrogen. Additionally, mycorrhizae increase their plant symbionts tolerance to adverse soil cond itions, influence their response to severe climatic conditions, and are essential in natural ecosystems (Quilambo 2003). There are two types of mycorrhizae: ect o and endomycorrhizae. Ectomycorrhizal fungi are identified by growth of hyphae around the root cells Endomychorrhizae are characterized by having both in terand intracellu lar hyphal fungal growth in the root cortex, forming structures referred to as ve sicles and arbuscles. Vesicles are spherical structures formed within the root cortex a nd are used for storage, whereas arbuscles are short-lived spherical stru ctures believed to play an esse ntial 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 na me of vescular arbuscular mycorrhizae. (VAM). Vescular 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, pres ent in 95% of the world's ex tant species of vascular plants (Quilambo 2003). Evidence is abundant that Glomalean fungi were present in pteridophyte 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 fo ssils 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 sp ecimens of early land plants. Rhynie material suggests a presence of similar structures to ar buscles, indicating a continuous 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 plants 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 va scular plants to develop during this time period, suggesting that funga l 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 developed with the colonization of land (Wang 2005). Archetypal data, fossil ev idence, and fungus-p teridophyte studies suggest that VAM fungi of th e order Glomales would be th e most probable symbiont of tropical ferns (Iqbal et al. 1980). In Monteverde, there are 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 b een conducted on fungus-pteridophyte relationships in Costa Rica, although there is much descriptiv e evidence from studies elsewhere that mycorrhizal relationships are important (Wagne r et al. 1983). In orde r to fill the gap on Costa Rica fern relationships, I conducted a br oad survey of the incidence of mycorrhizal 2
infections in epiphytic and te rrestrial ferns in premontane and lower montane wet forests in Monteverde, Costa Rica. Similar resear ch conducted on Hawa iian pteridophytes in tropical habitats showed that 83% of the te rrestrial species and 55% of the epiphytic species exhibited mycorrhizal assocations (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 with in five hundred meters of the Biological Station in Monteverde. Twenty-eight indivi duals were taken from the fifteen hectare wooded area surrounding the Ecolodge in San Luis, making a total count of 44 ferns. These 44 individuals included ten species of ferns found in eight genera and seven families, with four to six individuals per sp ecies 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 (Woodsiaceae), 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 epi phytic 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 fe rns from the wooded areas of San Luis (n = 4) and Monteverde (n=16). All pteridophyt es collected were young individuals, 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 sections 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 Sta tion in Monteverde. All were collected at breast height on one tree. Root samples from these bromeliads were stained using the same procedure with one slight difference: th e three cleared root samples were stained in Trypan blue dye for only twenty minutes before using light microscopy (Rowe 2001). A chi-squared goodness of fit test was used to determine whether the frequency of mycorrhizal associations in epiphytic and te rrestrial pteridophytes can by explained by chance. RESULTS Of the 44 individuals of pteridophytes examin ed, 39 were infected with some type of vescular arbuscular mycorrhizae, with eviden ce of mycorrhizae in all genera and families (Table 2). There were eight terrestrial indivi duals that only exhibite d infected vesicles, compared to zero epiphytic. Infected hyphae ha d a greater proportion in epiphytic species 3
with 13 individuals compared to a terrestrial three. Only five individuals were completely devoid of any type of infection. Of the ei ght genera with infected hyphae, only one lacked infected filaments; only one species with vesicles lacked infected hyphae (Table 1). Examination of bromeliad root samples under light microscopy displayed presence of vescular mycorrhizal fu ngi. Characteristic features of Glomus including dark staining and oval vesicles were pr esent in the slides examined. The Chi-square goodness of fit test show ed that the incidence of mycorrhizal association within epiphytic and terre strial pteridophytes is non-random ( 2 =15.884, df = 3, p 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, vesicles 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 th e frequency of mycorrhizal infection in pteridophytes in the Monteverde-San Luis ar ea. These data are important because they represent the first broad surv ey 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 previous studies that have displayed similar results (Figure 2) 4
(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 by hyphae (Figure 1). Through comparisons with root samples known to contain Glomus the order Glomales has been identified in this expe riment as the main symbiont with ferns. Samples from both Vreisea and the pteridophyte species, when examined with light microscopy, demonstrated 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 presen t in between root cortex cells in both Vreisea and the pteridophytes. Moreover, numerous studies have cited fossil evidence and genetic explanations for Glomales associations w ith vascular plants in the Devonian period. Visual evidence, comparative photogra phs, and fossil evidence support Glomales existence in tropical pteridophytes (Read et al. 2000). In addition to concluding that most pteridophyte genera show evidence of an association with Glomalean fungi, this study has pres ented thought-invoking data suggesting potentially facultative mycorrhizal relationships. It is interesting that five individuals did not have any evidence of myco rrhizal fungi (Figure 2) In the individuals lacking this association, 4 families with 4 diffe rent genera were represented. This trend is worth investigating to confirm the abse nce of mycorrhizae and to understand its assocation with pteridophytes of those genera. Potential reasons for this data include the possibility that the pteridophytes examined we re too young to develop a relationship with mycorrhizae. Other studies have presented data 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 facultati ve associations in the younger individuals (Cooke et al. 1998). The second hypothesis for an absence of mycorrhizae relies on the possibility that mi croscopic spores may not be present in the microhabitats of the pteridophytes. A third possi ble conclusion to be drawn is that the development of pteridophytes is not contig ent upon the nutrient upt ake ability provided by the mycorrhizal fungus. Another interesting pattern arises when considering the type of VAM structures present in differing microhabitats. For ex ample, vesicles are more common in Monteverde ferns, all of which are terrestri al whereas hyphal infection is predominant in in the San Luis epiphytic individuals. This trend may be explained by by age differences in epiphytic and terrestrial individuals. The epiphytic fern s 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 terre strial ferns were all approximately 12 cm long. In young individuals, hyphae should and did have a greater presence because these structures exist in early developm ental stages (Brundett 2002). Hyphae would benefit epiphytic individuals greatly as nutrient uptake in non-terrestrial species is more important because of lower levels of nut rient availability. The conditions for the epiphytic individuals in San Lu is 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. 2000). It would make sense that more epiphytic indi viduals would display hyphae to adapt to the nutrient poor lifestyle. Al ong the same lines, it is also possi ble that terrestrial individuals 5
would benefit by having a greater abundance of vesicles as oppos ed to hyphae. It is likely that storage vesicles are essent ial in tropical soils with limite d nutrient availa bility so as to best retain and utilize the minerals obtained by the hyphae. Another reason for the overwhelming pr esence of vesicles in Monteverde terrestrial individuals could be that an alte rnative kind of mycorrhiz al 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 pteridophyte 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 n eeds to be conducted on the intricacies of VAM associations within tropical pteridophyte ha bitats. 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 epiphytic individuals whose host tree did not exhibit the spongy ba rk capable of water uptake as Acnistus arborescens can. Yet, based on this resear ch, it is clear that there is a mycorrhizal association with tropical pteridophytes in Monteverde, Costa Rica. ACKNOWLEDGEMENTS I thank Karen Masters for her assistance with research and statistics, and her persistence in speaking to the Eco Lodge about facility us e. Thanks to Willow Zuchowski for help in identifying the ferns collected. I would like to thank Alan Masters 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 mycorrh izal fungi. Most of all, I would like to thank my parents for giving me the opportunity to be en trenched in a place of wondrous beauty and to c onduct a project in Costa Rica. LITERATURE CITED Brundett, M.C. 1996. Ectomycorrhizas. Mycorrhiza 6:509 Brundett, M. C. 2002. Coevolution of root s 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 Transi tion Zones. Restoration Ecology 6 (2) :214 Gemma, J.N.,Koske, R.E., Flynn, T.1992. Myco rrhizae in Hawaiian Pteridophytes: Occurence and Evolutionary Signifi cance. 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. 1985. Nutrient Cycling in Tropical Forest Ecosystems. Pg. 24. John Wiley and Sons, Great Britian. 6
Lellinger, D.B. 1985. Ferns and Fern-Allies. Pg. 15-33. Washington D.C. Smithsonian Institution Press. Mills, T. J. 2006. Presence of endomycorhiz ae on roots of epiphy tic orchids. Pg. 1-5. Tropical and Conservation Ecology (Summer). Moran, R.C. 2004. A natural history of fern s. Pg. 218-233. Portland, OR. Timber Press Inc. Clark, K., Lawton, R.O., Butler, P. R. 2000. Th e physical environment. In N. Nadkarni and N. Wheelwright (Eds.). In Montever de 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 (12) : 539-546 Read, D.J., Duckett, J.G., Francis, R., Li grone, R., Russell, A. 2000. Symbiotic fungal associations in "lower" la nd plants. The Royal Society: 355: 815-831 Rowe, A. 2001. Mycorrhizae in brom eliads. Pg. 731. UCEAP (Fall). Smith, S.E., Read, D.J. 1997. Mycorrhizal symbiosis. Pg. 312-313. San Diego, CA. Academic Press Inc. Taylor, T.N., Kerp, H., Hass,H. 2005. The r ynie chert and its flora. Mycologia: 97 : 269-285 Wang, Y.L. 2006. Phylogenetic distribution and e volution 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 vesicula r-arbuscular mycorrhizae in terrestrial and epiphytic ferns. Pg. 754. UCEAP (Fall). 7
0 2 4 6 8 10 12 14 Both vesicles and hyphae VesiclesHyphaeNone infected Infection Type Terrestrial Epiphytic FIGURE 1. The number of epiphytic or terrestrial individuals identified by incidence of infection in vesicles and hyphae. The incide nce of mycorrhizal st ructures seen in terrestrial and epiphytic species is non-random ( 2 = 15.884, df = 3, p 0.05). Terrestrial species have a greater incide nce of vesicles and epiphytic species are more prone to hyphal infection. 8
01234567Aspenium 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)SpeciesNumber of Individuals Hyphae VesiclesFIGURE 2. Number of individual s with infected vesicles or hyphae in each of the ten species..The incidence of mycorrh izal structures seen in terr estrial and epiphytic species is non-random ( 2 = 15.884, df = 3, p 0.05). 9