Effect of Algal Growth and Coverage on the Growth of American Eelgrass (Vallisneria Americana)

EFFECT OF ALGAL GROWTH AND COVERAGE ON THE GROWTH OF AMERICAN EELGRASS (VALLISNERIA AMERICANA) A PAPER SUBMITTED FOR COMPLETION OF SENIOR RESEARCH FOR THE COLLEGE OF ARTS AND SCIENCES STETSON UNIVERSITY BY Caitlin Brewer IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE(S) OF BACHELOR OF SCIENCE ENVIRONMENTAL SCIENCE ADVISOR Dr. Jason M. Evans, Ph.D. MAY 2016 i Table of Contents INTRODUCTION.…………………………………………………………………... 1 METHODOLOGY…………………………………………………………………... 5 Plant Material………………………………………………………………... 5 Sediment Composition………………………………………………………. 6 Algal Material……………………………………………………………….. 6 Experimental Condition and Analysis………………………………………. 7 RESULTS…………………………………………………………………………... 10 DISCUSSION……………………………………………………………………… 12 LITERATURE CITED…………………………………………………………….. 15 List of Illustrations Figure 1: Vallisneria americana diagram………………………………………… 5 Figure 2: Assigned algal concentrations………………………………………….. 7 Figure 3: Cage design…………………………………………………………….. 9 Figure 4: Experimental set up……………………………………………………. 9 Figure 5: Microscopic view of algae……………………………………………. 10 Figure 6: Algal treatment measurement graphs…………………………………. 11 Acknowledgements I would like to acknowledge Dr. Jason Evans and Dr. Kirsten Work, Blue Spring State Park, Florida Fish and Wildlife Commission Eustis Lab, and the Stetson Biology Department. ii Abstract Vallisneria americana, a once prominent SAV (submerged aquatic vegetation) species in freshwater ecosystems, have recently experienced declines by the masses in Volusia Blue Spring State Park. Alga can be a large contributor to the health of eelgrass populations. The intention of this study is to determine a relationship between algal growth and coverage on the growth of SAV. Two distinctly different types of alga, Oscillatoria and Synedra, were used to inoculate the plants. A total of 6 plants experienced each treatment. Physical changes to the macrophytes such as: weight, length, and asexual offspring were noted. These changes were then compared using a one-way ANOVA. Microscopic examination of leaves were conducted to count and identify the presence of algal cells. Any defining conclusions are unable to be made at this point in the research. 1 1. Introduction: American eelgrass was once a common submersed aquatic plant which populated a majority of freshwater systems (Schloesser et al. 2007). In recent years American eelgrass, or Vallisneria americana, has seen a decline in in abundance around the world. Australia has suffered a devastating loss of Vallisneria americana from shallow urban lakes (Morris et al. 2004). The Upper Mississippi River experienced a large deterioration of this once abundant aquatic plant (Kimber et al.. 1995). Pollution and urban runoff seems to have negatively affected the lower Detroit River (Schloesser et al. 2007). Hauxwell et al.. (2004) defined several lakes, rivers, and estuaries in Florida which have suffered from the loss of eelgrass. With this recent decline in Vallisneria americana and a rise in algal growth in Blue Spring1, the question arises; what effect does algae have on the growth and asexual reproduction of American eelgrass? Volusia Blue Spring can be described as a first-magnitude spring2 with a large circular pool which feeds into a spring run. An evaluation of the spring and spring run conducted by the Florida Geological Survey found minimal aquatic vegetation other than algae in October 2001 (Scott et al.. 2004). Blue Spring State Park is found along the St. John’s River, which Dunn et al. (2008) surveyed specifically for epiphytic algae. Although this algae does have many benefits to the ecosystem, it has been found that it reduces light availability for the plants on which it is living. They found around 122 infrageneric taxa3 of epiphytic algae growing on V. americana. This imbalance can be detrimental to not only the health of the spring, but the organisms which inhabit 1 Blue Spring State Park; Volusia County 2 First-magnitude spring: spring with a water discharge rate of at least 2800 liters of water per second 3 Infrageneric taxa: pertaining to division within a biological genus of organisms 2 it. It is possible that with this vast amount of algal growth the amount of light which penetrates the plants is being reduced. This leads me to believe that the plants without algae will have greater growth (Kurtz et al.. 2003). In fact, Boustany et al. (2009) demonstrated in an experiment that light does affect below ground biomass. This includes the roots of the plant, which are responsible for supplying the stems and leaves with nutrients needed to thrive. Algae found in Volusia’s Blue Spring can frequently be found cohabiting in areas with multiple types of algae. Two common forms of algae found in Blue Spring would be cyanobacteria, or blue-green algae, and diatoms. Cyanobacteria is a filamentous species which generally produce long chains. Diatoms are a distinctive species which typically grow as single cells or can form simple colonies. Not a lot is known about the relationship between algae and submerged aquatic macrophytes, such as Vallisneria americana. V. americana is a dioecious aquatic macrophyte which is entirely submerged and characterized by ribbon-like leaves which radiate from a central rosette (Gettys and Haller 2013). Although this species can reproduce both sexually and asexually, most new plants are a results of asexual reproduction (i.e., runners and winter buds). Occasionally large mats of eelgrass can occur as a result of the rapid clonal expansion (Hauxwell et al. 2007). These large mats provide many beneficial services to the spring itself and its inhabitants. Obvious contributions of these thick mats would be food and habitat for many organism. However, eelgrass can also act as a form of security and protection. The thick biomass which V. americana can produce provides an ideal nursery area for juvenile fish, macroalgae, epiphytic organisms, and benthic invertebrates such as crustacean, 3 bivalves, etc. (Hill 2002). The root system of eelgrass can also help to prevent erosion of the bottom of the spring, which is largely composed of limestone and sand. With such a drastic shift in what was once a thriving eelgrass population, the theory of alternative stable states comes to mind. Morris et al.. claims that the theory of alternative stable states predicts that high nutrient concentrations increase the probability of shallow lakes switching from a state dominated by vascular macrophytes to one dominated by phytoplankton and/or other algae. It was found that with moderate to high nutrient loading the possibility of a significant increase in algae biomass is likely. With this increase in algae biomass, there is reduced light penetration. The combination of reduced light penetration and concentrations of dissolved oxygen in the water column resulted in complete loss of V. americana within 4 months (Morris et al.. 2003). Applying this theory to Blue Springs, we are seeing similar results as Morris et al... In Florida’s springs, nitrate concentrations have been steadily increasing since the 1970s. With adequate phosphorus already in the water column, even low concentrations of nitrogen can cause a substantial shift in the balance of ecological communities, such as the degradation of submerged aquatic macrophytes due to the overgrowth of algae (Harrington et al.. 2010). The overall goal of both past research and the research that is still ongoing is to find the ideal conditions required for a successful reintegration of V. americana into Blue Spring. As stated by Lloyd et al. (2012), ecological restoration is important in maintaining a healthy and balanced ecosystem. Carter et al.. (1996) had a similar plan with the reintroduction of American eelgrass into the Chesapeake Bay and the tidal Potomac River. They accredited the decline to the light supply in the water column. Gosselain et al. (2005) also discussed that changes in light penetration played a large role in the biomass of epiphytes. However, Boustany et al.. did suggest that there 4 was a threshold level between 8% and 28% surface irradiance. With this small threshold it would be interesting to see if the algal growth experienced blocks enough light to fall within this threshold. Therefore, I have conducted a study on the effect of algal growth and coverage on the growth of Vallisneria americana. With the understanding of this relationship, we can eliminate another factor in the mystery of the decline of American eelgrass in Blue Spring. For example, if it is proven that algal growth does have a significant effect on the growth of V. americana, then an area with a lower prevalence of this algae can be considered for restoration. However, restoration is not completely dependent on this study. As stated earlier, it is just one of the many factors which need to be considered before reintroducing American eelgrass into the springs. Ultimately I am hopeful this will support the efforts to restore a healthy population of Vallisneria americana into Blue Spring, which may ultimately increase the function of the natural ecological system (Lloyd et al.. 2012). In order to understand this relationship, I will be completing a lab experiment rather than a field study. In this particular case it is important to complete at least the first stage as a lab experiment so others factors are able to be controlled; only the algae will be altered at this point. Since I am particularly interested in Volusia Blue Spring, water will be collected from the spring for this experiment. It would be ideal to work with plants directly from the spring; however, populations are extremely low so that will not be possible. Instead plants will be received from an outside lab. Statistical analysis and measurement will be taken to quantify the relationship between the algae and eelgrass. 5 2. Methodology: Plant Material A total of 18 American eelgrass plants, Vallisneria americana, were acquired from a previous study. All plants used were originally collected from the Florida Fish and Wildlife Commission Eustis Lab ensuring they are all the same ecotype since have been cultured for several generations. Dying leaves, runners, and other reproductive parts were removed. Their number of leaves, wet weight, root length, shoot length, and total length were also measured prior to the start of the experiment (Fig.1). The plants were rinsed in a bucket containing water collected from Volusia Blue Spring in order to remove any excess dirt or organisms caught in the roots. Along with the dirt and organisms, any algal growth attached to the leaves was removed in the bucket as well. Figure 1. Parts of V. americana which are important to note are the leaves (A), reproductive parts (B), roots (C), and the runners (D). A D C B 6 Sediment Composition Blends of Quikrete Premium fine playground sand and organic material collected from Blue Spring was used to create an artificial sediment mixture with a consistent level of organic matter. Around 36 cups (14,976 g) of sand was used in total. The total sand was then separated in to 9 equal units, each consisting of 4 cups (~1,664 g) of sand. The sediment blend consisted of 1.5% organic material. To find the proper ratio 3 small finger bowls were lined with previously weighed aluminum foil and filled with a 100mL mixture of saturated silt and water. The bowls were then placed into the drying oven at 100° F for a couple of days. After all the moisture has evaporated from the finger bowls they were removed from the drying oven and weighed again. The 3 weights were averaged together to find the weight of dried silt in 100 mL of the saturated silt and water mixture. This allowed us to determine how much organic material should be added to the substrate based on the total sand. Since there was approximately 1,664 g of sand in each unit then 1.5% would be ~25 g of organic material or 150 mL of the saturated silt mixture. Algal Material Two different types of algae were ordered from Carolina Biological Supply Company; cyanobacteria (blue green algae) and a diatom culture. Three 10mL tubes of Oscillatoria (cyanobacteria) and three tubes of Synedra (diatoms) were kept in the lab under fluorescent lighting for 2 days prior to inoculation. A hemocytometer was used to determine how many cells were contained in 1 mL for both Oscillatoria and Synedra in each tube. Once the number of cells were determined the highest cell count of Oscillatoria (54,000 cells) was used as the basis for volume added to each plant. The 3 treatments of Oscillatoria were as follows: 2 plants with 4.5 mL each, 2 plants with 7 1.5 mL each, and 2 plants with 1.0 mL each. The 3 treatments of Synedra were as follows: 2 plants with 0.4 mL each, 2 plants with 0.5 mL each, and 2 plants with 0.4 mL each. The plants were inoculated at the tip of the tallest leaf. All algal treatments were randomly assigned (Fig. 2). Figure 2. Algal Concentration Layout Experimental Condition and analysis The experimental units were held by a structured wooden cage (Fig. 3). In the cage there were 9 clear cylindrical fish bags measuring 60.96cm x 20.32cm. Each individual bag was securely fastened with staples to the four sides to ensure that the top stays open. The contents of each bag consisted of the organic material sediment composition, two American eelgrass plants, appropriate algal treatment, and spring water from Volusia Blue Spring to fill the remaining space (Fig. 4). After they were filled, the cages and bags rested on the ground. A thin layer of screen was placed between the top of the cage and the lighting fixture, with the intention of O 4.5 mL 4.5 mL C O 1.5 mL .5 mL C S 0.4 mL O 1.0 mL S 0.5 mL .5 mL C S 0.4 mL O: Oscillatoria O: Oscillatoria O: Oscillatoria O: Oscillatoria S: SynedraS: Synedra S: SynedraS: Synedra S: SynedraS: Synedra C: Control C: Control C: Control 8 disrupting light that could be perceived as too intense. Light was supplied by an aquarium T-5 high output fluorescent tube fixture which has an output measuring 67μmol m-2s-1. The light was set on a timer from 8 a.m. to 7 p.m. with the intention of replicating natural light conditions. The light remained constant among all treatments. They were housed in a classroom in Sage Hall at Stetson University so the temperature remained a constant 22°C. After about 39 days, the number of leaves, wet weight, root length, shoot length, and total length was measured again. In addition to the previously mentioned measurements, number of runners and runner weight were taken as well. These follow-up measurements will allow for a comparison of the growth which occurred during the time elapsed. To observe any algal growth on the leaves, a singular leaf was snipped at the base near the rosette and examined under an inverted microscope. Any free growing algae was also examined under the microscope. Experiments were conducted in the spring of 2016. A one-way ANOVA was used to test the significance of growth in the different concentration groups. 9 Figure 3. Cage designed by Robert Barber; used in light concentration studies Figure 4. Set up for V. americana algal growth study 10 3. Results: Contrary to what was expected, the algae which was introduced did not grow. Although there was algae present, it was of an unknown type. The structure of the unknown algae expressed substantial visual difference from both the Oscillatoria and Synedra (Fig. 5). A significant change in wet weight (p= 0.012) was expressed among the Synedra treatment. The number of leaves showed a statistically significant change among the control (p = 0.0015) and the Synedra treatment (p = 7.09*10-3). The most noteworthy change was observed in the root length; a statistically significant change was expressed among all three treatments: control (p = 0.014), Oscillatoria (p = 0.012), and Synedra (p = 0.004). As for shoot length, number of runners, and runner weight, no statistically significant change was expressed among any treatment. The growth which incurred among all treatments showed no bias towards the treatments without algae (Fig. 6). Figure 5. Microscope view at 35X of Oscillatoria, Synedra, and the unknown algae growth Oscillatoria Synedra Unknown 11 Figure 6. Effect algal growth on growth and asexual reproduction of Vallisneria americana. Bars represent average change from original condition and error bars represent ± SEM. An asterisk denotes any statistical significance. (A) Mean change in wet weight in grams. (B) Mean change in number of leaves. (C) Mean change in shoot length in cm. (D) Mean change in root length in cm. (E) Mean number of runners produced. (F) Mean weight of runners in grams. * * -1 0 1 2 3 4 5 6 ControlControlControlControl ControlControl Oscillatoria Oscillatoria OscillatoriaOscillatoria Synedra SynedraSynedra SynedraSynedra Average Change in Number Average Change in Number Average Change in Number Average Change in Number Average Change in Number Average Change in Number Average Change in Number Average Change in Number Average Change in Number Average Change in Number Average Change in Number Average Change in Number Average Change in Number Average Change in Number Average Change in Number Average Change in Number Average Change in Number of Leaves of Leaves of Leaves of Leaves of Leaves of Leaves of Leaves Algal TreatmentAlgal Treatment Algal Treatment Algal TreatmentAlgal TreatmentAlgal Treatment Algal Treatment Algal Treatment Algal Treatment * * * 3 4 5 6 7 8 9 10 ControlControlControlControl ControlControl Oscillatoria Oscillatoria OscillatoriaOscillatoria Synedra SynedraSynedra SynedraSynedra Average Change in Root Average Change in Root Average Change in Root Average Change in Root Average Change in Root Average Change in Root Average Change in Root Average Change in Root Average Change in Root Average Change in Root Average Change in Root Average Change in Root Average Change in Root Average Change in Root Average Change in Root Average Change in Root Length (cm)Length (cm)Length (cm)Length (cm)Length (cm)Length (cm)Length (cm)Length (cm) Algal TreatmentAlgal Treatment Algal Treatment Algal TreatmentAlgal TreatmentAlgal Treatment Algal Treatment Algal Treatment Algal Treatment 0.50.50.5 1 1.51.51.5 2 2.52.52.5 ControlControlControlControl ControlControl Oscillatoria Oscillatoria OscillatoriaOscillatoria Synedra SynedraSynedra SynedraSynedra Average Number of RunnersAverage Number of RunnersAverage Number of RunnersAverage Number of RunnersAverage Number of RunnersAverage Number of RunnersAverage Number of RunnersAverage Number of RunnersAverage Number of RunnersAverage Number of RunnersAverage Number of RunnersAverage Number of RunnersAverage Number of RunnersAverage Number of RunnersAverage Number of RunnersAverage Number of RunnersAverage Number of RunnersAverage Number of RunnersAverage Number of Runners Algal TreatmentAlgal Treatment Algal Treatment Algal TreatmentAlgal TreatmentAlgal Treatment Algal Treatment Algal Treatment 0.20.20.2 0.30.30.3 0.40.40.4 0.50.50.5 0.60.60.6 ControlControlControlControl ControlControl Oscillatoria Oscillatoria OscillatoriaOscillatoria Synedra SynedraSynedra SynedraSynedra Average Runner Weight (g)Average Runner Weight (g)Average Runner Weight (g)Average Runner Weight (g)Average Runner Weight (g)Average Runner Weight (g)Average Runner Weight (g)Average Runner Weight (g)Average Runner Weight (g)Average Runner Weight (g)Average Runner Weight (g)Average Runner Weight (g)Average Runner Weight (g)Average Runner Weight (g)Average Runner Weight (g)Average Runner Weight (g)Average Runner Weight (g)Average Runner Weight (g) Algal TreatmentAlgal Treatment Algal TreatmentAlgal Treatment Algal TreatmentAlgal TreatmentAlgal TreatmentAlgal TreatmentAlgal Treatment Algal TreatmentAlgal TreatmentAlgal Treatment -2 0 2 4 6 8 ControlControlControlControl ControlControl Oscillatoria Oscillatoria OscillatoriaOscillatoria Synedra SynedraSynedra SynedraSynedra Average Change in Shoot Average Change in Shoot Average Change in Shoot Average Change in Shoot Average Change in Shoot Average Change in Shoot Average Change in Shoot Average Change in Shoot Average Change in Shoot Average Change in Shoot Average Change in Shoot Average Change in Shoot Average Change in Shoot Average Change in Shoot Average Change in Shoot Average Change in Shoot Length (cm)Length (cm)Length (cm)Length (cm)Length (cm)Length (cm)Length (cm)Length (cm) Algal TreatmentAlgal Treatment Algal Treatment Algal TreatmentAlgal TreatmentAlgal Treatment Algal Treatment Algal Treatment Algal Treatment * 1.001.001.001.00 1.501.501.501.50 2.002.002.002.00 2.502.502.502.50 3.003.003.003.00 3.503.503.503.50 4.004.004.004.00 ControlControlControlControl ControlControl Oscillatoria Oscillatoria OscillatoriaOscillatoria Synedra SynedraSynedra SynedraSynedra Average Change in Weight Average Change in Weight Average Change in Weight Average Change in Weight Average Change in Weight Average Change in Weight Average Change in Weight Average Change in Weight Average Change in Weight Average Change in Weight Average Change in Weight Average Change in Weight Average Change in Weight Average Change in Weight Average Change in Weight Average Change in Weight (g)(g)(g) Algal TreatmentAlgal Treatment Algal Treatment Algal TreatmentAlgal TreatmentAlgal Treatment Algal Treatment Algal Treatment Algal Treatment A B C D E F 12 4. Discussion: In conclusion, the algae introduced to the individual Vallisneria americana plants did not express the growth that was expected. Although no credible conclusions about the effect algae has on the growth of Vallisneria americana can be made due to insufficient algal growth, it is important to continue on with this research. The relationship between algae and aquatic macrophytes is an important one to understand, especially as algae continues to grow in population. In Volusia Blue Spring, our proposed restoration site, a blue-green cyanobacteria (Lyngyba) has been observed in dense mats which smother vegetation (Hall 2010). With excessive algal growth, the light absorbed by V. americana would be limited. Carter et al. (1985 and 1996) determined SAV transplants with availability to higher light penetration are more likely to establish permanent populations. With the excessive algae observed in the St. Johns waterways, it is distressing that the reintroduction of V. americana could fail due to an overtaking of algae promoting excessive leaf coverage. Similarly, a previous study concluded that plants with high epiphytic algal loads and low light condition showed significant changes in total shoot length and number of new shoots (Asaeda et al. 2004). Expecting to see results comparable to the previously mentioned study, a lower light level was implemented. However, the “low light” level described by Asaeda et al.. (2004) was still higher than the light level used in this study. As for the inoculation process, it would be beneficial to revise the methods used. Either a greater volume of cells should be introduced at the beginning or repetitions of several smaller volumes could promote more significant growth which was lacking in this study. Furthermore, it would be beneficial to have a breakdown of not only the type of nutrients in the organic material but also the concentrations in which they occur. The macrophytes 13 exhibited positive visual growth among all treatments, while algae introduced was visually nonexistent. This difference in growth could be related to the abundance, or lack thereof, of nutrients in the organic material and the ability to be absorbed. Macrophytes commonly have a dense aggregation of roots which can explore the sediments and absorb nutrients (Chambers et al.. 1989). Alga, on the other hand, relies on nutrients suspended in the water column. Interestingly enough algae did form on the inside of all the bags, including the control. This suggests undetected algae was present in either the organic material or microscopic filaments remained in the water from time of collection. With this growth of unknown algae, it is not overtly clear why the introduced algae did not succeed. However, it is possible to speculate several factors revolving around nutrient concentrations could be responsible for the lack of growth. The macrophytes expressed root growth among all treatments which suggest there were sufficient nutrient levels present. However, macrophytes have the ability to navigate their root growth in order to absorb the proper nutrients. Algae, on the other hand, are immotile and require nutrients to be available in the water column. Another aspect worth considering is the source from where we received the alga cultures. The cultures were purchased from a biological supply company which grew them in a nutrient-rich medium. The change from such a nutrient dense medium to natural spring water could have caused the algal cells to die almost immediately. Overall, I would not deem this research a failure, more so a steppingstone for future studies. It is crucial to focus on the valuable information that can be collected with the objective of establishing healthy populations of American eelgrass. Vallisneria americana has several significant and useful benefits in an ecological system. It is important to be able to monitor the health of springs, or any productive body of water, for any contaminants. V. americana is a valuable natural indicator of contaminants and surrounding environmental conditions (Lovett et 14 al.. 1994). Different aspects of the plant will reveal almost a timeline of different issues. Lovett et al.. (1994) describes the success field managers have experienced with tracking and documenting the environmental quality in areas of concern in the Great Lakes. 15 Literature Cited Asaeda, T., M. Sultana, J. Manatunge, and T. Fujino. 2004. The effect of epiphytic algae on the growth and production of Potamogeton perfoliatus L. in two light conditions. Environmental and Experimental Botany 52:225-238. Boustany, R., T. 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