Abundance of mycorrhizae in epiphytic and terrestrial orchid roots from genus Epidendrum Rebecca Sanchez 9 June 2017 U niversity of C alifornia, Santa Barbara UCEAP Tropical Ecology and Conservation Spring 2017 ______________________________________________________________________________ ABSTRACT Orchids are one of the most diverse organisms in Costa Rica with over 1 460 documented species. They are unique in their germination and early juvenile life stage in that their survival is dependent on mychorriza e symbiosis. Though the symbiosis is only thought to be obligatory in the juvenile life stage, mature orchids are often found to maintain mychor rizal associati ons. I used lacto phenol blue dye to compare the mychorrizae colonization points in Epidendrum radicans a terrestrial orchid, and Epidendrum piliferum an epiphytic orchid, in Monteverde Costa Rica. The intention of my study was to determine if mychorrhizae colonization in orchid roots was significantly different in epiphytic orchids with arboreal soil and terrestrial orchids. I found expec ted, the terre strial orchids had a higher abundance of orchid mychorrizae colonization points. I also compared the presence of hyp hal structures from the family G lomeraceae in each orchid species. My results showed no significant difference in the presence of branched o r blurred hyphal structures between the two species despite the difference in the abundance of orchid mychorrizae. The difference in abundance of mychorrizae between the terrestrial and epiphytic orchid is likely attributed to a difference in nutrient avai lability. ______________________________________________________________________________ A bundancia de micorrizas en orqudeas epifita s y terrestre s del g nero Epidendrum RESUMEN Las orqudeas son muy diversa s en Costa Rica con ms de 1 460 especies documentadas. Las orqudeas son nicas durante su germinacin y en la etapa temprana de la vida cuando su supervivencia depende de la simbiosis mic orriza. Aunque se piensa que la simbiosis solo es obligatoria en la etapa temprana de la planta a menudo se encuentran orqudeas maduras que mantienen micorrizas. Utilic azul de lactofenol para co ntar los puntos de colonizacin de micorrizas en Epidendrum radicans una orqudea terrestre, y Epidendrum piliferum una orqudea epfita. La intencin de mi estudio fue determinar si la colonizacin de micorri zas en races de orqudeas era diferente en tre orqudeas epfitas con suelo arbreo y orqudeas terrestres. Encontr una diferencia significativa de la cantidad de pun tos de colonizacin para las races de cada especie Como esperaba la especie de orqudeas terrestres tuv o mayor abundancia de puntos de colonizacin de micorriza Tambin compar la presencia de estructuras de hifas de hongos de la familia Glomeraceae en cada especie de orqudea. Mis resultados no mostraron diferencias significativas en la presencia de estructuras de hifas ramificadas o borrosas entre las dos especies a pesar de la diferencia en la abundancia de orqudeas micorrizas La d iferencia en la abundancia de micorrizas entre la orqudea terrestre y epfita se podra atribuir a diferencia s en la disponibilidad de nutrientes.
Presence of Mychorrhiza in Mature Epidendrum sp. Sanchez 2 Orchidaceae is one of the largest and most diverse plant families. There are 1460 orchid species described in Costa Rica alone, with an expectation of 100 200 left undescribed (Ossenbach et al 2007) Orchids are both terrestrial and epiphytic and share a distinct root system. F irst there is a layer of spongy tissu e around the true root, called the velamen. The velamen is important for water and mineral uptake in many species, and in dryer areas, can aid in light protection (Fanfani and Rossi 1988). Terrestrial orchids can also be seen with thin root systems and rhizomes designed to stay dormant during the dry s eason. Still, even with these adaptations most orchids cannot germinate without a symbiotic fungus, mycorrhizae. This is due to the small size of orchid seeds which render the plants unable to feed from their own food supply (Dressler, 1993). Mycorrhizae typically works as a mutualist symbiont of orchids, with a few exceptions where mature orchids are believed to parasitize the fungi (Brundrett 2002) Mycorrhizal roots have been shown to be more efficient in nutrient absorption for carbon, phosphorous, zin c and nitrogen. As was mentioned, this nutrient uptake is crucial to the beginning germination and growth of orchids, as it provides the young plants with the carbon necessary to hydrolyze insoluble carbohydrates from the soil (Smith and Read, 1997) Howev er, it ha s also been shown that orchids are capable of cell lysis and inducing hyphal collapse to prevent fungal invasion during its mature life stage (Smith and Read, 1997). Though this control has been observed it still remains that many orchid species carry on th is symbiotic mutualism far beyond the juvenile life stage. This continued mutualism is presumably to receive the minerals that would be more difficult to obtain with non mycorrhizal root systems. Still, few studies have question ed how the substrate on wh ich orchids grow correlates with the abundance of mycorrhizae in their roots. I observe d the abundance and morphotypes of mycorrhizae in Epidendrum radicans and Epidendrum piliferum I chose the genus Epidendrum because it is one of few abundant genera in the Orchidaceae family with both terrestrial and epiphytic species in the Monteverde region. Considering the difference in nutrient availability between the two substrates it i s likely for there to be more my chorrizal associations in one species than the o ther. The goal of this study is to determine if the presence of mycorrhizal roots differs significantly between epiphytic and terrestrial orchids. METHODS In total I collected roots from 11 E. piliferum plants and 12 E. radicans plants I collected the E. radicans from three locations. The first location was across from the Hotel Belmar along the opening of the path to the TV towers. The second location was along the roadside that leads to the Biological Station and the third was in the garden at the Bio logical Station I collected the E. piliferum roots along the main trail at the Biological Station I collected three or four root samples from each plant depending on how many I could find. From three of the root samples I cut 4cm long segments from the end of the root. The fourth root was extra to replace any of the other three roots should the clear ing process not work properly. Af ter collecting my samples I began the process of dying the mycorrhizae by rinsing them in tap water several times. I then placed them in 100 mL beakers and added 10 20 mL 10% KOH solution. The beakers were then heated in a bath of boiling water for six minutes. This process cleared the roots and prepared them for the dye. After six minutes the roots were rinsed in tap water again and placed in 10 20 mL of 5% lactophenol blue vinegar solution. The roots while in the dye solu tion were then plac ed in a boiling water bath for three minutes. After the
Presence of Mychorrhiza in Mature Epidendrum sp. Sanchez 3 three minutes the roots were rinsed in tap water and placed in a 1% vinegar solution (Vierlheilig et al 1998 ) I then cut the roots in two 1cm segments from the end of the ro ot. I sliced open the roots with a longitudinal cut and mounted it on a slide I then observed the root slices under a compound microscope under 10x/20 ocular pieces and a 10x objective. From the center of each root segment I counted the number of observed colon ization points out of an estimated 100 cells. I also took note of the different types of hyphae that I saw on each root segment. I then conducted a t test to see the significance of my data comparing the colonization points for the two orchid species. I also used a C hi square test to determine the significance of the presence of the two hyphal morphotypes in the root segments. RESULTS In total I observed 55 root segments from E.radicans and 57 root segments from E.piliferum The mean number of colonization points in E.radicans was 73.1 + 3.59 SD The mean number of colonization points in E.piliferum segments was 39.00 + 3.62 SD The re was a significant difference in colonization points between E. radicans and E. piliferum (F igure 1, t 109 = 6.69, p<0.0001 ). I also compared two morphotypes of hyphae that were viewed in the root segments. The first morphotype was branched intraradical hyphae and the secon d was a long intraradical hyphal blur which had no visible branched parts and remained intraradical between the cells In the E. radicans 19 of the 55 segments had blurred intraradical hyphae and 26 had branched hyphae. In the E. piliferum 24 of 57 segments had blurred intraradical hyphae and 2 0 had branched hyphae. I compared th e presence of the morphotypes for each orchid and there were no significant difference in the numbres for E. radicans and E. piliferum (Table 1 ). (a)
Presence of Mychorrhiza in Mature Epidendrum sp. Sanchez 4 (b) (c) DISCUSSION The results from my data are consistent with my expected observations and answer the question of whether the abundance of mycorrhizae differs in terrestrial and epiphytic orchids. As can be observed in F igure 1 the presence of colonization points in the E radicans root segments was on average significantly higher than the number of colonization points in the E. piliferum root segments. This difference in abundance i s likely best attributed to higher nutrient availability for epiphytic orchids. According t o Lesica and Antibus (1990), levels of NO3 NH4+, and P04 have been recorded as higher in canopy soils than terrestrial soils at La Selva. Costa Rica. Of these NO3 and NH4+ have been proven to be useful compounds for orchid Branched Blurred no. segments % of segments no. of segments % of segments E.r adicans 26 47.3 19 34.5 E.p iliferum 20 35.1 24 42.1 Figure 1 (a) C omparison of the mean number for each orchid species in a range of 0 to 100 colonized cells in each examined root segment (t 109 = 6.69, p<0.0001) (b) E. radicans histogram distribution of colonization points. The x axis represent the number of colonization points per 100 cells. There is an upward trend indicating that more root segments had 60 100 colonization points per 100 observed cells. (c) E. piliferum histo gram distribution of colonization points. The x axis represent the number of colonization points per 100 cells. There is a downward trend indicating that majority of root segments had 10 40 colonization points per 100 observed cells Table 1. N umber of branched and blurred segments. For E. radicans there were a total of 55 segments and v alues for E. piliferum are from a total of 57 root segments.
Presence of Mychorrhiza in Mature Epidendrum sp. Sanchez 5 nutrient uptake ( Hew C.S. et al 1993, Wang 2007, Wu et al 2013 ). As leaves fall from the canopy, epiphytic orchids have an advantage of collecting the organic matter as well as atmospheric deposition such as dust, mist, and rain (Lesica and Antibus, 1990). Though the nutrient availa bility in canopy soils seems to be a strong environmental factor, further research is still ne eded to better understand the influence nutrient availability has o n mychorrizae orchid symbiosis for epiphytic orchids. M ycorrhizal root systems have allowed for orchid growth in a wider range of habitats for terrestrial orchids such as E. radicans (Nurfadihla et al ., 2013). For terrestrial orchids nutrient availability is l ikely more difficult due to poor soil nutrient access In en vironments that receive heavy rainfall such as in Monteverde it is common for important nutrients such as carbon to become difficult to obtain due to saturation Therefore it would be more beneficial to continue sharing carbohydrates with mycorrhizae in exchange fo r their uptake of carbon, nitrogen and phosphorous (Smith and Read 1997 Peterson and Massicotti 2004 ) In my study I also compared the presence of two morphot ypes of arbuscular mycorrhizae hyphae within the root segments for each orchid species. The goal in observing the presence of each morphotype of hyphae was to see if there were any associations between the abundance of colonization points within an orchid species and the presence of hyphae within the root segment. T here was no significant different between t he two orchid species when comparing the presence of the two morphtoypes of hyphal structures in t heir root segments. Though I could not specifically identify the genus of the fungi present I was able to identify the hyphal structures as a part of the Glomeraceae family (INVAM). It would have been good to identify the specific genus of the fungi to determine the specificity of the fung us orchid relationship. According to Hadley (1970) Epidendrum radicans is non specific in its germination phase and is known to form a symbiosis with a vari ety of genera from Glomeraceae. Epiphytic orchids have been shown to have possible specificity with specific species on fungi. In the Andean cloud forest epiphytic orchids Stelis concinna, S. hallii, S. superbiens and Pleurothallis lilijae were all shown to favor species from the fungal genus Tulasnelloid ( Surez et al 2006) All four orchids are from the same tribe as my study orchids, Epindendreae. No clear pattern of selective symbiosis has been determined yet but with continued research it is possible there will be a more defined trend of species specific selection in the f uture. Overall this study provided new insights to the relationship between mycorrhizae and mature orchids. It has been well documented that juvenile orchids depend on the nutrient transfer from mychorriza e to advance to their next life stage ( Dressler, 1993) but mature orchid interaction s with mycorrhizae still remain largely unknown. This study creates a template for future research of adult orchid mychorrizae symbioses For example, colonization points in terrestrial and epiphytic orchid s co uld be observed for different genera, localities and diff erent methodologies A study could be conducted with a larger sample size and with cross sections of each root segment rather than longitudinal slicing to compare with my results. This study showed a continued symbiosis amongst mature terrestrial orchids and mycorrhizae but does not yet categorize the relationship as mutualistic or parasitic. As Hijner and Arditti (1973) discuss, though there is a known nutrient transfer between the two contributors of the symbiosis it is not clear the extent to which they aid each other. It is known that orchids evolved to depend on mycorrhizal associations due to the obligatory symbiosis that occurs during germination of orchids (Leake, 1994) However, we still do not know how the carbohydrates presumably being transferred to the fungus during the mature stage of orchids compares to the amount of nutrients being provided by the mycorrhizae ( Hijner and Arditti,
Presence of Mychorrhiza in Mature Epidendrum sp. Sanchez 6 1973) The potential for mature orchid parasitism remain s unclear as researchers continue to accumulate information indicating that orchid mychorrizae are not an obligatory symbiont yet identify a clear exchange of nutrient between the symbionts (Brundrett, 2002). ACKNOWLEDGEMENTS I would like to thank Sofia Arce Flores for her advice and guidance throughout this study. I would like to thank Eladio Cruz for sharing his time and aiding in orchid identification. I also am extremely grateful to Laura Aldrich Wolfe for lending her knowledge and expertise in mycho rrizal associations Finally I would like to thank Bridget Banuelos for providing me with the encouragement and confidence to continue my study LITERATURE CITED Brundrett, M. C. (2002), Coevolution of roots and mycorrhizas o f land plants. New Phytologist 154: 275 304. doi:10.1046/j.1469 8137.2002.00397.x Dressler, Robert L. Field Guide to the Orchids of Costa Rica and Panama. Cornell University Press. 1993. Print. Inc. 1988. Print. H S pecificity 1970. H ew C. S., Lim L. Y. and Low C. M. ical orchids. Environmental an Experimental B otany 33, 273 28. 1993 Hijner Production and Requirements by American Journal of Botany vol. 60, no. 8, 1973, pp. 829 835. JSTOR www.jstor.org/stable/2441176 INVAM: International Culture Collection of Vesicular Arb uscular Mychorrizal Fungi, West Virginia Univeristy. www.invam.wvu.edu Leake J. R. (1994), The biology of myco ) plants. New Phytologist, 127: 171 216. doi:10.1111/j.1469 8137.1994.tb04272.x Lesica, P., & Antibus, R. (1990). The Occurrence of Mycorrhiz ae in Vascular Epiphytes of Two Costa Rican Rain Forests. Biotropica, 22 (3), 250 258. doi:10.2307/2388535 Nurfadilah, S., Swarts, N. D., Dixon, K. W., Lambe rs, H., & Merr itt, D. J. (2013). Variation in nutrient acquisition patterns by mycorrhizal fungi of r are and common orchids explains diversification in a global biodiversity hotspot. Annals Of Botany 111 (6), 1233 1241. Ossenbach, C., F. Pupulin & R. L. Dr essler. Orqudeas del istmo centroamericano: catlogo y estado de conservacin/Checklist and conservation status: Orchids of the Central American. 2007. Peterson, R. Larry and Hugues B. Massicotte. "Exploring Structural Definitions of Mycorrhizas, with Emphasis on Nutrient Excha nge Interfaces." Canadian Journal of Botany vol. 82, no. 8, Aug. 2004, pp. 1074 1088. EBSCO host doi:10.1139/1304 071. Smith S.E. and Read D.J. Mycorrhizal Symbiosis: Second Edition. San Diego. Academic Press, Inc. 1997. Print. Surez Juan Pablo Michael Wei, Andrea Abele, Sigisfredo Gar nica, Franz Oberwinkler, Ingrid Kottke, Diverse tulasnelloid fungi form mycorrhizas with epiphytic orchids in an Andean cloud forest, Mycological Research, Volume 110, Issue 11 2006.
Presence of Mychorrhiza in Mature Epidendrum sp. Sanchez 7 Vierheilig Staining Technique for Arbuscular Microbiology. 1998. Wang Y T. High NO3 N to NH4 N ratios promote g rowth and flowering of a hybrid Phalaenopsis grown in two root substrates, Hortscience 2008, vol. 43 (pg. 350 353) Wu, Jianrong Ma, Huancheng Xu, Xingliang Qiao, Na Guo, Shitan Liu, Fang Zhang, Donghua and Zhou Liping; Mycorrhizas alter nitrogen acquisition by the terrestrial orchid Cymbidium goeringii Ann Bot 2013; 111 (6): 1181 1187. doi: 10.1093/aob/mct062