Elizabeth Schultheis, Fall 2009 The role of plant-­soil feedbacks in the biological invasion of Acer platanoides, and implications for native forests. Biological invasions are a major concern for scientists and land managers because they can have a wide array of impacts on natural areas, including reducing native biodiversity (Vitousek et al. 1997) and altering ecosystem functions such as hydrology and nutrient dynamics (Vitousek 1990). For this reason, there is great motivation to understand the invasion process and the factors contributing to the success of invasives. The Escape From Enemies Hypothesis states that release from enemies (pathogens, predators, and competitors) in their native range may be a large factor driving the invasiveness of certain plants in their exotic range (Wolfe 2002). This hypothesis stems from the Janzen-­‐Connell hypothesis (Janzen 1970, Connell 1971), which predicts negative density dependence effects caused by the accumulation of pathogens and predators surrounding parental trees. For invasive plant species, escape from enemies results in lower pathogen and predator loads, leading to higher densities in exotic ranges. The dislocation from coevolved relationships experienced by a novel invader could explain how invasive species are seemingly able to overcome usual population controls such as density dependence and competition tradeoffs (Hallett 2006). In this context, my research will focus on how enemy escape contributes to the invasive nature of Acer platanoides in Michigan, and answer the following questions: (1) Is initial enemy escape experienced by an invasive species lessened over time as native pathogens and predators evolve to exploit the novel resource? (2) How does enemy escape alter competition between an invasive species and other members of the invaded community? The role of plant-­soil feedbacks in the invasion process: While above ground community members may play a key role during invasion, attention is being drawn to below ground pathogens, predators, and mutualists, which have the potential to alter the performance of different plant species (Klironomos 2002). Plant soil feedbacks (PSFs) occur when plants interact with the microbial community through root exudates, root chemistry, and susceptibility to predators and mutualists (Kulmatiski et al. 2008). These processes alter microbial composition, which in turn feeds back to influence plant growth and abundance (Klironomos 2002). Positive feedbacks result when plants receive relatively more benefit from soil mutualists than enemies, and negative feedbacks result when the opposite is true (Bever et al. 1997). Through feedbacks, microbial community composition has the potential to regulate plant diversity and community composition (van der Heijden et al. 2008). Mutualists tend to be generalists with more cosmopolitan ranges, and therefore while introduced species may escape specialist predators and pathogens, they will still be facilitated by mutualisms in their new range (Richardson et al. 2000). Previous research has shown enemy escape and exploitation of native mutualisms may play a key role in the invasion process, yet few have tracked how the relationship of an invasive species and its invaded community changes over time. Fewer still have looked at the below ground microbial community and how soil enemies and mutualists affect invasive species growth and competition with native species. With current molecular tools, it is now possible to determine the mechanism behind feedbacks between soil communities to the plants they interact with. Study System Acer platanoides (Norway Maple) was introduced into the United States from Europe in 1756 (Nowak and Rowntree 1990) and has since invaded intact forests across the US, lowering understory diversity and inhibiting native tree species regeneration (Reinhart et al. 2005). A. platanoides has been found to have higher survival under lowlight conditions and outcompete native tree species for key resources (Morrison and Mauck 2007, Kloeppel and Abrams 1995). Enemy escape is thought to play a large role in its invasion, possibly explaining how it is able to apparently overcome many of the tradeoffs that plague native species. In support of this hypothesis, a cross continental study found that A. platanoides experienced three times less leaf herbivory in the invaded range compared to native populations (Adams et al. 2009), and Reinhart and Callaway (2004) found that density of A. platanoides was higher in its invasive range, which may be due to soil biota facilitating invasion in North America through positive plant soil feedbacks. However, other work has found that herbivory and foliar disease was similar in A. platanoides and the native Acer saccharum, and concluded that success of this species was not due to enemy escape. Overview of Methods: To investigate how enemy escape and PSF influence the biological invasion of A. platanoides, I will measure the strength of PSFs across forests that differ in age of invasion in a series of greenhouse studies. Additional competition experiments between the exotic A. platanoides and it’s most common native competitor, A. saccharum, will be used to identify how these factors influence effects on native community members. Is initial enemy escape experienced by an invasive species lessened over time as native pathogens and predators evolve to exploit the novel resource? Understanding how invasive species acquire enemies over the course of their invasion will help to better predict the long-­‐term effects of these invasions (Mitchell et al. 2006). Previous studies have not shown clear patterns of enemy acquisition over time; results may have been confounded by factors such as range size and phylogenetic effects (citations in Mitchell et al. 2006), and many experiments only focused on above ground predation, like herbivory (ex. Carpenter and Cappuccino 2005). Below ground predators and pathogens are recognized as key drivers of plant diversity and productivity (van der Heijden et al. 2008) and should be included in studies addressing the Escape From Enemies Hypothesis. I predict that over the course of an invasion, the initial escape from below ground enemies experienced by A. platanoides should erode as native soil pathogens and predators shift from native hosts to exploit the new resource. I will compare PSFs across a chronosequence of forests that have been invaded by A. platanoides for different periods of time (old > 50 years, mid = 20-­‐50 years, young < 20 years). Within a controlled greenhouse study I will be able to isolate the effects of the microbial community on seedling growth and see how it changes over the course of an invasion. I have selected 6 forests with varying ages of invasion (this includes Kleinstuck, KK in Figure 1), and sampled 10 trees of both Acer species in each. I took diameter at breast height (DBH) measurements as an approximation of tree age, and used the largest DBH to represent my initial time of invasion for the forest. Because density may be correlated with age of invasion, I estimated density by averaging the distance to the three nearest conspecific trees over 2m in height (preliminary data, Figure 1). From each tree I collected three soil cores 10cm deep, and used these samples to inoculate pots in a greenhouse study. Soil samples from each tree were homogenized and sifted to remove large particles. Each homogenized sample was used to inoculate 1 A. platanoides and 1 A. saccharum seedling. Seedlings are currently growing for three months in the Kellogg Biological Station greenhouses in Hickory Corners, MI. After three months, I will take height measurements, harvest above and below ground biomass, dry, and weigh leaves, shoots, and roots. Figure 1: Preliminary data collected Summer 2009. DBH measurements collected for each forest represent mean age for A. platanoides (ACPL) and A. saccharum (ACSA). Highest DBH values for ACPL in each forest will be used to estimate age of invasion. Density measurements for forests will be included as a covariate in analysis. Forests with oldest invasion dates tend to have highest density levels. Kleinstuck is forest KK in figures and represents a mid-­‐invasion time; other forests include MSU landholdings (BW & SN) and parks in Ann Arbor, Michigan (AR, BH & KL). Data analysis: By analyzing seedling growth as a function of canopy species identity, tree age, and the species by age interaction, I will be able to determine how soil enemy acquisition occurs over the course of an invasion. I predict that PSF will change along temporal scales, with PSF dropping from positive to negative over the time span covered in my study. All data will be analyzed with ANCOVA where seedling height and weight are included as response variables and forest, canopy tree species, tree age, and density are included as predictor variables. How does enemy escape alter competition between an invasive species and other members of the invaded community? Through feedbacks, the microbial community has the potential to alter plant species composition by altering the outcome of competition between species (van der Heijden et al. 2008). Soil communities alter the performance of plants, yet do not have the same effects across species (Klironomos 2002). If some species receive positive PSF, while others experience negative PSF, the outcome of competition could be altered (Bever 2003). For example, mycorrhizal mutualists have been shown to alter the outcome of competition between plant species by increasing competitive ability for soil resources (Zabinski et al. 2002). I predict that A. platanoides will benefit from escaping specialist predators while still being able to interact with generalist mutualists, and therefore will have Figure 2: Greenhouse experiments testing direction and intensity of positive PSF in its invasive range. PSF, and soil training for I will conduct this study using a soil training competition experiment. experiment in the greenhouse (Figure 2). I am currently using seedlings of A. platanoides and A. saccharum to train the microbial community within pots, and have left some pots without seedlings to act as a control (60 of each, n=180). All pots were inoculated with whole microbial communities, consisting of combined soil from forests used in the previous experiment (again, including soil from Kleinstuck). For three months the seedlings will grow, changing the microbial community through root chemistry and susceptibility to different microbes. After the three months I will collect soil samples from each pot for molecular analysis. I will then harvest the tree seedlings and plant in new seeds. The experimental design is 3x2 factorial, manipulating soil training species (A. platanoides, A. saccharum, control) and seedling (A. platanoides and A. saccharum). I will allow these seeds to grow for a further three months in the greenhouse, and after this period measure survival, above and below ground biomass, and leaf number. PSF between a native and invasive congeneric pair will be estimated as the effects of seedling species, soil inocula, and the species by inoculate interaction on seedling growth. By contrasting the growth of two plant species in their own and each others soil communities, I will be able to predict whether these species experience positive feedbacks, which lead to loss of diversity, or negative, which maintains it (Bever et al. 1997, Bever 2003). This is done using the model designed by Bever (2003), which encapsulates direct and indirect effects of invasive and native species on the soil community, and on each other. I predict that A. platanoides will experience positive soil feedbacks (i.e., they will grow best in soil trained by a conspecific), while A. saccharum may experience negative PSF, as it trains a soil community that is detrimental to its own growth, culturing higher densities of root pathogens and predators. These soil feedbacks could contribute to the ability of A. platanoides to outcompete native trees (Reinhart et al. 2005), by facilitating its own growth while inhibiting the growth of other species. Using the collected greenhouse soil I will determine abundance and ratio of different microbial groups using quantitative PCR. This information will allow me to determine the mechanism behind changes in PSF caused by A. platanoides invasion. I predict that I will find a higher ratio of bacteria to fungi in the pots trained by A. platanoides, as invasive species tend to have higher acquisition of soil resources and provide higher quality resources for microbes, thus favoring bacterial populations with higher turnover rates and more efficient decomposition (Wardle et al. 2004). I will further use primers to specifically identify mycorrhizal fungi, and use quantitative PCR to determine abundance of these species. I predict that mycorrhizal fungi will be more abundant in soil samples trained by A. saccharum, as they have evolved to interact with this native species. Research Implications Over 50,000 exotic species are present in the US today. Even if only a fraction of these species become invasive, huge impacts on native ecosystems are expected (Pimentel et al. 2000). Invasive species are one of the major drivers of biodiversity loss (Vitousek et al. 1997, Pimentel et al. 2000), and it is estimated that invasive species cost over $137 billion in damage per year, globally (Pimentel et al. 2000). For this reason, it is important to identify the processes facilitating invaders. However, given the economic costs and the potential ecological consequences of invasive species removal, it is also important to understand how exotic species become integrated into natural communities and the consequences of invasion over longer time scales. In order to make these predictions, we must first understand the mechanisms behind what makes a small proportion of introduced species become invasive. Displacement from coevolved relationships and the Enemy Release Hypothesis provides a testable prediction about the behavior of a species when introduced to a new range -­‐ species once controlled by predators and pathogens in their native range will be able to reach high abundances once released from this control (Reinhart et al. 2003). Plants with a more negative PSF in their native range may benefit most from enemy release, and have the potential to become invasive in an exotic range (Kulmatiski et al. 2008). Beyond the impacts of introducing a new species, the alteration of the microbial community by an invasive plant causes large and lasting impacts on invaded communities (Wardle et al. 2004). Positive feedbacks have the potential to create positive density dependence, reducing diversity of the native plant community (Bever et al. 1997). The results of my study will help us to better understand how an invasive species alters the microbial and plant community over the course of its invasion. If A. platanoides is able to culture the microbial community to its own benefit, this may result in positive density dependence and the creation of monocultures where diverse forests were once present. Further, changes to the microbial community could have long lasting effects (Wardle et al. 2004), and once changes have been made, managers may need to restore the microbial community reestablish soil processes and plant assemblages. Alteration of the microbial community is an environmental, as well as human problem, as microbes are responsible for many of the ecosystem services on which humans rely, such as decomposition and conversion of nutrients to forms usable by plants. Nutrient cycles are vulnerable to microbe community composition (Kandeler et al. 1996), and invasive species have been shown to alter nutrient cycling through PSF (Ehrenfeld et al. 2001). Therefore, the total effects of an invasion will not only include displacement of native species by the invader, but also changes to microbial interactions with plants, nutrient cycling, and diversity (Wardle et al. 2004). Literature Cited Adams, J.M., et al. (2009) A cross-­‐continental test of the Enemy Release Hypothesis: leaf herbivory on Acer platanoides (L.) is three times lower in North America than in its native Europe. Bever, J.D. et al. (1997) Incorporating the soil community into plant population dynamics: the utility of the feedback approach. Journal of Ecology. 85, 561-­‐573. Bever, J.D. (2003) Soil community feedback and the coexistence of competitors: conceptual framework and empirical tests. New Phytologist. 157, 465-­‐473. Carpenter, D. and N. Cappuccino (2005). 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