Community composi-on and carbon isotopes in testate amoebae: poten-al new tools for global climate change research Ryan P. Herbert ([email protected]), Travis Andrews, & Robert K. Booth Department of Earth and Environmental Sciences, Lehigh University Introduc-on Results and Discussion Understanding changes in the ability of natural ecosystems to sequester carbon in a changing climate is cri-cal to an-cipa-ng rates of climate change, determining when the cri-cal 2°C threshold of warming will be crossed, and informing energy policy and poli-cal decisions. Northern peatlands are an important component of the earth’s carbon cycle, sequestering nearly 550 Gt of carbon during the past 20,000 years (Yu et al. 2010). Although the higher evapora-on rates caused by an overall warming of the Earth would be expected to lead to increased decomposi-on in peatlands, recent studies have shown carbon accumula-on may have increased during past warm periods in some regions, possibly because primary produc-on increased more than decomposi-on (Charman et al. 2013). The increased rate of carbon accumula-on during past warm periods may have been due to decreased cloudiness, with more solar radia-on s-mula-ng primary produc-on. However, there are currently no reliable proxies for past cloudiness to test this hypothesis. One group of organisms that may be useful as a cloudiness proxy are testate amoebae, a group of protozoa that form shells which are preserved in peat sediments (Mitchell et al. 2008). These organisms are key microbial consumers in peatlands, feeding on a broad range of food sources including bacteria, algae, fungi, and ciliates. However, several common species are also mixotrophic, combining heterotrophy with autotrophy by using endosymbio-c zoochlorellae (Figure 1). Therefore, the intensity of light at the peatland surface may affect community composi-on and the δ13C isotopic signature of mixotrophic shells by influencing compe--ve outcomes and the rela-ve importance of metabolic pathways among mixotrophic and heterotrophic species. Figure 1: Four of the most abundant testate amoebae from our samples (length: 40μm-­‐200μm). From leo to right, they are as follows: Archerella flavum, Hyalosphenia papilio, Heleopera sphagni, and Hyalosphenia elegans. The three amoebae on the leo are mixotrophs, and H. elegans is a heterotroph. Note the green-­‐colored zoochlorellae, which are endosymbio-c algae that perform photosynthesis for the amoebae. Photos (from leo to right) sourced from: Microworld.com (hep://www.arcella.nl/Archerella-­‐flavum); Microworld.com (hep://www.arcella.nl/hyalosphenia-­‐papilio); Moor-­‐impressionen.at (hep://moor-­‐impressionen.at/Testaceen.htm); Microworld.com Community Change Considerable community changes were observed during the course of the experiment, par-cularly in the moderate-­‐shading and high-­‐shading plots (Figure 5). Interes-ngly, mixotrophic species increased in rela-ve abundance at the moderate-­‐shading and high-­‐shading sites (Figure 6). However, this increase was primarily due to a significant decrease in Hyalosphenia elegans at these shaded sites. Although H. elegans is a heterotroph, observa-ons on food vacuole content suggest that this species may be herbivorous, feeding on algae (R. Meisterfeld, personal communica-on). Mixotrophic species may have been impacted less than this herbivorous species by the shade reduc-on because these species were able to feed on a wider variety of food sources. Furthermore, addi-onal -me may be needed for popula-on dynamics to reach a stable state in the experimental treatments. We will analyze samples collected aoer a second growing season this year, and we predict that mixotrophic species may decline in the shaded plots as more generalist heterotrophs expand. Preliminary Isotopic Data At present, eight δ13C values for Hyalosphenia papilio and Heleopera sphagni have returned from the isotopic lab. Samples from Bog D show no effect from shading; however, the samples from Bog B show changes associated with shading (Figure 7). H. papilio shows a marked decrease in δ13C from the shaded treatment at Bog B, consistent with a shio toward more heterotrophy (Figure 2). Even so, H. sphagni, another mixotroph, has a more posi-ve δ13C value in the shaded plot at Bog B. The reasons for the differences in δ13C values at the two bogs, and the lack of any experimental effect at Bog D, are unclear. However, our samples likely integrate both living and dead testate amoebae over the past couple years, and this integra-on may decrease our ability to detect changes aoer a single growing season. More data are needed to evaluate the significance of δ13C changes. H. sphagni H. papilio H. sphagni Hypothesis: I conducted an experimental study to test two hypotheses: (hep://www.arcella.nl/hyalosphenia-­‐elegans) H. papilio 1)  Variability in light intensity at the peatland surface will lead to differences in community composi-on by altering compe--ve outcomes among heterotrophic and mixotrophic species. 2)  Lower light intensi-es cause mixotrophic testate amoebae to u-lize more heterotrophy, which should lead to more nega-ve δ13C levels (Jassey et al. 2011, Figure 2). Higher light intensi-es would cause mixotrophic testate amoebae to use photosynthesis as a greater por-on of their energe-c budget, leading to more posi-ve δ13C levels (Jassey et al. 2013). Heterotrophs Figure 5 (leo): The top graph in black illustrates the measured light reduc-on in our shade plots. Below are the changes in abundances of the most abundant testate amoeba species between our two sampling periods. Species-­‐specific abundances are shown for the three mixotrophs and the heterotroph illustrated in Figure 1. Other species without dedicated graphs are s-ll included in the “all mixotrophs” and “all heterotrophs” averages. Mixotrophs Figure 2: Previous measurements of δ13C ranges for heterotrophic and mixotrophic species under normal condi-ons (Jassey et al. 2013). The range of values for heterotrophic species is derived from from Euglypha compressa, Nebela 4ncta, Bullinularia indica, and Arcella vulgaris. The mixotrophic range of values is derived from Hyalosphenia papilio. We expect that, under moderate to high shading, mixotrophs will employ more heterotrophy to fulfill their energe-c needs, and this should shio their δ13C composi-on closer to that of pure heterotrophs. Acknowledgements Study Design and Methods Funding for this project came from an EI/STEPS Undergraduate Student Research Grant Fellowship and from a Lehigh University Faculty Research Grant to Robert Booth. I am very thankful to my project advisors, Robert Booth, Gray Bebout, and Stephen Peters, and to Travis Andrews for his contribu-ons. Nineteen 1.5mx1.5m plots were established in three peatlands of the Northern Highlands Lake District of northern Wisconsin in spring of 2014 (Figures 3, 4). All sites were Hloating Sphagnum peatlands; Hloating peatlands were speciHically chosen because they have a constant water table depth (relative to the surface) throughout the growing season (Ireland & Booth, 2011). Control plots (no shading) and plots utilizing different shade cloth weaves were established. Data loggers recorded light levels every 15 minutes in the middle of each plot, and the actual light reduction at each experimental plot was calculated relative to the controls. Testate amoeba samples were collected before the experimental shading (early May 2014) and after one season of growth (early October 2014). Samples were collected from each of four quadrants in a plot, and the community data collected were averaged for each plot. Standard techniques were employed to extract and analyze testate amoeba communities (Booth et al. 2010). Amoebae were prepared for isotope analysis through separations with nested sieves, and 350-­‐600 individuals of two mixotrophic species – Hyalosphenia papilio and Heleopera sphagni – were collected individually using a micropipette at two control sites and two high-­‐shade sites within two peatlands.
Bog B Bog C Bog D Woodruff, WI Figure 6 (above): Box plots showing mixotroph abundance changes for each treatment. Moderate shade and high shade (p<0.01, t-­‐test) caused significant increases in mixotroph abundance, although this may have been due to decreases in the abundance of H. elegans. Literature Cited Figure 3 (leo): Loca-on of our sampling sites in northern Wisconsin. In the leo map, the central loca-on is Crystal Lake, a site monitored by the Trout Lake Field Sta-on of UW-­‐
Madison. The bogs we inves-gated (B, C, and D) are all shown. Figure 4: Pictures of fieldwork from May of 2014. Booth, R. K., M. Lamentowicz, and D. J. Charman. 2010. Prepara-on and Analysis of Testate Amoebae in Peatland Palaeoenvironmental Studies. Mires and Peat 7, 1-­‐7. Charman, D. J., D. W. Beilman, M. Blaauw, R. K. Booth, S. Brewer, F. M. Chambers, J. A. Christen, A. Gallego-­‐Sala, S. P. Harrison, P. D. M. Hughes, S. T. Jackson, A. Korhola, D. Mauquoy, F. J. G. Mitchell, I. C. Pren-ce, M. Van Der Linden, F. De Vleeschouwer, Z. C. Yu, J. Alm, I. E. Bauer, Y. M. C. Corish, M. Garneau, V. Hohl, Y. Huang, E. Karofeld, G. Le Roux, J. Loisel, R. Moschen, J. E. Nichols, T. M. Nieminen, G. M. Macdonald, N. R. Phadtare, N. Rausch, Ü. Sillasoo, G. T. Swindles, E.-­‐S. Tuicla, L. Ukonmaanaho, M. Väliranta, S. Van Bellen, B. Van Geel, D. H. Vie, and Y. Zhao. 2013. Climate-­‐related Changes in Peatland Carbon Accumula-on during the Last Millennium. Biogeosciences 9, 14327-­‐14364. Ireland, Alex W., and Robert K. Booth. 2011. Hydroclima-c Variability Drives Episodic Expansion of a Floa-ng Peat Mat in a North American Keelehole Basin. Ecology 92, 11-­‐18. Jassey, Vincent E. J., Caroline Meyer, Chris-ne Dupuy, Nadine Bernard, Edward A. D. Mitchell, Marie-­‐Laure Toussaint, Marc Me-an, Auriel P. Chatelain, and Daniel Gilbert. 2013. To What Extent Do Food Preferences Explain the Trophic Posi-on of Heterotrophic and Mixotrophic Microbial Consumers in a Sphagnum Peatland? Microbial Ecology 66, 571-­‐80. Jassey, Vincent E.J., Satoshi Shimano, Chris-ne Dupuy, Marie-­‐Laure Toussaint, and Daniel Gilbert. 2011. Characterizing the Feeding Habits of the Testate Amoebae Hyalosphenia Papilio and Nebela Tincta along a Narrow "Fen-­‐Bog" Gradient Using Diges-ve Vacuole Content and 13C and 15N Isotopic Analyses. Pro4st 1-­‐14. Mitchell EAD, Charman DJ, Warner BG. 2008. Testate amoebae analysis in ecological and paleoecological studies of wetlands: past, present and future. Biodiversity and Conserva4on 17, 2115-­‐2137. Yu, Zicheng, Julie Loisel, Daniel P. Brosseau, David W. Beilman, and Stephanie J. Hunt. Global Peatland Dynamics since the Last Glacial Maximum. Geophysical Research Le<ers 37. Figure 7: δ13C values for two species of mixotrophic testate amoebae in control and experimental (moderate to high shade) condi-ons aoer one growing season of the experiment. Samples from Bog B are shown with solid lines, and samples from Bog D are shown with dashed lines; H. papilio samples are colored red, and H. sphagni samples are colored blue. The experimental samples from Bog D appear to show no significant change. However, the Bog B sample of H. papilio becomes more nega-ve in δ13C with shading, and the Bog B sample of H. sphagni becomes more posi-ve in δ13C with shading. Addi-onal data will allow us to evaluate sta-s-cal significance, and we will also analyze isotopic changes aoer a second growing season. Future Work A second set of samples will soon be sent out to the isotope lab for δ13C measurements. This set will also include a sample of the heterotroph Hyalosphenia elegans, which may support our predic-on that it is an herbivore. Another set of samples will be collected from our plots in October 2015 so that we can assess both community and isotopic changes aoer a second growing season. These data will provide sufficient δ13C measurements for an analysis of sta-s-cal significance. Our preliminary data indicate that light intensity likely influences the microbial environment on the peat surface in complex ways, and more data are needed to fully assess whether testate amoebae can serve as past cloudiness indicators in paleoecological studies of peatland carbon accumula-on. .
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