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1 Species Diversity of Aquatic Invertebrates in St. Olaf Ponds Heather Bouma-Johnston, Cassie Davis, Ambele Mwamelo Abstract We conducted this experiment to study the aquatic invertebrate diversity in three St. Olaf College ponds: Regents Pond, Big Pond, and Baseball Pond. Aquatic biodiversity is important to ecology because it is essential to both the health of our environment and to human life. We collected invertebrate samples at each of the ponds, documented the the number and abundance of the species found, and used this data to calculate the species diversity of the ponds with Shannon’s and Simpson’s Diversity Indices. Our results indicated that larger ponds (Baseball and Big Ponds) had more diversity than smaller ponds (Regents Pond) and that ponds with more shoreline development (Regents Pond) had less biodiversity than ponds with more natural surroundings (Baseball and Big Pond). We also found a negative correlation between temperature and species diversity, with Regents Pond (the warmest pond) having the least species diversity. Overall, we found that there are three main factors that influence diversity in St. Olaf ponds-- temperature, pond size, and development around the pond. Introduction Aquatic biodiversity affects both human life and the health of the environment, making it a valuable area of study. We depend on many aquatic plants and animals, and their ecological functions, for our survival. For example, plants such as wild rice, wasabi, watercress, taro, and lotus, all only grow in freshwater lakes and streams and are important for some human diets (Edible Water Plants: Aquatic Vegetables, 2008). Furthermore, many organisms (such as wood frogs and salamanders) have important life stages in ponds, but also interact with other ecosystems; thus, the loss of pond species diversity could have negative impacts on many other habitats, as well (Yeager and Brittingham 2014). Understanding what factors influence pond biodiversity can help with conservation measures and protecting aquatic ecosystems for the future. 2 Warmer aquatic temperatures are often associated with a decrease in species diversity. In streams in Malaysia, studies show that water temperature is negatively correlated with two species of shredders (Salmah et al. 2014). Similarly, studies in Oman attributed low faunal diversity in streams to high water temperature (Boulaaba et al. 2014). Possible explanations for a decrease in diversity with higher water temperatures include a corresponding decrease of oxygen in the water (Suski et al. 2006, Horne 1971), a decrease in egg hatching and breeding activity due to lost temperature cues (Boulaaba et al. 2014, Horne 1971), and inhibition of some organisms’ metabolic functions (Suski et al. 2006). Thus, we predicted that the St. Olaf pond with warmest water temperature will have lowest species diversity. Larger areas provide a more extensive habitat, with more resources, and thus are often associated with increased species diversity. While there is not complete consensus that this is true for all aquatic ecosystems (Oertli et al. 2002), there are studies showing a positive correlation between pond size and species diversity for many populations in ponds, including dragonflies (Kadoya et al. 2004), snails (Brönmark 1985), and diving beetles (Lundkvist et al. 2001). Thus we predicted that the larger St. Olaf ponds (Baseball Pond and Big Pond) would have higher species diversity than Regents pond, which is very small. Shoreline development has been shown to have a negative impact on many aquatic communities. In comparisons between forested and agricultural ponds (surrounded by natural vegetation) and urban ponds, studies show that urban ponds have significantly lower biodiversity. In this study, the most ideal landscape, with the highest biodiversity, was a mosaic of forest and open habitats surrounding wetlands (Gagne and Fahrig 2007). Vegetation surrounding ponds actually plays an important buffering role, impeding direct runoff of nutrients into the water. This is increasingly important in areas where nutrient flow includes toxic 3 substances, such as pollutants dioxin, polychlorinated biphenyls, and road de-icing salts (Trombulak and Frissell 2000). Influx of these nutrients and toxins can suppress growth and cause mortality. In one study, tadpoles exposed to road de-icing salts experienced significantly lower survivorship, decreased time to metamorphosis, reduced weight and activity, and increased physical abnormalities with increasing salt concentration (Sanzo and Hecnar 2006). Since Regents Pond is urbanized, with shoreline development of roads and parking lots, we predicted that it would have the lowest species diversity. Baseball Pond and Big Pond, however, are located in a more natural setting. Big Pond in particular was in the ‘ideal’ setting of forest surrounded by wetlands (described above), and should have a correspondingly higher species diversity. In our experiment, we determined which St. Olaf ponds had the highest aquatic invertebrate species diversity: Regents Pond, Big Pond, or Baseball Pond. We hypothesized that temperature, pond area, and shoreline development would have the largest effects on pond biodiversity, and that ponds with a colder temperature, a bigger area, and less shoreline development would contain the highest species diversity. Methods In order to determine the environmental characteristics of Regents Pond, Baseball Pond, and Big Pond, we measured the turbidity of the water (with a Secchi Tube), the water temperature, and the depth of the shore. Furthermore, we observed the surroundings of the ponds and the vegetation that grew in and around them. Using a Dnet, we collected 18 samples from each pond, with the goal of studying different types of invertebrates and their populations in varied habitats. We placed the collected samples into buckets that were 1/5 filled with the respective ponds’ water and brought them to the lab for analysis. In order to view the samples 4 more clearly, it was important to separate the water samples into smaller containers. Then we collected organisms of interest and identified them under a microscope. Once identified, we estimated the population of each invertebrate by counting different invertebrates in small containers and extrapolating the total population in the whole sample from that small sample. We used Anova One-way statistical tests to analyze the difference in the species richness between the three ponds. We then used a pairwise comparison of means to find which specific ponds had significant differences in species richness. We also used Anova One-way statistical tests to analyze differences in the species diversity (as determined by both Simpson’s and Shannon’s Diversity Index) between the three ponds. We then used a pairwise comparison of means to find which specific ponds had significant differences in species diversity. Finally, we calculated linear regression to analyze potential correlation between depth and temperature and species diversity (Shannon’s and Simpson’s diversity index) for the three ponds. We used R commander to run all statistical analyses. Results There is a significant difference in the species richness between Regents Pond, Big Pond, and Baseball Pond (Oneway- ANOVA, p=.0268). There is a significant difference in species richness between Big Pond and Regents Pond (Pairwise comparison of means, p=0.0237). There is not a significant difference in species richness between Big Pond and Baseball Pond (Pairwise comparison of means, p=0.6726) or Regents Pond and Baseball Pond (Pairwise comparison of means, p=0.1077). Big Pond had the highest species richness, followed by Baseball Pond and then Regents Pond with the lowest species diversity (Fig. 1). There is a significant difference in species diversity between the three ponds, measured by both Shannon’s diversity index (OnewayANOVA, p= 0.00252; fig. 2) and Simpson’s 5 diversity index (OnewayANOVA, p=0.00192; fig. 3). With indexes, Baseball pond and Big pond had high species diversity, while Regents pond had much lower species diversity. In both Shannon’s and Simpson’s species diversity index, there was a significant difference between Big Pond and Regents Pond (Pairwise comparison of means, Shannon p=.00539, Simpson p=0.00254) and Regents Pond and Baseball Pond (Pairwise comparison of means, Shannon p=0.00442, Simpson p=0.00654). There was not a significant difference in species richness between Big Pond and Baseball Pond for either index (Pairwise comparison of means, Shannon p=0.99345, Simpson p=0.86554). There is no correlation between the depths of all the ponds and species diversity according to their Shannon diversity index values (Linear Regression, p=0.22473). There is also no correlation between the depths of all the ponds and species diversity according to the Simpson diversity index values, either (Linear Regression, p=0.2763). There is a correlation between the temperature of all the ponds and species diversity according to both the Shannon diversity index (Linear Regression, p=0.0312; Fig. 4) and the Simpson diversity index (Linear regression, p=0.0312; Fig. 5). In both cases, an increase in temperature is correlated with a decrease in species diversity. This is illustrated by a negative correlation between temperature and diversity index in Shannon’s index (Fig. 4) and a positive correlation between temperature and diversity index in Simpson’s index (Fig. 5) Figures 6 Fig. 1: Plot of Average Species Richness vs. Pond Site. There is a significant difference in species richness between the three ponds (Oneway-ANOVA, p=.0268). Big Pond had the highest species richness, followed by Baseball Pond and then Regents Pond with the lowest species diversity. Error bars represent standard error of the means. Fig. 2: Plot of Species Diversity (Shannon’s Index) vs. Pond Site. There is a significant difference in species diversity between the three ponds in relation to the Shannon’s diversity index (Oneway-ANOVA, p=0.00252). The Baseball Pond had the most species diversity very closely followed by the Big Pond and then Regents Pond which dips down to have the least amount of species diversity. Error bars represent standard error of the means. 7 Fig. 3: Plot of Species Diversity (Simpson’s Index) vs. Pond Site. There is a significant difference in species diversity between the three ponds according to the Simpson’s diversity index (Oneway-ANOVA, p=0.00192). According to this index, Regent’s Pond has the least amount of diversity, while Baseball Pond and Big Pond have the most species diversity. Error bars represent standard error of the means. Fig. 4. Linear Regression of Shannon Diversity Index vs. Temperature. There is a significant relationship between the ponds’ temperatures and their Shannon diversity indexes (Linear Regression, p=0.0312). The green line indicates the line of best fit for the correlation. There is a negative correlation, showing that with an increase of temperature, there is a decrease in diversity in all three ponds. Fig. 5 Linear Regression of Simpson’s Diversity Index vs. Temperature. There is a significant relationship between the species diversity (as measured by the Simpson diversity index) and temperature (Linear Regression, p=0.0312). The green 8 line indicates the line of best fit for the correlation. There is a negative correlation, indicating that with an increase in temperature, there is less diversity. Fig. 6. St. Olaf College Natural Lands Map. The three ponds used in our study include Regents Pond, Baseball Pond, and Big Pond. This map indicates their location, surrounding environment, and size. Discussion We found that three main factors affect the aquatic invertebrate diversity in St. Olaf College ponds: temperature, size of the pond, and the amount of development around the pond. Our results supported our hypothesis on all accounts: increased pond area, decreased development, and colder temperatures lead to an increase in aquatic invertebrate diversity. 9 In terms of shoreline development, we expected the diversity to be the highest in Baseball Pond and Big Pond because of their locations in the Natural Lands surrounded by plant life. Regents Pond, because of its location in a parking lot and its composition of mainly runoff, we expected to have the lowest species diversity. This hypothesis was supported, because our results indicate significantly higher species diversity in Baseball Pond and Big Pond than Regents Pond. These results are consistent with prior research indicating that urbanized ponds with shoreline development have significantly lower species diversity (Gagne and Fahrig 2007). It is possible that Regents Pond’s loss of an essential ‘vegetation buffer,’ which controls the amount of nutrient influx in runoff, contributes to an increase in nutrients, including harmful chemical pollutants such as dioxin, polychlorinated biphenyls, and road de-icing salts (Trombulak and Frissell 2000). Studies show that an increase in such pollutants can lead to a decreased survivorship of some species (Sanzo and Hecnar 2006), and thus a decrease in species diversity. We expected to find a greater species diversity in Baseball Pond and Big Pond, the larger ponds, than Regents Pond, the smaller pond. A larger pond is a bigger habitat for plant and animal life, and has a greater availability of resources, which increases habitat diversity. This is consistent with our results and with most of the previous research on this topic. Some of the pond populations which show similar results to our own –more species diversity in a larger pond area—are dragonflies (Kadoya et al. 2004), diving beetles (Lundkvist et al. 2001), and snails (Brönmark 1985). As for temperature, we expected the pond with the highest temperature to have the lowest species diversity. Our results confirm that there is indeed a negative correlation between higher 10 water temperature and species diversity, with Regents Pond having the highest temperature and lowest species diversity. This is consistent with other studies, as well, where higher stream temperatures in Malaysia (Salmah et al. 2014) and Oman (Boulaaba et al. 2014) resulted in lower aquatic species diversity. There are a range of reasons for why higher aquatic temperature results in lower species diversity. Warm water holds less oxygen and can lead to hypoxic conditions that make it difficult for species to survive. In a study completed on fairy shrimp and phyllopods, Horne (1971) attributed seasonal variation in shrimp diversity to changes in temperature and corresponding hypoxic conditions which limit survival. Another explanation is a decrease in breeding activity of adults (Boulaaba et al. 2014) as well as a decrease in egg hatching (Horne 1971) due to changes in temperature cues, both of which contribute to decreased population growth rates. Finally, higher temperatures can inhibit metabolic functions such as the replenishment of white muscle energy stores in largemouth bass as well as impede physiological functions by elevating plasma cortisol concentrations (Suski et al. 2006), all of which decrease survival. Aquatic biodiversity is essential not only to our environment but also to humans’ everyday lives. 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