| Summary: | The purpose of this study was to examine how trophic interactions have changed over the last 500 years in response to weather events and human impacts. Past trophic levels of Twin Ponds in Brookfield, Vermont was determined by analyzing a short sediment core. The ephippia in this mesotrophic pond were analyzed for abundance, size, and pigmentation using image analysis. These changes in characteristics were compared to known land use changes such as deforestation and reforestation, recent changes in climate, weather events, and European settlement. The ephippia abundance also helped determine the past abundance of fish and phytoplankton due to them having an inverse relationship with ephippia. This research and analysis provides a better understanding of how Twin Ponds ecosystem has changed over time, and will allow for future predictions. The short core was analyzed to complete ongoing research of a Holocene core that was also collected from Twin Ponds at a similar depth. Winner of the 2021 Friends of the Kreitzberg Library Award for Outstanding Research in the Senior Science/Technical category. 1 Using Daphnia Ephippia Pigmentation and Size to Determine Past Trophic Levels of Twin Ponds, Vermont. Lauren Graham Department of Earth and Environmental Science Norwich University, Northfield VT Introduction Zooplankton are an indicator of an entire food chain within a freshwater ecosystem. Their abundance is controlled by the quantity of planktivorous fish, which in turn impacts phytoplankton abundance. The zooplankton, Daphnia, produces resting eggs, known as ephippia, that preserves in sediment and can be used to reconstruct the past environment of a freshwater ecosystems. The abundance, size, and pigmentation of ephippia have previously been correlated with the predation from planktivorous fish (Jeppsen et al., 2001, Tellier et al., 2016). Ephippia preserve through time in lake sediment, therefore, have the potential to record past abundance of several organisms within the aquatic food cycle. The purpose of this study was to examine how trophic interactions have changed over the last 500 years in response to weather events and human impacts. Past trophic levels of Twin Ponds in Brookfield, Vermont was determined by analyzing a short sediment core. The ephippia in this mesotrophic pond were analyzed for abundance, size, and pigmentation using image analysis. These changes in characteristics were compared to known land use changes such as deforestation and reforestation, recent changes in climate, weather events, and European settlement. The ephippia abundance also helped determine the past abundance of fish and phytoplankton due to them having an inverse relationship with ephippia. This research and analysis provides a better understanding of how Twin Pond’s ecosystem has changed over time, and will allow for future predictions. The short core was analyzed to complete ongoing research of a Holocene core that was also collected from Twin Ponds at a similar depth. Previous Work The use of ephippia to understand past changes in fish abundance has been conducted in other northern regions, including, Canada and Denmark (Jeppsen et al., 2001, Tellier et al., 2 20160). In Canadian boreal lakes, research on ephippia pigmentation was used to determine exactly when brook trout had gone extinct (Tellier et al., 2016). The researchers identified a change from light to dark ephippia that reflected the extinction of brook trout. This work was done by image manipulation software that showed the darkest pigmentation and largest size of ephippia were found in lakes without brook trout. Similarly, research on ephippia preserved in Danish lakes by Jeppesen et al., (2001), shows that ephippia size is negatively correlated with the abundance of planktivorous fish. Past declines in planktivorous fish were thought to reflect a decline in nutrient food source or removal due to fishing. Both of these studies suggest the importance of top-down (fish-driven) changes in the food chain Previous research on Twin Ponds productivity (Grigg and Magdon, 2019; Graham and Grigg 2020) shows that during the past 3000 years, the abundance of ephippia is negatively correlated with the abundance of diatoms, a diverse group of phytoplankton. This study suggests that Daphnia have been effective grazers of phytoplankton over centennial-time scales and hypothesizes that periodic declines in Daphnia and increased phytoplankton were caused by a reduction in fish predation. This research suggests a long-term relationship between ephippia, phytoplankton, and fish that is ultimately driven by abiotic changes in the pond. Grigg and Magdon’s research did not analyze the last 100 years and therefore were analyzed in this study. It is important to evaluate modern sediment to understand how an environment has responded to recent changes. Human interaction with the ecosystem as well as major weather events must be considered when analyzing past changes. Humans have caused deforestation and fishing pressure (Foster, 1992). An increase in fishing due to settlement may have caused a decrease in piscivorous fish and an increase in planktivorous fish, which may be reflected by a decrease in the abundance, pigmentation, and size of ephippia. Deforestation in the late 1800’s produced a greater amount of surface water runoff and erosion and may have increased the input of nutrients in the lake. The more recent reforestation of the watershed would have reversed this trend, however, an increase in annual precipitation over the past 40 years (Beckage et al., 2008) in Vermont must also be considered. Other major historic events, such as, the hurricane of 1938 caused for millions of fallen trees and heavily impacted New England forests and watersheds (Spurr, 1956). 3 Physical Setting The location of research was Twin Ponds in Brookfield, Vermont (Fig. 1). Twin Ponds is located within the CaCO3-rich Waits River formation (Ratcliffe et al., 2011). Twin Ponds drains into a small stream located on the south side of the east pond which is a tributary of the Second Branch of White River. The short core was taken from the deepest point of the west pond from a depth of about 7.5m. The pond is mesotrophic and immediately surrounded by a fringing wetland and by northern hardwood rich-forest (Thompson et al., 2019). There are also two small waterfront areas sued by property owners. The shallow (0-3m) part of the pond is a littoral bench and is dominated by the macrophage, Chara. The deeper part of the pond is plankton dominated. The drainage area is 0.68 square miles based on the US Geological Survey’s stream stats (https://streamstats.usgs.gov/ss/, Dec 2020). Figure 1: Image of Twin Ponds in Brookfield VT on the left. Yellow star indicates where the short core was collected. Image on right is a location map of Vermont. Methods This research was conducted by collecting a short sediment core from Twin Ponds in Brookfield, Vermont. A universal gravity corer was used to collect the sediment. The core was sampled at 1cm intervals by using an instrumental core extruder. The samples were collected in labeled Whirl-Pak bags. A total of 35cm was collected. 4 In the lab, samples were analyzed in 5cm intervals. Additional samples were studied as needed. 5mL of the wet sediment samples were wet sieved at 150 microns. The organic matter left in the sieve was transported into a petri dish using deionized water. The petri dish samples were analyzed under a dissecting microscope for ephippia (magnification 10-80X). The total count of ephippia for each sample was recorded. A microscope camera was used to take black and white images of 20 ephippia per sample. In samples with more than 20 ephippia, ephippia were randomly selected for image analysis. In samples with less than 20 ephippia, additional sediment was processed to reach this number but the extra ephippia were not included in the counts. The ephippia images were examined using ImageJ, an image processing program, for pigmentation and size. The area was calculated in ????????² by drawing a line around the perimeter with the polygon tool. The length of an ephippia was calculated in ????????² by using the line tool to draw a line along the ephippium’s longest side. The pigmentation was measured by calculating the color within the ephippia based on thresholds set from the digital light intensity scale 0-225 (Tellier et al., 2016). Pigmentation was determined by measuring the area of pixels less than 75 and less than 125. The pigment threshold for 75 and 125 were divided by the calculated average area for that ephippia and multiplied by 100 to create a percent pigment threshold area. Results Sediment from the short core showed a similar concentration (>20/5mL) of ephippia throughout the entire core. The highest abundance is seen between 19-20 cm and the lowest is 0.00 20.00 40.00 60.00 80.00 100.00 0.0 5.0 10.0 15.0 20.0 25.0 30.0 75% Threshold Area Depth (cm) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.0 5.0 10.0 15.0 20.0 25.0 30.0 Average Area (mm2) Depth (cm) Figure 2: Calculated 75% threshold ephippia area versus core depth. Blue dots are the 20 measured ephippia per sample. The orange line is the average area per sample. Figure 3: Calculated average ephippia area versus core depth. Blue dots are the 20 measured ephippia per sample. The orange line is the average area per sample. 5 seen from 13-14cm (Fig. 5). Pigmentation was measured by the average percent area greater than 75 digital light value and is greatest at 11cm and drastically decreases at 12cm and then rises again at 13cm (Fig. 2). At depths less than 13.5cm, there is a small increase in pigmentation and then a gradual decrease. Average area of ephippia rarely changed from 1-30 cm (Fig 3). There were slight increases and decreases throughout the short core, but there was little overall change in average area. Highest average area was 0.337 mm² and the lowest was 0.28mm². Percent pigmentation and area do not show expected correlation. In Figure 4, this correlation is shown in three segments. The oldest ephippia from 21-30cm show less size variability and are lighter. An increase in size variability showing larger and darker ephippia and dark and small ephippia is seen at depths of 0-20cm. The short core was also analyzed for biogenic silica abundance (Fig. 5). The oldest part of the short core shows an inverse relationship between biogenic silica and ephippia abundance which indicates low nutrients. From 30-20cm, ephippia abundance gradually increases and biogenic silica abundance decreases. From 19.5-13.5cm, ephippia abundance decreases while biogenic silica abundance increases. 12.5-9.5cm shows an increase in abundance between 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.00 20.00 40.00 60.00 80.00 100.00 Area (mm2) Percent Pigment Area Figure 4: Correlation between percent pigment area and average area. Blue dots indicate averages between depths from 0-10cm. Orange dots indicate averages between depths from depth 0f 11-20cm. Gray dots indicate averages between depths from depths of 21-30cm. Figure 5: Averages of ephippia abundance, biogenic silica abundance, and percent area pigmentation versus depth. Arrows show interpretations of high or low nutrients 0 1 2 3 4 5 6 7 0 10 20 30 40 50 60 70 1800 1850 1900 1950 2000 2050 % Biogenic Silca >75% Pigmented Area and #ephippia Year Ephippia Abundance 75% Pigmented Area Biogenic Silica Abundance6 both ephippia and biogenic silica and indicates high nutrients. At the depth of 12.5cm was when the lightest pigmented ephippia occur and then followed by the highest calculated pigmented area at 11.5cm. After 9.5cm, biogenic silica abundance decreases and ephippia abundance generally increases and indicates low nutrients. The analysis from the short core was combined with the analysis from the Holocene core. Figure 6 shows that the ephippia abundance from the short core is relatively low compared to the past and follows the general decrease of abundance seen at the top of the Holocene core. Figure 7 shows that the % area pigmentation <75 from the short core was generally high. The short core followed a general increase in pigmentation at the top of the Holocene. Ephippia area was low compared to the Holocene (Fig. 8). Discussion Abundance 0 50 100 150 200 250 300 350 0 100 200 300 400 500 Ephippia Abundance Depth (cm) Figure 6: Ephippia abundance versus depth of short core in orange and Holocene core in blue. 0.000 10.000 20.000 30.000 40.000 50.000 60.000 70.000 0 100 200 300 400 500 Average 75% Area Pigmentation Depth (cm) Figure 7: 75% pigmented ephippia area versus depth of short core in orange and Holocene core in blue. 0.000 0.200 0.400 0.600 0.800 1.000 0 100 200 300 400 500 Average Area (mm2) Depth (cm) Figure 8: Average ephippia area versus depth of short core in orange and Holocene core in blue. 7 Biogenic silica analysis for the short core and the Holocene core were dated and aligned (Chang, 2021) and provided an opportunity to examine the relationship between Daphnia and its primary food source, phytoplankton. During the Holocene, it was shown by the data from Grigg and Magdon (2019) that the relationship between ephippia and biogenic silica was inverse with the exception of some time periods. This inverse relationship between biogenic silica and ephippia represents a predator prey cycle between Daphnia and diatoms (a type of phytoplankton). In the shore core, this inverse relationship was apparent between 1500-1830 years (Fig. 9). The increase in biogenic silica abundance after 1750 followed by an increase in ephippia abundance at 1800 years coincides with the increase in deforestation and farming by 1780 as suggested by pollen data (Grigg et al., 2020). Deforestation would have increased surface erosion and provided an influx of nutrients entering the pond and increased the abundance of phytoplankton and zooplankton. This explanation is supported by concurrent increases in sediment accumulation rates (Grigg and Magdon, 2019). The decrease in both biogenic silica and ephippia during the early to mid-1800’s is due to a decline in rates of deforestation and the Figure 9: Historic comparison of ephippia abundance to biogenic silica abundance. Comparison to ephippia pigmentation and average ephippia area. All comparisons are graphed versus age in years. The darkest colors represent data from the Holocene core and the lighter colors are data from the short core. 8 start of farmland abandonment in 1850 (Foster 1992). Farms were no longer being used and runoff of nutrients would have been less likely to occur. During this recovery phase there is a small increase in ephippia, suggesting that Daphnia likely sustained a low abundance of diatoms. From 1930 to 1950, both ephippia and biogenic silica abundance increase with the biogenic silica showing a larger increase overall. From 1950 to present, ephippia abundance is stabilized at low levels and biogenic silica declines. The New England hurricane of 1938 is the likely cause for the increase in both ephippia and biogenic silica abundance. During this hurricane, 3 billion feet of tree were blown down in New England (Spurr, 1956). This amount of tree loss caused for an increase of fallen trees and storage of the logs on the ponds, including Twin Ponds (Lew Stowell, personal communication). The increase in downed trees would cause more erosion and surface runoff, while the storage of logs on Twin Ponds resulted in an increase in organic carbon. Both of these events would cause for an influx of nutrients and an increase productivity. Following the impacts of the 1938 hurricane, Daphnia abundance remained low which may reflect increasing temperatures particularly during the 1990’s which was the hottest decade in history (Beckage et al., 2008). An increase in air and water temperatures has been shown to cause a decrease in Daphnia abundance due to the mismatch of predator and prey cycles (Winder and Schindler, 2004). Declining Daphnia abundance may also have been due to climate driven changes in nutrients (Wojtal-Frankiewicz, 2011). Warmer water temperatures cause for a decrease in mixing and less nutrient availability and will decrease the food for phytoplankton and then for Daphnia. Another factor that may decrease Daphnia abundance is the quality of phytoplankton as a food source for Daphnia. Previous studies have shown that an increase in the temperature of a lake or pond can cause these bottom-up changes to the food chain (Fischer et al., 2011). A decrease in edible phytoplankton lead to less Daphnia. Pigmentation and Size The pigmentation and size of the short core did not indicate fish predation of Daphnia as expected from previous work completed on Twin Ponds (Grigg and Magdon, 2019). This is determined by the lack of correlation between ephippia size and pigmentation (Fig. 9). Previous 9 work suggests that in the presence of fish predation, there would be small and light ephippia and in the presence of little to no fish predation, large and dark ephippia (Jeppsen et al., 2001, Tellier et al., 2016). However, this study shows that during the historic times, ephippia were small and dark as well as large and dark. The small average ephippia size indicates fish presence, but the darker color indicates a lack of predation and is not a clear signal of fish predation. These results suggest that another factor other than fish predation is impacting the Daphnia population at Twin Ponds. Research has suggested that predation is not the only factor for the characteristic change of ephippia food (Wojtal-Frankiewicz 2011 & Nevevalinen et al., 2016). The change in the ephippia size and pigmentation may have been caused by a change in the predator prey trends due to a decrease in edible phytoplankton (Wojtal-Frankiewicz, 2011). Currently, Twin Ponds contains low levels of chlorophyll A and Daphnia (DeNault, 2020). Low levels of chlorophyll A means less food for the rest of the food chain. This low level of phytoplankton, zooplankton, and fish may explain the currents trends in size and pigmentation of the ephippia. The small size of ephippia may be from predation pressures. However, it is likely that the fish have another food source such as copepods, which are better adapted to changes in the food chain (DeNault, 2020) Daphnia prefer to produce dark ephippia when not under pressure because it increases durability (Mellors, 1975). Another factor that causes a decline in size of ephippia is the increase in relative temperature and inedible algae (Wojtal-Frankiewicz, 2011). This may contribute to why ephippia are the smallest during the present day (Fig. 8). Conclusions The short core shows that Daphnia ephippia pigmentation may no longer be tied to fish predation. In comparison with the Holocene core, the most recent ephippia are the darkest of the record but do not show expected correlation with size and abundance that would suggest a response to fish predation. The comparison between the changes with the Holocene and the short core shows how the ecosystem has changed. Analyzing ephippium changes allowed for an understanding of how land use changes impact productivity. Deforestation caused an increase in nutrients and then an increase in productivity. Land use and other human impacts have shifted Daphnia’s ability to recover fully. The impacts have also caused for a shift to bottom-up control of Daphnia. 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