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Asimetra de las estructuras florales y la posibilidad de auto-polinizacin en Oerstedella exasperata (Orchidaceae)
Asymmetry of floral structures and the possibility of self-pollination in Oerstedella exasperata (Orchidaceae)
The purpose of this study was to investigate the causes of the pervasive asymmetry found in the neotropical orchid Oerstedella exasperata. Previous studies have found that high levels of asymmetry decrease reproductive success, and can be caused by a high amount of homozygosity within a population. This study tested the ability of O. exasperata to self-pollinate, a condition that indicates the possibility of a high level of homozygosity. In addition, the relationships between three degrees of asymmetry and floral color, floral mass, plant size and sun exposure were investigated to determine whether higher asymmetry is correlated with lower reproductive success or reduced overall plant fitness. Results showed an overall trend suggesting that self-pollination is possible, which implies that O. exasperatas high level of asymmetry could be caused by homozygosity. Floral asymmetry was not related to any of the other examined factors, implying that it is a neutral trait that does not affect the overall fitness of this species of orchid. These results contradict other studies findings that asymmetry is detrimental to plant health, indicating that O. exasperata could be an exception to the general rule that symmetry indicates superior genetics in plants.
El propsito de este estudio fue investigar las causas de asimetra en la orqudea neotropical Oerstedella exasperata. Estudios anteriores han reportado que los niveles elevados de asimetra bajan el xito reproductivo, y pueden ser causados por homocigosis alta en una poblacin. Este estudio probo la capacidad de O. exasperata de auto-polinizarse, una condicin que indica la posibilidad de un nivel alto de homocigosis.
Text in English.
Orchids--Costa Rica--Puntarenas--Monteverde Zone
Orqudeas--Costa Rica--Puntarenas--Zona de Monteverde
Orqudeas polinizacin--Costa Rica
Tropical Ecology 2008
Oerstedella exasperata--Species fitness
Ecologa Tropical 2008
Oerstedella exasperata--Estado fsico de las especies
t Monteverde Institute : Tropical Ecology
Asymmetry of floral structures and the possibility of self pollination in Oerstedella exasperata Orchidaceae Lindsay Brownell Department of Biology, Davidson College ABSTRACT The purpose of this study was to investigate the causes of the pervasive a symmetry found in the Neotropical orchid Oerstedella exasperata . Previous studies have found that high levels of asymmetry decrease reproductive success, and can be caused by a high amount of homozygosis within a population. This study tested the ability of O. exasperata to self pollinate, a condition that indicates the possibility of a high level of homozygosis . In addition, the relationships between three degrees of asymmetry and floral color, floral mass, plant size and sun exposure were investigated t o determine whether higher asymmetry is correlated with lower reproductive success or reduced overall plant fitness. Results showed an overall trend suggesting that self pollination is possible, which implies that O. exasperata Â€s high level of asymmetry co uld be caused by homozygosis . Floral asymmetry was not related to any of the other examined factors, implying that it is a neutral trait that does not affect the overall fitness of this species of orchid. These results contradict other studiesÂ€ findings t hat asymmetry is detrimental to plant health, indicating that O. exasperata could be an exception to the general rule that symmetry indicates superior genetics in plants. RESUMEN El propÃ³sito de este estudio fue investigar las causas de la asimetrÃa en la orquÃdea neotropical Oerstedella exasperata . Estudios anteriores han reportado que niveles elevados de asimetrÃa bajan el Ã©xito reproductivo, y pueden ser causados por homozigosidad alta en una poblaciÃ³n. Este estudio probÃ³ la capacidad de O. exasperat a de auto polinizarse, una condiciÃ³n que indica la posibilidad de un nivel alto de homozigosidad. TambiÃ©n, las relaciones entre tres grados de asimetrÃa y color floral, masa floral, tamaÃ±o de la planta y exposiciÃ³n al sol fueron investigados para determina r si la alta asimetrÃa se correlaciona con un bajo Ã©xito reproductiv o . Los resultados mostraron una tendencia que surgiere que la auto polinizaciÃ³n ocurre, lo que implica que el alto nivel de asimetrÃa en O. exasperata puede ser causado por homozigosidad. L a asimetrÃ a floral no fue relacionada a ni ngÃºn otro factor examinado, lo implica que puede ser un carÃ¡cter neutro que no afecta la salud general de esta especie de orquÃdea. Estos resueltos contradicen otros estudios que dicen que la asimetrÃa es mala para la salud de plantas, e indica que O. exasperata pueda ser una excepciÃ³n a la regla general que simetrÃa indica una genÃ©tica superior en plantas. INTRODUCTION Symmetry is a characteristic used by many organisms to evaluate the genetic quality of other in dividuals. Potential mates with relatively high levels of fluctuating asymmetry have been shown to be less fit than more symmetrical individuals, and therefore tend to be selected against Palmer and Strobeck 1986. Symmetry is also an important factor in plant pollination systems, as flowers provide a visual signal to pollinators in order to disperse their pollen. Various studies have shown that pollinators prefer symmetrical flowers over asymmetrical ones, and that bees in particular show a preference for symmetrical flowers independent of pollinator rewards, floral color, odor and size MÃ¸ller 1995, MÃ¸ller and Eriksson 1995, MÃ¸ller and Sorci 1998. Giurfa et al. 1999 also note that flowers with lower levels of fluctuating asymmetry enjoy higher
visitati on rates, which result in greater pollen removal and deposition, as well as the receipt of pollen of superior quality. Given these findings, it is surprising that the Neotropical montane orchid Oerstedella exasperata shows a persistently high level of fluct uating asymmetry, both within and between individuals Snayd 2007. However, Snayd 2007 has found that asymmetry does not have a negative effect on the reproductive success of O. exasperata as measured by pollinia removal. In addition, Snayd found that t here was no correlation between the degree of bilateral asymmetry and the degree of fringe symmetry, which contradicts earlier studies MÃ¸ller 1995 and Neal et al. 1998 that indicate that asymmetry in flowering plants results in an overall decrease in fit ness, which is the ability to survive and transmit genes to the next generation Jenkins 1990. It is possible, therefore, that asymmetry is a selectively neutral trait for O. exasperata , and its persistence in the species is simply due to a lack of natura l selection against it. It is also known that individuals showing high levels of homozygosity due to inbreeding can be prone to higher levels of fluctuating asymmetry MÃ¸ller and Eriksson 1995. Higher levels of homozygosity are found in organisms that ar e capable of self pollination, and evidence of selfing ability in O. exasperata would be an important step toward confirming homozygosity as a cause of its persistent asymmetry. In addition, resource constraints have been found to affect flower production in the Neotropical orchid Brassavola nodosa Murren and Ellison 1996, so it may be possible that O. exasperata also expresses asymmetry as a response to environmental conditions. The purpose of this study was to shed light on the cause or causes of the h igh asymmetry seen in O. exasperata by determining whether the orchid is capable of the self pollination that would imply inbreeding and homozygosity, and whether floral asymmetry is correlated with different floral characteristics, plant size or sun expos ure. I hypothesized that, like many montane species, O. exasperata would show self compatibility because of low pollinator ability due to harsh environmental conditions. I predicted that floral asymmetry would not be correlated with other floral traits, as implied by SnaydÂ€s previous study. I also hypothesized that asymmetry would be correlated with plant size and sun exposure, because of past studies that suggest that environmental factors affect floral development. METHODS Study Organism Oerstedella ex asperata is a terrestrial or epiphytic orchid distributed throughout the mountains of Costa Rica and Panama between 1000 and 2500 m, and is common along roadsides. It is one of the largest orchids in Costa Rica, reaching up to three m in size Zuchowski 20 05. It has adventitious roots and tolerates various conditions of elevation and rainfall, which helps account for its ubiquity. Its stems are highly branched, and can reach over one cm in diameter at the base, with leaves ranging from five to fifteen cm l ong. Its petals are usually greenish at the base fading to yellow toward the terminal end, and are more oblique in shape than the oblanceolate sepals HÃ¡gsater 1992. The column and labellum are flanked by prominent, lobate fringes, which have a variable n umber of lobes. The white fringes, labellum and callus are usually highlighted with purple markings, and the area of this coloration is also highly variable.
Study Site This study was conducted between October 30 th and November 16 th , 2008 near the Mont everde Cloud Forest in Costa Rica. I examined nineteen O. exasperata plants growing along the TV Tower Road leading to Cerro de los Amigos. All individuals were found in the clay bank of the west side of the road. Plants were marked with flagging tape and given a unique identification letter. Self Pollination Experiment I selected 40 young flowers with intact pollinia on nine O. exaperata plants on which to conduct my self compatibility experiment. I self pollinated 20 flowers and cross pollinated 20 flo wers, using a pin to remove pollinia from the pollinaria and deposit them onto the adhesive surface within the column. I then put pollinator bags around the pollinated flowers to prevent pollinator interference. Pollination was performed from November 2 nd to November 4 th . I removed the pollinator bags on November 13 th and recorded the presence or absence of a swollen pedicel and column that would presumably indicate fruit formation and therefore fertilization. I entered the data into a contingency table and analyzed my findings with a chi square test. Asymmetry Experiment I sampled 124 individual flowers from 19 plants at the study site, labeling each flower with its parent plant letter and a unique number. I evaluated fringe asymmetry and bilateral asymm etry following SnaydÂ€s methods of taking the absolute value of the difference between the number of fringes on each side and the lengths of each side of the labellum. I also evaluated petal asymmetry by taking the absolute value of the difference in the l engths of the petals. I measured the length of each side of the labellum and the lengths of each of the petals using calipers. In addition to the three asymmetry measurements, I created a scale to measure the amount of relative coverage of purple on the la bellum, callus and fringes, and rated each flower from one minimal purple to five saturation. I also weighed each flower to determine its mass. In addition to the floral characteristics, I determined the size of each plant by counting the number of lea ves and measuring the length of the longest leaf Murren and Ellison 1996. To evaluate the effects of sun exposure on plant development, I categorized each plant as being in the shade or sun, ÂshadeÂ‚ being defined as having other plants growing above it a nd preventing it from absorbing as much light as an uncovered plant. Bivariate regressions were performed to evaluate the relationship between the three different degrees of floral asymmetry, floral mass and color; between the average floral asymmetry, siz e and number of flowers on each plant; and between the number of flowers and plant size. RESULTS None of the pollinated flowers showed swollen pedicels, but as of the eleventh day of the experiment, 20 flowers did show swelling in the column, of which 35 % were flowers that had been selfed. This implies that outcrossing may be a more common occurrence. The numbers of outcrossed flowers showing swelling and selfed flowers lacking swelling both exceeded the expected value of ten, with thirteen flowers each. Seven selfed flowers had swelling and seven outcrossed flowers lacked swelling, both of which are lower numbers than the expected ten
flowers. However, these differences are not statistically significant Figure 1. There was no significant difference betw een the average floral asymmetry of plants growing in different sunlight conditions, though fringe asymmetry was considerably higher than lip or petal asymmetry Figure 2. Based on statistical tests, asymmetry is not related to any of the plant characteri stics tested Table 1, nor are the three degrees of asymmetry correlated with each other Figure 3. In addition, no correlations were found between the three degrees of floral asymmetry and flower mass Figure 4 or flower color Figure 5. However, larg er plants were observed to produce significantly more flowers than smaller ones Figure 6. Figure 1: Difference between observed and expected values for presence or absence of swelling in the columns of selfed and outcrossed Oerstedella exasperata flowers in Monteverde, Costa Rica. Expected value for all = 10. N = 40 manually pollinated flowers. Chi squared value = 3.6; df = 1; critical value = 3.84. 0 2 4 6 8 10 12 14 Selfed/Swelling Outcrossed/Swelling Selfed/No Swelling Outcrossed/No Swelling Flower Condition Number of Flowers Observed Expected
Figure 2: Mean value and standard error of three degrees of floral asymme try with respect to sun conditions. Lip and petal values and corresponding error bars were multiplied by 10 to improve the resolution. N of sun plants = 11, n of shade plants = 8. Table 1. Statistical results of bivariate regressions comparing fringe, l ip and petal asymmetry to flower mass, flower color, size of plant and number of flowers. Values are F p value and R 2 . Fringe Asymmetry Lip Asymmetry Petal Asymmetry Flower Mass 2.259 0.135 0.018 0.752 0.388 0.006 1.559 0.214 0.013 Flower Color 0.281 0.597 0.002 0.202 0.654 0.002 1.134 0.289 0.009 # of Leaves 2.589 0.126 0.132 0.160 0.695 0.009 1.547 0.230 0.083 Longest Leaf 3.490 0.079 0.170 1.252 0.279 0.069 0.778 0.390 0.044 # of Flowers 0.0002 0.988 0.00001 1.758 0.202 0.094 0.126 0.728 0.007 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Sun Shade Sun Conditions Mean Asymmetry Fringe Lip Petal 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0 1 2 3 4 Fringe Asymmetry Lip Asymmetry
Figure 3: Three regressions showing: a relationships between fringe asymmetry absolute value of [# of fringes on left side Âƒ number of fringes on right side] and lip asymmetry absolute value of [length of left s ide of labellum Âƒ length of right side of labellum] R 2 = 0.010, p = 0.265; b relationships between fringe asymmetry and petal asymmetry absolute value of [length of left petal Âƒ length of right petal] R 2 = 0.012, p = 0.220; c relationships between l ip and petal asymmetry R 2 = 0.003, p = 0.580. For all, n = 124 flowers. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 1 2 3 4 Fringe Asymmetry Petal Asymmetry 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.1 0.2 0.3 0.4 Lip Asymmetry Petal Asymmetry
Figure 4: Mass of flower related to: a. fringe asymmetry R 2 = 0.018, p = 0.135; b. lip R 2 = 0.0049, p = 0.338 and petal asymmetry R 2 = 0.0127, p = 0.214 related to flor al mass. N = 124 flowers. 0 0.5 1 1.5 2 2.5 3 3.5 0.1 0.15 0.2 0.25 0.3 0.35 Mass Fringe Asymmetry 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0.1 0.15 0.2 0.25 0.3 0.35 Mass Lip/Petal Asymmetry Linear petal Linear lip
Figure 5: Relationship between number of leaves and: a. average degree of fringe asymmetry R 2 = 0.132, p = 0.126; b. average degrees of lip R 2 = 0.009, p = 0.695 and petal asymmetry R 2 = 0.083, p = 0.230. N = 19 plan ts. 0 0.5 1 1.5 2 2.5 3 5 15 25 35 45 55 Number of Leaves Avg Fringe Asymmetry -0.01 0.01 0.03 0.05 0.07 0.09 0.11 0.13 5 15 25 35 45 55 Number of Leaves Avg Lip/ Petal Asymmetry Linear Lip Linear Petal
Figure 6: Relationship between number of flowers and number of leaves per plant. R2 = 0.509 , p <0.05 . N = 19 plants. DISCUSSION This study is the first to document manual pollination attempts on the Monteverde population of O. exasperata , as well as the first to examine the effects of sunlight on its floral morphology. Although the pollination data were not statistically significant, seven selfed individuals did show swelling, which suggests that O. exasperata is indeed self compatible, though out crossing seems to result in higher pollination success. Given that this species is usually found in montane conditions at mid to high elevations, it could be that pollinators are often in short supply, and therefore self pollination is utilized by this sp ecies when outcrossing is not possible. A repetition of the experiment with a larger sample size may provide significant support of this trend. It is of interest to note that the observed swelling did not occur in the ovary area of the pedicel as expected , but rather in the column itself. This characteristic may be a precursor of fruit formation, and the time constraints of this study did not allow for the observation of subsequent swelling in the pedicel. However, it may also be that the swelling of the c olumn is a response of the plant to successful pollination, but that no fruit developed because fertilization is prevented by self incompatibility, in which case O. exasperata would not be capable of self pollination Richards 1996. The pollination system of this species is still poorly understood, and deserves more in depth study. In order to avoid pollinating flowers that had already received pollen, only flowers with intact pollinia were used in this experiment, as the previous arrival of a pollinator would probably have dislodged the pollinia. However, they were not bagged as buds and then allowed to flower, which would have eliminated the chance of previous pollination. Future studies should employ this method to obtain more accurate results. 0 5 10 15 20 25 30 35 40 0 10 20 30 40 50 60 Leaves Flowers
If O. e xasperata is found to be self incompatible, it may still experience a high level of homozygosis due to its adventitious roots, which allow vegetative reproduction. This could result in patches of individuals who are essentially clones of each other, which would reinforce homozygosis if its pollinators generally forage in a small area. In this case, outcrossing and selfing would both contribute to an increased level of homozygosis , as there would be no effective genetic variation between neighboring indivi duals. It would be beneficial to perform genotyping on this population to determine whether it does indeed have the high level of homozygosis that can result in increased fluctuating asymmetry. The fact that floral asymmetry is not correlated with floral color or mass implies that it does not have a negative impact on the reproductive success of O. exasperata , as more colorful and larger flowers would presumably be more attractive to pollinators. This finding contradicts past studies on other species of f lowering plants but agrees with SnaydÂ€s previous study on the same population. It could be that this species of orchid or this population specifically, is an exception to the general rule that symmetrical flowers confer a reproductive advantage. Perhaps i ts primary mode of reproduction is vegetative, and therefore it does not invest energy in producing symmetrical flowers to attract pollinators. It could also be that its pollinator, as yet unknown, is a species or genus that is not attracted by floral symm etry. Neal et al 1998 state that the environmental factors that trigger asymmetry may result in overall reduced fitness. If this condition were true for O. exasperata , decreased sunlight would be expected to result in both smaller plant size and increase d asymmetry, as a plant starved of solar energy would not be able to invest extra resources in growth and the production of symmetrical flowers. This would lower this speciesÂ€ fitness in terms of reproductive ability and overall health. The strong correlat ion between the number of leaves and flowers present on plants indicated that plant size was a good measurement of both overall fitness and potential reproductive success. However, floral asymmetry was not correlated with sun exposure or plant size, which implies that higher asymmetry, is not an indicator of poor plant quality, nor is it affected by environmental conditions. Additional studies evaluating the effects of other environmental factors such as substrate, rainfall and elevation on asymmetry are nee ded to confirm this prediction. In addition, as Snayd 2007 observed, one would expect a correlation between all measures of asymmetry if environmental factors do affect plant health. However, this study found no correlations between fringe, lip and petal asymmetry, which further suggests that increased asymmetry does not decrease this speciesÂ€ overall fitness. In conclusion, floral asymmetry in O. exasperata appears to be a neutral trait, showing no negative, predictable relationship with other floral c haracteristics, plant size or levels of sunlight. This conclusion contradicts previous studies that have found that symmetrical flowers have higher reproductive success, which implies that O. exasperata may be an exception to the general rule that floral a symmetry is a detrimental trait in plants. The high level of asymmetry in this population could be perpetuated due to a high level of homozygosis caused by either self fertilization or low levels of genetic variation as a result of vegetative reproduction . Future studies should focus on the pollination system of this orchid, its genetic makeup and the effects of different environmental conditions on floral asymmetry. ACKNOWLEDGMENTS I am eternally grateful to the EstaciÃ³n BiolÃ³gica Monteverde for the o pportunity to perform this study; Karen Masters for her orchid expertise and patient help with statistics; Alan Masters for creating this truly unique study abroad experience; Chanchos de Monte for memorable evenings;
Pablo Allen and Moncho CalderÃ³n for su pplies, snacks, humor and additional assistance with statistics; my classmates for companionship and commiseration; and whichever higher power, scientific or spiritual, that created the magical place that is the tropical rainforest. LITERATURE CITED Giur fa, M, A. Dafni and P. R. Neal. 1999. Floral Symmetry and Its Role in Plant Pollinator Systems. International Journal of Plant Sciences 160: S41 S50. HÃ¡gsater. 1992. Oerstedella exasperata . In: Orchids of Costa Rica, Part 2 , D.E. Mora de Retana and J.T . Atwood eds. The Marie Selby Botanical Gardens, Sarasota, FL, pp.1460 1461. Jenkins, J. B. 1990. Human Genetics . Harper and Row, New York, NY. As cited on Webref.org. Mitton, J.B. and M. C. Grant. Association s Among Protein Heterozygosis , Growth Rate and Developmental Homeostasis. Annual Review of Ecology and Systematics 15: 479 499. MÃ¸ller, A.P. 1995. Bumblebee Preference for Symmetrical Flowers. Proceedings of the National Academy of Sciences of the Unit ed States of America 92: 2288 2292. MÃ¸ller, A.P. and G. Sorci. 1998. Insect Preference of Symmetrical Artificial Flowers. Oecologia 114: 37 42. MÃ¸ller, A.P. and M. Eriksson. 1995. Pollinator Preference for Symmetrical Flowers and Sexual Selection in Pla nts. Oikos 73: 15 22. Murren, C.J. and A.M. Ellison. 1996. Effects of Habitat, Plant Size, and Floral Display on Male and Female Reproductive Success of the Neotropical Orchid Brassavola nodosa . Biotropica 28: 30 41. Neal, P.R. et al . 1998. Floral Sy mmetry and Its Role in Plant Pollinator Systems: Terminology, Distribution, and Hypotheses. Annual Review of Ecology and Systematics 29: 345 373. Palmer, A.R. and C. Strobeck. 1986. Fluctuating Asymmetry: Measurement, Analysis, Patterns. Annual Review of Ecology and Systematics 17: 391 421. Richards, J.A. 1996. Breeding Systems in Flowering Plants and the Control of Variability. Folia Geobotanica & Phytotaxonomica 31: 283 293. Snayd, M. 2007. Relationship between Floral Advertisement and Pollinia Removal in Oerstedella exasperata Orchidaceae. EstaciÃ³n BiolÃ³gica Monteverde, Costa Rica. Biology. Fall 2007, pp. 35 41. Zuchowski, W. 2005. A Guide to Tropical Plants of Costa Rica. Zona Tropical, Miami, FL, pp. 420 421.