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Copyright Maria Magdalena Meza-Lopez 2015 Abstract Nutrient enrichment and climate warming effects on plant and herbivore invasions in wetland communities by Maria Magdalena Meza-Lopez Ecosystems invaded by one exotic species often contain abundant populations of other exotic species. This may reflect facilitative exotic species interactions, common responses to anthropogenic factors (nutrient enrichment and/or climate warming), or result from common paths of introduction. My dissertation asked 1) Do exotic species on multiple trophic levels reciprocally facilitate each other’s invasions of freshwater wetlands in southeast Texas?, 2) Do nutrient enrichment and/or climate warming increase exotic plant performance in the presence or absence of exotic herbivores?, 3) Do nutrient enrichment, warming, plant invasions, and their interactions affect exotic herbivore performance? A factorial field mesocosm experiment that manipulated the order of exotic plant and exotic herbivore invasions found no evidence of reciprocal exotic species facilitation that would lead to an invasional meltdown in native wetland communities invaded by Pomacea maculata (apple snail) and Alternanthera philoxeroides (alligator weed). Pomacea maculata may facilitate A. philoxeroides invasions because they preferentially consumed native plants but P. maculata invasions did not depend on the presence of A. philoxeroides. Greenhouse experiments showed that elevated nutrients (nitrogen and phosphorus) had no direct impact on P. maculata survival and Eichhornia crassipes (water hyacinth) and A. philoxeroides are not ideal food sources for P. maculata. However, P. maculata growth increased with elevated nutrients via changes in plants but was independent of plant identity. In field mesocosm experiments, elevated nutrients increased plant invasions independent of warming by more strongly increasing growth of exotic plants. However, warming can influence the magnitude of nutrient enrichment effects on exotic species. In addition, P. maculata performance was insensitive to elevated nutrients but warming increased the reproductive output of P. maculata four-fold. Together these results suggest that exotic herbivores that have high impacts on native plants may facilitate plant invasions, that nutrient enrichment and warming may each increase the performance of exotic herbivores, and plant invasions are more very sensitive to nutrient enrichment than warming. Furthermore, these studies suggest that it is critical to investigate the effects of multiple anthropogenic factors on native and exotic species at the community level. Acknowledgments I would like to thank my advisor Evan Siemann for giving me the opportunity to learn and growth as scientist and for believing in me. Thank you for your patience and for having the energy to work with me endlessly on many things that included fellowship applications, experimental design, setting up experiments, data analysis, and manuscript preparations. I also want to thank you for always pushing me to exceed limits throughout graduate school. You have made me a stronger person in many ways and have prepared me for the challenges that lie ahead. Thank you for all of your help. I want to thank my committee members Rudolf Volker, Qilin Li, and Anna Armitage for guidance with experimental designs and for challenging me to think critically and broadly. In addition, I want to thank Amy Dunham for her support and guidance as a committee member. Special thanks to Anna Armitage for her support and for not hesitating to step in when I needed her help. Thanks to all of the graduate students that welcomed me when I joined the department, those that joined the department later, and to the Chinese visiting students in our lab for making graduate school enjoyable. Special thanks to Leiyi Chen (a.k.a. Jenny) and Ling Zhang for their help in the field and in lab and for always willing and ready to grab a beer at the end of the day regardless of what was going on. I also want to give special thanks to Stephen Hovick for his advice and mentoring during my first year of graduate school, to Chris Roy for being a good officemate, to my labmates Chris Gabler, Juli Carillo, and Thomas Ye for v their advice and useful comments on experimental designs, fellowship applications, and manuscripts. I am also very grateful for everyone that provided field and laboratory assistance throughout the years including lab technicians, graduate and undergraduate students. Thank you Candice Tiu, Nick Hill, Abby Lamb, Daniel McDermott, Qiang Yang, Gang Liu, Ariel Nixon, Erica Soltero, Hannah Chen, Cassandra Corrales, Jordan Bunch, Josh Hwang, Hayden Ju, Peter Sylvers, Boris Zhong, and Michelle Shapiro. Special thanks to Dana Galvan for volunteering and assisting me in field and in the lab for three years. I also want to thank Sarah Margolis who volunteered and provided immense help in the field during my last field season. I want to thank my family for motivating me and giving me the drive to continue and overcome the challenges that I have encountered along the way. Thanks to my husband William Lopez for his love, patience, advice and support along this journey. Thank you also for enduring long hot days at South Campus to assist me with my field experiments. Thanks to my mother and siblings for their understanding, support, and love along the way. I also want to give a special shout out to my fur babies Boots and Buddha for their unconditional love, for keeping me company and assisting me at South Campus. Thanks to the numerous organizations that have funded me and my work, including the Environmental Protection Agency, Ford Foundation, Houston Conchology Society, Society of Wetland Scientist, Kroger, Ecology and Evolutionary Biology department at Rice University, and Rice University Shell vi Center for Sustainability. I also want to thanks Chef Derrix at Rice University for providing large quantities of romaine lettuce to sustain apple snail populations in the lab. I want to give special thanks to AGEP and Ford Foundation. Thanks Theresa Chatman and Richard Tapia for your support, advice, and mentoring. I also want to thank Chris O’Brien for her support, advice, motivations, and for her sincere friendship. Thanks to all of friends that I have acquired throughout the years through these programs and organizations. Special thanks to Joseph Young, Toni Tullius, and Nabor Reyna for their advice, mentoring, and friendship. I dedicate this work to my nieces Leticia, Crystal, Karen, Vanessa, Daniela, Gabriela, Jazmin, Wendy (2), Brenda, Dayana, and Alondra, and my nephew Cesar and grandnephew Landen to encourage them to pursue their dreams and goals. vii Contents Abstract ............................................................................................................... ii Acknowledgments ............................................................................................. iv Contents ............................................................................................................ vii List of Figures ..................................................................................................... x List of Tables .................................................................................................... xii List of Appendix Figures................................................................................. xiii List of Appendix Tables .................................................................................. xiv Ch 1 Introduction .............................................................................................. 15 Ch 2 Experimental test of the Invasional Meltdown Hypothesis: an exotic herbivore facilitates an exotic plant, but the plant does not reciprocally facilitate the herbivore ..................................................................................... 21 2.1. Abstract .................................................................................................... 21 2.2. Keywords.................................................................................................. 22 2.3. Introduction............................................................................................... 22 2.4. Methods.................................................................................................... 26 2.4.1. Study system ................................................................................ 26 2.4.2. Establishing native wetland communities ..................................... 27 2.4.3. Exotic species............................................................................... 28 2.4.4. Experimental manipulation ........................................................... 28 2.4.5. Data analyses ............................................................................... 29 2.5. Results ..................................................................................................... 30 2.5.1. Reciprocal facilitation between exotic species (IMH) .................... 30 2.5.2. Exotic species impacts ................................................................. 31 2.6. Discussion ................................................................................................ 32 2.6.1. Reciprocal facilitation between exotic species (IMH) .................... 32 2.6.2. Exotic species impacts ................................................................. 34 2.7. Acknowledgments .................................................................................... 35 Ch 3 Nutrient enrichment increased exotic snail Pomacea maculata growth independent of native and exotic wetland plant identity .............................. 41 3.1. Abstract .................................................................................................... 41 viii 3.2. Keywords.................................................................................................. 42 3.3. Introduction............................................................................................... 43 3.4. Methods.................................................................................................... 46 3.4.1. Study system ................................................................................ 46 3.4.2. Experimental set-up...................................................................... 48 3.4.3. Exp.1: Nutrients’ direct and indirect effects on P. maculata mortality and shell length ...................................................................................... 48 3.4.4. Exp. 2: Nutrient enrichment effects on P. maculata, plants, and plant-herbivore interactions .................................................................... 49 3.4.5. Data analyses ............................................................................... 50 3.5. Results ..................................................................................................... 51 3.5.1. Exp. 1: Nutrient enrichment’s direct and indirect effects on P. maculata mortality and shell length .................................................... 51 3.5.2. Exp. 2: Nutrient enrichment effects on P. maculata, plants, and plant-herbivore interactions .................................................................... 52 3.6. Discussion ................................................................................................ 52 3.7. Acknowledgments .................................................................................... 57 Ch 4 Nutrient enrichment effects on invasions depend on climate warming: an experimental test with freshwater wetland plant communities ............... 62 4.1. Abstract .................................................................................................... 62 4.2. Keywords.................................................................................................. 63 4.3. Introduction............................................................................................... 63 4.4. Methods.................................................................................................... 69 4.4.1. Study system ................................................................................ 69 4.4.2. Experimental setup ....................................................................... 70 4.4.3. Data analyses ............................................................................... 72 4.5. Results ..................................................................................................... 73 4.6. Discussion ................................................................................................ 75 4.7. Acknowledgements .................................................................................. 79 Ch 5 Warming increased exotic snail reproduction independent of nutrient enrichment and plant invasions in wetland communities ............................ 88 5.1. Abstract .................................................................................................... 88 5.2. Keywords.................................................................................................. 89 ix 5.3. Introduction............................................................................................... 89 5.4. Methods.................................................................................................... 92 5.4.1. Study system ................................................................................ 92 5.4.2. Experimental setup ....................................................................... 95 5.4.3. Data analyses ............................................................................... 96 5.5. Results ..................................................................................................... 97 5.5.1. Snails ............................................................................................ 97 5.5.2. Plants ........................................................................................... 98 5.6. Discussion ................................................................................................ 99 5.7. Acknowledgements ................................................................................ 105 References ...................................................................................................... 113 x List of Figures Figure 2-1 (a) Alternanthera philoxeroides dry mass and (b) mass as percent of total plant mass in treatments with exotic plants introduced in June 2011. Means + 1 SE. Means with the same letter were not significantly different in post-hoc tests. P = exotic plant, S – P = exotic snail invasion followed by exotic plant invasion, P & S = simultaneous exotic plant and exotic snail invasion ........................................................................................ 38 Figure 2-2 (a) Native plant dry mass and (b) diversity in each invasion treatment. Means + 1 SE. Means with the same letter were not significantly different in post-hoc tests. C = native plants and native snails, P = exotic plant, S = exotic snail, P – S = exotic plant invasion followed by exotic snail invasion, S – P = exotic snail invasion followed by exotic plant invasion, P & S = simultaneous invasion by exotic plant and exotic snail ..................... 39 Figure 2-3 (a) Pomacea maculata operculum length, (b) adult mass, (c) reproductive output (total egg volume) and (d) juvenile abundance were independent of A. philoxeroides invasions when snails were introduced in June 2011. Means + 1 SE. S = exotic snail, P – S = exotic plant invasion followed by exotic snail invasion, P & S = simultaneous invasion by exotic plant and exotic snail ....................................................................................... 40 Figure 3-1 The dependence of exotic P. maculata (a) shell length on nutrients and plants and (b) percent mortality on plant species in experiment 1. Means+1SE. Means with the same letter were not significantly different in post-hoc tests. E. c. = E. crassipes and A. p. = A. philoxeroides .................................................................................................... 59 Figure 3-2 The dependence of exotic P. maculata operculum length on (a) nutrients and (b) plants in experiment 2. Means +1SE. Means with the same letters were not significantly different in post-hoc tests. L. s. = L. spongia, H. u. = H. umbellata, A. p. = A. philoxeroides, E. c. = E. crassipes .............. 60 Figure 3-3 The dependence of wet plant mass on (a) snails and (b) plants in experiment 2. Means +1SE. Means with the same letters were not significantly different in post-hoc tests. L. s. = L. spongia, H. u. = H. umbellata, A. p. = A. philoxeroides, E. c. = E. crassipes ............................... 61 xi Figure 4-1 The dependence of plant mass in communities with (a) exotic plants only or (b) native plants only ,and (c) exotic plant mass and (d) native plant mass in communities with both native and exotic plants. ....... 85 Figure 4-2 The dependence of percent cover for each native and exotic plant species on temperature and nutrients in communities with both native and exotic plants ................................................................................... 86 Figure 4-3 The dependence of (a-b) native and (c-d) exotic plant mass and diversity on nutrients and community composition. Means +1SE. Means with the same letters were not significantly different in post-hoc tests. Np = native plant, ep = exotic plant, np + ep = native and exotic plants in community ........................................................................................................ 87 Figure 5-1 Exotic P. maculata snail egg clutch mass dependence on (a) nutrients, (b) temperature, and (c) plant addition treatments over time. Means ±1SE..................................................................................................... 109 Figure 5-2 Exotic P. maculata snail mass dependence on nutrient and temperature treatments. Means +1SE. Means with the same letters were not significantly different in post-hoc tests ........................................................ 110 Figure 5-3 Native P. acuta snail abundance dependence on nutrient treatments. Means +1SE. Means with the same letters were not significantly different in post-hoc tests.............................................................................. 111 Figure 5-4 (a) Total plant mass dependence on nutrients together with plant addition and (b) on temperature together with plant addition treatments. (c) Native plant mass dependence on nutrients together with plant addition and (d) on temperature together with plant addition treatments. (e) Exotic plant mass dependence on nutrients and (f) on temperature. Means +1SE. Means with the same letters were not significantly different in post-hoc tests ................................................................................................................. 112 xii List of Tables Table 2-1 Description of experimental treatments. Each tank received topsoil, native plants and native snails in June 2010 and additional plants and snails in April 2011 and June 2011. Exotic plant or snail additions in each treatment are shown in bold................................................................... 37 Table 3-1 The dependence of exotic P. maculata snail shell length and percent mortality in experiment 1 and P. maculata operculum length and wet plant mass in experiment 2 on nutrients, plants, snails, and their interactions in ANOVAs. Significant results are shown in bold ................... 58 Table 4-1 The dependence of community composition using plant mass in native plant only, exotic plant only, and communities with both native and exotic plants on nutrients, temperature, and their interaction in PERMANOVAs. Significant results are shown in bold .................................. 81 Table 4-2 The dependence of native and exotic plant mass and diversity (Shannon Diversity Index) on plant composition, nutrients, temperature, and their interactions in ANOVAs. Significant results are shown in bold ... 82 Table 4-3 The dependence of exotic to native plant mass ratio on nutrients, temperature and their interaction, in contrasts of expected ratio from native only and exotic only plant communities and the observed ratio from communities with both native and exotic plants. Significant results are shown in bold ................................................................................................... 83 Table 4-4 The dependence of species cover on nutrients, temperature, and their interactions in repeated measures ANOVAs using proportional cover for each species in communities with both native and exotic plants. Significant results are shown in bold ............................................................. 84 Table 5-1 The dependence of exotic snail P. maculata egg clutch number, egg clutch mass, adult mass, and native snail P. acuta abundance on nutrients, temperature, plant addition, and their interactions in ANOVAs or repeated measures ANOVAs. Significant results are shown in bold ......... 107 Table 5-2 The dependence of total, native, and exotic plant masses on nutrients, temperature, plant addition, and their interactions in ANOVAs. Significant results are shown in bold ........................................................... 108 xiii List of Appendix Figures Appendix Figure 3-1 The dependence of P. maculata shell length on (a) nutrients and (b) plants in experiment 1. Means +1SE. Means with the same letters were not significantly different in post-hoc tests. E. c. = E. crassipes, A. p. = A. philoxeroides……………………………………….…………………...126 Appendix Figure 3-2 The dependence of (a) L. spongia, (b) H. umbellata, (c) E. crassipes, and (d) A. philoxeroides wet plant mass on snails in experiment 2. Means +1SE. Means with the same letters were not significantly different in post-hoc tests…………………………………...……127 Appendix Figure 4-1 The dependence of exotic (a-b) E. crassipes, (c-d) A. philoxeroides, and (e-f) P. stratiotes and native (g-h) P. cordata, (i-j) L. spongia, (k-l) H. umbellata, and (m-n) T. latifolia in relation to nutrient and temperature in exotic or native only communities and communities with both native and exotic plants. Symbol size represent species dry plant mass…………………………………………………………………………………..128 Appendix Figure 4-2 (a) Native L. spongia and (b) exotic E. crassipes mass dependence on community composition and nutrients. Bars represent +1SE. Means with the same letters were not significantly different in posthoc tests. Scale is different for each species…………………………………129 Appendix Figure 5-1 Exotic P. maculata snail (a) egg clutch number and (b) egg mass dependence on the interactive effect of temperature, nutrients, and plant addition treatments over time. Means ± 1SE……………...………130 Appendix Figure 5-2 Pomacea maculata mass dependence on plant addition, nutrients, and temperature treatments. Means +1SE…………….131 Appendix Figure 5-3 (a) P. acuta and (b) P. trivolvis dependence on plant addition, nutrients, and temperature treatments. Means+1SE…………….132 Appendix Figure 5-4 Native (a) H. umbellata (no significant interactive result), (b) L. spongia, (c) P. cordata, and (d) T. latifolia mass dependence on nutrients and plant addition treatments. Means +1SE. Means with the same letters were not significantly different in post-hoc tests. Note that the y-axis scales differ among species………………………………………….…..133 Appendix Figure 5-5 Exotic (a) E. crassipes, (b) A. philoxeroides, and (c) P. stratiotes mass dependence on nutrient treatments. Means +1SE. Means with the same letters were not significantly different in post-hoc tests. Note that the y-axis scales differ among species…………………….……………..134 xiv List of Appendix Tables Appendix Table 3-1 The dependence of native L. spongia and H. umbellata and exotic A. philoxeroides and E. crassipes wet plant mass in experiment 2 on nutrients, snails, and their interactions in ANOVAs. Significant results are shown in bold………………………………………………………..…………135 Appendix Table 4-1 The dependence of percent cover on community composition, nutrients, temperature, and their interaction in repeated measures ANOVAs. Significant results are shown in bold…………………136 Appendix Table 4-2 The dependence of exotic plant masses on nutrients, temperature, and their interaction in ANOVAs. Significant results are shown in bold…………………………………………………..……………………137 Appendix Table 5-1 Total and native plant mass and individual native species mass dependence on plant addition, nutrient, and temperature. Exotic plant mass and individual exotic species mass dependence on nutrients and temperature. Means±SE……………………………………….....138 Appendix Table 5-2 The dependence of native plant masses on nutrients, temperature, plant addition, and their interactions in ANOVAs. Significant results are shown in bold…………………………………………………………139 Appendix Table 5-3 The dependence of exotic plant masses on nutrients, temperature, and their interaction in ANOVAs. Significant results are shown in bold……………………………………………..…………………………140 15 Chapter 1 Introduction Ecosystems are often invaded by multiple exotic species. This may reflect exotic species’ common paths of introduction (Padilla & Williams, 2004; Westphal et al., 2008), common responses to anthropogenic factors (Mainka and Howard 2010; Montoya and Raffaelli 2010) or facilitative interactions among exotic species (Simberloff and Von Holle 1999; Simberloff 2006). Because many ecosystems are often already invaded by exotic species, it is critical to understand how established exotic species can affect future exotic species invasions and the overall impact of multiple exotic species in native ecosystems. In addition, ecosystems that are invaded by numerous exotic species are simultaneously threatened by multiple anthropogenic factors. Studies have shown that anthropogenic factors such as nutrient enrichment and climate warming in isolation from one another can increase the probability of species invasions. Studies investigating single anthropogenic factors effects on exotic species 16 increase our knowledge of invasion ecology, but limit our ability to predict the interactive effect of anthropogenic factors on species invasions and their impacts on native species. However, studies investigating the effect of multiple anthropogenic factors is increasing, emphasizing that it is critical to simultaneously investigate multiple anthropogenic factor effects on species invasions and their effect on native species because these effects may be complex. Anthropogenic factors may interact with each other increasing, reducing, or have no effect on other anthropogenic factors. Therefore, it is critical to investigate 1) how established exotic species may influence the performance and invasion success of other exotic species, 2) interactive effects of multiple anthropogenic factors on exotic species performance and their impact on native species, and 3) the effect of species invasion and multiple anthropogenic factors (nutrient enrichment and warming) on native freshwater wetland communities. I conducted two greenhouse and three field mesocosm experiments to evaluate the role of species interactions (reciprocal invader facilitation) and the effect of multiple anthropogenic factors on exotic aquatic plants and an exotic herbivore and on native aquatic plants and native snails at the population and community level. The native and exotic plants and consumers that were included in these studies co-occur in southeast Texas. The exotic plants include were Alternanthera philoxeroides (alligator weed - Amaranthaceae), Eichhornia crassipes (water hyacinth - Pontederiaceae), Pistia stratiotes (water lettuce Araceae), and the exotic snail Pomacea maculata (island apple snails - 17 Ampullariidae; synonym P. insularum) (Hayes et al. 2012). These species heavily invade local wetlands in southeast Texas. These exotic species co-occur with native wetland plants that include Hydrocotyle umbellata (water pennywort Araliaceae), Limnobium spongia (frog's bit - Hydrocharitaceae), Pontederia cordata (pickerelweed - Pontederiaceae), Typha latifolia (cattail - Typhaceae), and also native snails that include Physa acuta (bladder snail - Physidae) and Planorbella trivolvis (marsh ramshorn snail - Planorbidae). These native and exotic plants were selected for these studies based on their clonal reproduction, functional group (i.e. submerged, emergent, or floating) and co-occur with each other in the introduced range. Exotic Pomacea maculata, island apple snails, are voracious generalist plant consumers (Rawlings et al. 2007) with high impacts on native wetland communities (Meza-Lopez and Siemann 2015) compared to native snails. These exotic plants and herbivore are sympatric in their native (South America) and introduced range. To understand the effect of anthropogenic factors on exotic plants and P. maculata, I first investigated nutrient enrichment direct and indirect effects on P. maculata performance. I found that nutrients tended to increased P. maculata size when they were fed exotic E. crassipes or A. philoxeroides, but P. maculata size was larger with cut lettuce independent of nutrient treatment, suggesting nutrient enrichment did not directly affect P. maculata performance. In addition, P. maculata mortality was lower with lettuce and highest with A. philoxeroides, suggesting that exotic plants are not an ideal food source for P. maculata. 18 To further understand the effects of nutrient enrichment on P. maculata and exotic plant performance, I investigated nutrient enrichment effects on exotic plants E. crassipes and A. philoxeroides mass and exotic snail P. maculata operculum length. Elevated nutrients increased P. maculata operculum length via native and exotic plants independent of plant identity. In addition, P. maculata reduced exotic plant mass, but native plant mass was lower independent of P. maculata invasions, suggesting that exotic plants would benefit from P. maculata invasions. Even though we found that P. maculata may benefit A. philoxeroides invasions and that elevated nutrients increased exotic plant and herbivore performance, it is critical to understand how nutrient enrichment may alter species invasions and influence the effect of other anthropogenic factors on multitrophic exotic species and their effects on native communities. I investigated the interactive effect of nutrient enrichment and warming on plant invasions and their effect on native wetland plant communities. Nutrient enrichment increased plant invasions and warming had no effect. The effect of nutrient enrichment was higher in communities with exotic plants than in communities with both native and exotic plants. In addition, the magnitude of nutrient enrichment effects on exotic species dependent on warming. Furthermore, nutrient enrichment increased exotic plant mass but only a few species benefitted from an increase in nutrients. In contrast, warming had no effect on diversity, suggesting that warming would not influence plant invasions. Additional studies are necessary to increase our understanding of climate warming effects on plant invasions with longer field studies. In addition, native 19 and exotic plant mass and diversity was higher in native or exotic plant communities than in communities with both native and exotic plants with elevated nutrients. Furthermore, the exotic to native plant mass ratio in communities with both native and exotic plants was lower than expected from native or exotic plant communities. Together these results emphasize that it is critical to include native plant communities to better predict the effect of multiple anthropogenic factors on plant invasions. Our results suggest that warming will not influence plant invasions but will influence the effect of nutrient enrichment on exotic species. Finally, I investigated the interactive effect of anthropogenic factors on multitrophic exotic species and how these anthropogenic factors influenced exotic species effects on native plants and snails in wetland communities. Warming increased P. maculata reproduction four-fold, but had no effect on exotic plants, suggesting that warming effects can vary depending on exotic species identity or trophic level. Elevated nutrients increased P. maculata operculum length and warming increased P. maculata reproduction, suggesting that the interactive effect of nutrient enrichment and warming cannot be predicted based on their individual effects. In addition, elevated nutrients increased plant performance, but had greater effects on exotic plants and also increased native snail abundance. Native and exotic plant interactions reduced the effect of elevated nutrients on each other. These results suggest that nutrient enrichment effects can vary with trophic level and species identity. Therefore, it is critical to understand the effect of multiple anthropogenic factors on native and exotic 20 plants and native and exotic consumers and how they influence exotic species effects on native species. These studies increased our understanding of role of species interactions, the effect of multiple anthropogenic factors on plant invasions and an exotic herbivorous snail, and their impacts on native aquatic plants and snails at the population and community level. These results showed that warming together with elevated nutrients may increase the probability of aquatic herbivore invasions, that native plants influence the effect of elevated nutrients on plant invasions, and that the effect of nutrient enrichment and warming can vary with species trophic level and species identity. Together these results showed that nutrient enrichment and warming influence the performance of exotic plants and exotic herbivores in freshwater wetland communities and that their interactive effect on plant invasions cannot be predicted based on their individual effects without considering native communities. Chapter 2 Experimental test of the Invasional Meltdown Hypothesis: an exotic herbivore facilitates an exotic plant, but the plant does not reciprocally facilitate the herbivore Meza-Lopez, M. M. and Siemann, E. (2015), Experimental test of the Invasional Meltdown Hypothesis: an exotic herbivore facilitates an exotic plant, but the plant does not reciprocally facilitate the herbivore. Freshwater Biology, 60: 1475–1482. doi: 10.1111/fwb.12582 2.1. Abstract Ecosystems with multiple exotic species may be affected by facilitative invader interactions, which could lead to additional invasions (Invasional Meltdown Hypothesis). Experiments show that one-way facilitation favours exotic species and observational studies suggest that reciprocal facilitation among exotic species may lead to an invasional meltdown. We conducted a mesocosm 21 22 experiment to determine whether reciprocal facilitation occurs in wetland communities. We established communities with native wetland plants and aquatic snails. Communities were assigned to treatments: control (only natives), exotic snail (Pomacea maculata) invasion, exotic plant (Alternanthera philoxeroides) invasion, sequential invasion (snails then plants or plants then snails) or simultaneous invasion (snails and plants). Pomacea maculata preferentially consumed native plants so A. philoxeroides comprised a larger percentage of plant mass and native plant mass was lowest in sequential (snail then plant) invasion treatments. Even though P. maculata may indirectly facilitate A. philoxeroides, A. philoxeroides did not reciprocally facilitate P. maculata. Rather, ecosystems invaded by multiple exotic species may be affected by oneway facilitation or reflect exotic species’ common responses to abiotic factors or common paths of introduction. 2.2. Keywords invasional meltdown, reciprocal facilitation, Pomacea maculata, Alternanthera philoxeroides, experimental wetlands 2.3. Introduction Ecosystems are often invaded by multiple exotic species. This may reflect exotic species’ common paths of introduction (Padilla and Williams 2004; Westphal et al. 2008), common responses to abiotic factors (Mainka and Howard 2010; Montoya and Raffaelli 2010) or facilitation among exotic species (Simberloff and 23 Von Holle 1999; Simberloff 2006). Because many ecosystems are often already invaded by exotic species, it is critical to know how established exotics affect future invasions and to understand the overall impact of multiple exotic species in an ecosystem. An established exotic species may increase the susceptibility of ecosystems to future invasions by altering biotic and/or abiotic characteristics of an invaded habitat (Simberloff and Von Holle 1999; Grosholz 2005). If such facilitation of subsequent exotic species is common, this may lead to an invasional meltdown where exotic species facilitate other exotic species by increasing their likelihood of survival and/or their ecological impacts, including the magnitude of their impacts, as proposed by the Invasional Meltdown Hypothesis (IMH hereafter) (Simberloff and Von Holle 1999). Simberloff (2006) suggested that various degrees of facilitation, ranging from simple facilitation (one exotic species aiding the invasion of another) to reciprocal facilitation (exotic species increasing each other’s performance), could lead to an invasional meltdown. Reciprocal facilitation is a type of facilitation and an aspect of the IMH that remains controversial because experimental studies have yet to show evidence of reciprocal facilitation among exotic species that leads to an invasional meltdown (Simberloff 2006; Gurevitch 2006). Studies supporting the IMH show evidence of one-way facilitation between exotic species (Ricciardi et al. 2013) that may occur via consumptive or nonconsumptive mechanisms. For instance, exotic red deer (Cervus elaphus) and fallow deer (Dama dama) indirectly facilitated exotic Douglas fir (Pseudotsuga menziesii) by preferentially consuming the native evergreen conifer Austrocedrus 24 chilensis (Relva et al. 2009). In addition, crayfish (Orconectes rusticus) may facilitate the invasion of exotic Eurasian watermilfoil (Myriophyllum spicatum) by increasing plant fragmentation (non-consumptive) (Maezo et al. 2010). Likewise, exotic plants may facilitate exotic herbivores by providing a higher quality food source (Engelkes and Mills 2013) or by supplying higher quality substrata for reproduction (Burks et al. 2010) or shelter (Sessions and Kelly 2002). These studies showed that exotic species can facilitate other invaders; however, they did not investigate whether reciprocal facilitation occurred. An invasion meltdown may occur via reciprocal facilitation among exotic species as suggested by field surveys (O’Dowd et al. 2003; Green et al. 2011) or via one-way facilitation among exotic species, increasing the frequency of additional invasions (Simberloff, 2006; Helms, Hayden & Vinson, 2011). A recent study evidenced a newly formed mutualism between two exotic species where the yellow crazy ant Anoplolepis gracilipes protects exotic scale insects and scales produce honeydew that ants consume (O’Dowd et al. 2003). Exclusion of A. gracilipes reduced the abundance of exotic scale insects (Abbott and Green 2007), but the effects of the scale insect on A. gracilipes are yet to be investigated. In addition, an exotic grass (Cynodon dactylon) increased the abundance of exotic honeydew-producing mealybug (Antonina graminis) that in turn may increase the abundance of exotic fire ants (Solenopsis invicta) that tend A. graminis; however, A. graminis did not affect the performance of C. dactylon (Helms et al. 2011). These studies suggest that exotic species may facilitate 25 other exotic species, contributing to an invasional meltdown, without their effects being reciprocal. The effects of established exotic species on subsequent invasions may influence their impact on native species (Johnson et al. 2009; Green et al. 2011). For instance, the exotic rusty crayfish Orconectes rusticus and exotic Chinese mystery snail Bellamya chinensis had weak negative effects on each other, but together they drove a native snail to extinction and reduced the abundance of another snail by 95% (Johnson et al. 2009). These interactive effects were due to facilitative interactions between invaders. Similarly, exotic scale insects (Tachardina aurantiaca and Coccus spp.) may facilitate A. gracilipes invasions, reducing native red land crab (Gecarcoides natalis) abundance and increasing the probability of exotic giant African land snail Achantina fulica invasions (O’Dowd et al. 2003; Green et al. 2011). These studies suggest that the co-occurrence of multiple exotic species could have greater consequences on native species than their individual impacts (Johnson et al. 2009). Here, we investigated whether reciprocal facilitation between exotic species contributes to an invasional meltdown in wetland mesocosm communities. Specifically, we evaluated whether an exotic plant (Alternanthera philoxeroides) and an exotic generalist herbivorous snail (Pomacea maculata) reciprocally facilitate each other and whether their impact on native plants is non-additive. 26 2.4. Methods 2.4.1. Study system In south east Texas, Alternanthera philoxeroides (alligator weed Amaranthaceae) and Pomacea maculata (island apple snails – Ampullariidae; synonym P. insularum) heavily invade local wetlands, co-occurring with native wetland plants Hydrocotyle umbellata (water pennywort – Araliaceae), Limnobium spongia (frog's bit – Hydrocharitaceae), Myriophyllum pinnatum (cutleaf watermilfoil - Haloragaceae) and Pontederia cordata (pickerelweed – Pontederiaceae) with native snails Haitia acuta (Physidae – synonyms include Physa acuta and Physella acuta). Alternanthera philoxeroides was introduced to the USA in ballast water from the Parana River region of South America (Julien et al. 1995). It was first recorded in Mobile, Alabama in 1897; by 1963, it had invaded coastal states from North Carolina to Texas (Spencer and Coulson 1976). In aquatic habitats, it anchors to banks and reproduces clonally, but does not produce viable seeds in its introduced range (Spencer and Coulson 1976; He et al. 2014). Alternanthera philoxeroides can form extensive interwoven mats from plant fragments that can become independent free-floating monocultures (Spencer and Coulson 1976; Sainty et al. 1997). Thought to be introduced via the aquarium trade, Pomacea maculata was discovered in 1989 in south east Texas with source populations probably from the lower Parana River Basin in La Plata region of Argentina (Hayes et al. 2012). 27 Its native range extends north to the Amazon basin of Brazil, north of Manaus, and also occurs in Uruguay and Paraguay (Hayes et al. 2008; Karatayev et al. 2009; Hayes et al. 2012). Pomacea maculata populations have been reported along the Gulf Coast from Texas to Florida and along the Atlantic coast to South Carolina (Rawlings et al. 2007; Byers et al. 2013). Unlike native aquatic snails that primarily consume periphyton, P. maculata is a generalist voracious plant consumer (Rawlings et al. 2007). Pomacea maculata’s impact on native ecosystems is currently unknown. 2.4.2. Establishing native wetland communities In June 2010, we established forty wetland communities in 473 L tanks (Poly tank, round flat-bottom nestable tanks, Litchfield, MN, USA) in an open greenhouse structure at the Rice University South Campus Field Station (29.6 N, 95.4 W). To prepare the greenhouse structure, three layers of poly cloth floor were attached to boards that were secured on the bottom perimeter of the greenhouse frame. To each tank, we added 15 cm of topsoil, municipal water, 115 native snails and native plants (H. umbellata, L. spongia, M. pinnatum and P. cordata – one individual of each). Water was allowed to sit for a week before plants and snails were added. Dechlorinator was added to subsequent water additions, which were necessary to maintain water levels constant for the duration of the study. Plants that did not survive the initial planting were replaced to ensure that all species were included in the communities. Native plants and snails were allowed to establish for 10 months. Native Utricularia inflata (bladderwort) invaded some tanks, perhaps from the waterfowl that occasionally 28 landed in tanks; therefore, it was added to all uninvaded tanks to reduce variation among communities. After plants were established, the open greenhouse structure was enclosed with fiberglass screen (Metro Screenworks, Englewood, CO, USA) attached to secured boards to tightly seal the entire greenhouse structure. The fiberglass screen reduced incoming radiation by fifty percent. 2.4.3. Exotic species Pomacea maculata egg clutches and A. philoxeroides plants were collected in Armand Bayou in Pasadena, Texas, USA (29.6 N, 95.1 W). Females emerge from water at night to lay egg clutches on solid objects above the water level. To simulate natural hatching conditions in the laboratory, collected egg clutches were placed on top of a plastic mesh with small holes above a container partially filled with water. This allowed P. maculata to fall into water as they hatched. They were reared on commercial romaine lettuce until they reached the desired size for the study (Morrison and Hay 2011). 2.4.4. Experimental manipulation In April 2011, established communities were randomly assigned to the following treatments: (1) control (only natives) [C], (2) invasion by plants (A. philoxeroides) [P] or snails (P. maculata) [S], (3) simultaneous invasion of plants and snails [P & S] or (4) sequential invasion: plants then snails [P - S] or snails then plants [S - P]. Single invasion treatments were replicated eight times, while other treatments were replicated six times. Replicates were limited by the number of tanks available. Every tank received plants collected from the same location in 29 April and June 2011, so that plant invasion treatments only manipulated plant origin (native or exotic) (Table 2-1). Plant additions were always 60 g of plant mass independent of plant origin. Snail invasion treatments were either three P. maculata, simulating natural densities in lotic waters in south east Texas (Howells et al. 2006), or 20 native snails. Pomacea maculata on average weighed 27 g with an average operculum length of 25 mm, while native snails were much smaller (~5 mm). Weekly P. maculata egg clutch surveys were conducted throughout the study to measure and quantify egg clutches and record the location of egg clutch oviposition. Total egg clutch volume per tank was estimated as the sum of the products of length, width and thickness of each egg clutch. In August 2011, we removed, sorted by species and oven-dried aboveground plant mass. Native plant diversity was calculated using the Shannon Diversity Index. Adult P. maculata were collected, weighed and measured. Native snail and juvenile P. maculata abundances were estimated by collecting as many snails as possible in fifteen minutes. Such incomplete sampling of snails could increase the possibility of a type II error. Collected snails were sorted by size and counted in the laboratory. 2.4.5. Data analyses Analysis of variance (ANOVA) was used to test the dependence of exotic plant variables (exotic plant dry mass and plant mass as a percent of total mass, excluding native plant mass), exotic snail performance (operculum length, adult mass, total egg clutch volume and juvenile abundance), native plant variables 30 (plant dry mass and diversity, excluding exotic plant mass) and native snail abundance on invasion treatments (P<0.05). Data met the assumptions of ANOVA. Exotic plant ANOVAs included only treatments with plant invasions in June 2011 ([P], [S - P] and [P & S]). Exotic snail ANOVAs only included treatments with snail invasions in June 2011 ([S], [P - S] and [P & S]). [P & S] invasion treatments were included in both exotic plant and exotic snail ANOVAs. Tukey-Kramer multiple comparison tests were conducted to determine the differences between treatment levels (P<0.05) for significant variables with more than two levels. We performed additional two-way ANOVAs to examine whether the impacts of A. philoxeroides and P. maculata on native plant mass and diversity and native snail abundance were non-additive (i.e. non-independent). These interactive ANOVAs included only [C], [P], [S] and [P & S] invasion treatments and exotic snail presence, exotic plant presence and their interaction as predictors. A significant interaction between predictors indicates non-additive impacts (i.e. the combined effect of the species is different from the sum of each species’ individual impacts). All statistical analyses were performed using StatView (v 5.0). 2.5. Results 2.5.1. Reciprocal facilitation between exotic species (IMH) Pomacea maculata in sequential [S - P] and simultaneous [P & S] invasion treatments reduced A. philoxeroides dry mass (F2,17 = 9.5, P = 0.0017; 31 Figure2-1a), but A. philoxeroides mass as percent total plant mass was highest in sequential [S – P] invasion treatments (F2,17 = 4.0, P = 0.0373, Figure 2-1b). By contrast, native plant dry mass was lowest in sequential [S – P] invasion treatments (F5,34 = 11.8, P < 0.0001; Figure 2-2a). Alternanthera philoxeroides did not facilitate the invasion of P. maculata. Alternanthera philoxeroides did not affect P. maculata’s operculum length (F2,17 = 1.8, P = 0.1931; Figure 2-3a), adult mass (F2,17 = 0.5, P = 0.6349; Figure 2-3b), total egg clutch volume (F2,17 = 0.5; P = 0.6378; Figure 2-3c) or juvenile abundance (F2,17 = 0.9, P = 0.4095; Figure 2-3d). 2.5.2. Exotic species impacts Pomacea maculata together with exotic plant invasions had greater effects on native plants than did A. philoxeroides invasions alone. Native plant diversity was lowest in simultaneous [P & S] invasion treatments (F5,34 = 14.3, P < 0.0001; Figure 2-2b). Pomacea maculata significantly reduced native plant mass (F1,24 = 15.2, P < 0.0007) and diversity (F1,24 = 35.5, P < 0.0001), while A. philoxeroides did not affect native plant mass (F1,24 = 2.5, P = 0.13) or diversity (F1,24 = 0.5, P = 0.50). Together these exotic species had additive impacts on native plant mass (snail*plant: F1,24 < 0.1, P = 0.8379) and diversity (snail*plant: F1,24 = 0.7, P = 0.4161) as indicated by non-significant interaction terms. No factors influenced native snail abundance (all P > 0.59). 32 2.6. Discussion 2.6.1. Reciprocal facilitation between exotic species (IMH) Multiple exotic species in these wetland communities are not a result of reciprocal facilitation. Alternanthera philoxeroides did not affect the size, survival, reproduction or population growth of P. maculata. In addition, P. maculata directly limited the abundance of A. philoxeroides. However, P. maculata’s preferential consumption of native plants may indirectly facilitate A. philoxeroides invasions by reducing native plant competition. Other studies show that exotic herbivores, including exotic slugs (Hahn et al. 2011; Hahn et al. 2011), deer (Nuñez et al. 2008; Relva et al. 2009), gypsy moths (Lymantria dispar) (McEwan et al. 2009) and snails (Motheral and Orrock 2010), may facilitate plant invasions by preferentially consuming native plants. This suggests that exotic herbivore facilitation of exotic plants is a common pattern that may reflect the traits of exotic plants, the traits of exotic herbivores or more complex mechanisms (Pearse et al. 2013). Exotic herbivores may also directly facilitate exotic plants via non-consumptive mechanisms. For instance, plant fragmentation may increase the dispersal of exotic plants (Maezo et al. 2010). A greenhouse study showed that mechanically generated A. philoxeroides’ single-node fragments survived and grew in a moist soil mix, so fragmentation may increase the rate of spread of this clonal species (Dong et al. 2012). Here, P. maculata reduced A. philoxeroides’ mass, but also generated plant fragments that could increase 33 the spread of A. philoxeroides to new patches in open, natural conditions, or contribute to existing patches. In the introduced range, the only herbivore other than P. maculata that commonly feeds on A. philoxeroides is the alligator weed flea beetle Agasicles hygrophila (Schooler et al. 2006). The beetle preferentially feeds on the young leaves and stem tissue of A. philoxeroides, but does not generate nodal fragments (Schooler et al. 2006). Our experiment did not consider local negative effects or distant positive effects of dispersal from A. philoxeroides fragmentation by P. maculata. It is possible that P. maculata’s feeding may facilitate A. philoxeroides’ spread and invasions by creating nodal fragments that could contribute to existing patches and the establishment of new patches. Exotic plants did not directly facilitate exotic herbivore invasions in native plant dominated wetlands. Pomacea maculata’s survival, growth (operculum length and adult mass), reproduction (total egg clutch volume) and juvenile abundance were all independent of A. philoxeroides invasions. These results are not a strong test of the direct effects of A. philoxeroides on P. maculata when the exotic plant is abundant because P. maculata preferentially consumed native plants more than A. philoxeroides. Even though A. philoxeroides did not directly facilitate P. maculata invasions, other exotic plants that co-occur with P. maculata in the introduced range may facilitate P. maculata invasions. For instance, a feeding trial showed that P. maculata could survive and grow when offered the exotic plants Eichhornia crassipes, C. esculenta or M. spicatum, although it consumed little E. crassipes and C. esculenta (Burks et al. 2011). In 34 contrast, a feeding trial in China showed that P. canaliculata, another apple snail, had low survival and did not grow or reproduce when reared on E. crassipes, M. aquaticum or C. esculenta (Qiu and Kwong 2009). Studies on the facilitative effects of exotic plants on exotic herbivores through consumptive mechanisms are inconclusive. However, exotic plants may facilitate exotic herbivorous snails through non-consumptive mechanisms. For example, C. esculenta may indirectly facilitate P. maculata by providing substrata for egg oviposition (Burks et al. 2010). In our study, A. philoxeroides did not seem to be a preferred oviposition substratum because all P. maculata egg clutches were oviposited on the sides of tanks or on native P. cordata leaves and stems. Although the invasion of P. maculata was independent of A. philoxeroides, the wide variety of plant architecture and plant chemistry represented in exotic aquatic plants that co-occur with P. maculata leaves open the possibility that other exotic plants may facilitate or inhibit P. maculata invasions (Pearse et al. 2013). 2.6.2. Exotic species impacts Exotic herbivores affected native plants in all treatments by decreasing native plant mass and diversity independent of A. philoxeroides invasions. Similarly, a feeding trial showed that P. maculata consumed native plants (Ceratophyllum demersum, Hymenocallis liriosme, Ruppia maritima and Sagittaria lancifolia) and exotic plants (C. esculenta, A. philoxeroides and E. crassipes), but consumption of native plants was higher (Burlakova et al. 2009). However, another study showed that P. maculata did not differentially consume native and exotic plants (Qiu and Kwong 2009). Pomacea canaliculata has had severe impacts on native 35 plants (Lowe et al. 2004), transforming native wetlands in Thailand into open water habitats without plants (Carlsson et al. 2004). Our recorded effects of P. maculata on wetlands are comparable to those of P. canaliculata because P. maculata significantly reduced native plant mass and diversity in wetland communities. In contrast, A. philoxeroides only affected native plants in the presence of P. maculata. Other studies have shown that A. philoxeroides reduces native plant diversity in weedy habitats (Lin and Qiang 2006), native plant cover in aquatic habitats in New Zealand (Bassett et al. 2012) and species richness, diversity and evenness in India (Chatterjee and Dewanji 2014). The duration of a study and the initial exotic plant mass may influence the outcome of native and exotic plant interactions. Indeed, a longer experiment might provide a more conclusive test. Even though A. philoxeroides’ effects on native plants were greater with prior P. maculata invasions, their combined effect in the simultaneous [S & P] invasion treatment was additive. Johnson et al. (2009) also found that the effect of multiple exotic species on native species is greater than the effect of a single exotic species and greater effects of invaders from higher trophic levels. This suggests that exotic consumers with large consumptive effects on native species can facilitate lower trophic invaders, leading to multiply invaded ecosystems. 2.7. Acknowledgments We thank Steve Pennings for tanks; Candice Tiu, Nick Hill, Dana Galvan, Ariel Nixon, Erica Soltero and Leiyi Chen for assistance in the field and lab; Xin-Min Lu 36 for helpful comments; Romi Burks, Jenn Rudgers, Amy Dunham, Volker Rudolf and Anna Armitage for helpful discussions and two anonymous reviewers for comments. This work was supported by a Ford Foundation Predoctoral Fellowship (to MML), an Environmental Protection Agency Science to Achieve Results Fellowship for Graduate Environmental Study (to MML), a Constance E. Boone Graduate Student Research grant from the Houston Conchology Society (to MML), a Society of Wetland Scientist Graduate Research grant (to MML) and a Kroger Community Donation (to MML). Table 2-1 Description of experimental treatments. Each tank received topsoil, native plants and native snails in June 2010 and additional plants and snails in April 2011 and June 2011. Exotic plant or snail additions in each treatment are shown in bold Treatment April 2011 June 2011 Control [C] 60 g native plants 20 native snails 60 g native plants 20 native snails Plant invasion [P] 60 g native plants 20 native snails 60 g exotic plants 20 native snails Snail invasion [S] 60 g native plants 20 native snails 60 g native plants 3 exotic snails Simultaneous invasion-plants and snails [P&S] 60 g native plants 20 native snails 60 g exotic plants 3 exotic snails Sequential invasion-plants then snails [P–S] 60 g exotic plants 20 native snail 60 g native plants 3 exotic snails Sequential invasion-snails then plants [S–P] 60 g native plants 3 exotic snails 60 g exotic plants 20 native snails 37 30 (a) single a sequential simultaneous b b 20 10 0 P S-P Invasion treatment P&S A. philoxeroides mass (% of total) A. philoxeroides dry mass (g) 40 10 (b) sequential b 8 6 single a simultaneous a 4 2 0 P S-P P&S Invasion treatment Figure 2-1 (a) Alternanthera philoxeroides dry mass and (b) mass as percent of total plant mass in treatments with exotic plants introduced in June 2011. Means + 1 SE. Means with the same letter were not significantly different in post-hoc tests. P = exotic plant, S – P = exotic snail invasion followed by exotic plant invasion, P & S = simultaneous exotic plant and exotic snail invasion 38 (a) Native plant dry mass (g) 800 a single sequential ab bc 600 simultaneous c c d 400 200 0 C (b) P S P-S S-P P&S Native plant diversity 1.5 a 1.2 a b b b 0.9 c 0.6 0.3 0.0 C Native snail abundance (c) P S P-S S-P P&S P S P-S S-P P&S 800 600 400 200 0 C Invasion treatment Figure 2-2 (a) Native plant dry mass and (b) diversity in each invasion treatment. Means + 1 SE. Means with the same letter were not significantly different in post-hoc tests. C = native plants and native snails, P = exotic plant, S = exotic snail, P – S = exotic plant invasion followed by exotic snail invasion, S – P = exotic snail invasion followed by exotic plant invasion, P & S = simultaneous invasion by exotic plant and exotic snail 39 150 (a) single 40 sequential 30 20 (b) single simultaneous Adult mass (g) Adult operculum length (mm) 50 simultaneous 100 50 10 0 0 S P-S P&S S Invasion treatment 200 (c) (d) sequential single 150 100 P&S simultaneous simultaneous 250 200 P-S Invasion treatment Juvenile abundance 300 Egg clutch volume (ml) sequential 150 single 100 sequential 50 50 0 0 S P-S P&S Invasion treatment S P-S P&S Invasion treatment Figure 2-3 (a) Pomacea maculata operculum length, (b) adult mass, (c) reproductive output (total egg volume) and (d) juvenile abundance were independent of A. philoxeroides invasions when snails were introduced in June 2011. Means + 1 SE. S = exotic snail, P – S = exotic plant invasion followed by exotic snail invasion, P & S = simultaneous invasion by exotic plant and exotic snail 40 Chapter 3 Nutrient enrichment increased exotic snail Pomacea maculata growth independent of native and exotic wetland plant identity 3.1. Abstract Ecosystems invaded by many exotic species are often simultaneously exposed to other anthropogenic factors. Invaders may have common positive responses to anthropogenic factors that directly and indirectly increase exotic herbivore and exotic plant performance and also alter plant-herbivore interactions. We conducted two freshwater mesocosm experiments. In experiment one, we investigated nutrient enrichment effects on exotic herbivorous snails (Pomacea 41 42 maculata) fed detached lettuce leaves or grown with a single exotic plant species (Eichhornia crassipes or Alternanthera philoxeroides). Snail shell length varied with nutrients (control, low, or high), plant type, and their interactions. Nutrients did not affect shell length of snails fed lettuce, suggesting no direct nutrient enrichment effects on snail growth. However, snail mortality varied with plant species, suggesting that snail performance also depends on plant identity. In experiment two, we investigated: 1) nutrient enrichment effects on snail operculum length fed a single native (Limnobium spongia or Hydrocotyle umbellata) or exotic (E. crassipes or A. philoxeroides) plant species and 2) the effect of nutrient enrichment and exotic snail interactions on native and exotic plants. Elevated nutrients increased snail operculum length, but nutrients did not affect plant mass. Snails reduced plant mass, but plant mass was higher for exotic plants than native plants independent of snail and nutrient treatments, suggesting that these exotic plants may survive and grow in habitats with varying environmental conditions. Together these results suggest that nutrient enrichment will increase exotic herbivore growth and may contribute to multiple plant invasions, resulting in multiply invaded freshwater wetland ecosystems. 3.2. Keywords nutrient enrichment • Pomacea maculata • Alternanthera philoxeroides • Eichhornia crassipes • indirect effects • plant-herbivore interactions 43 3.3. Introduction Ecosystems invaded by many exotic species are often simultaneously exposed to other anthropogenic factors. Multiple invasions may be a result of exotic species’ common responses to anthropogenic factors (Mainka and Howard 2010; Montoya and Raffaelli 2010). For instance, plant invasions are often associated with nutrient enrichment (Tyler et al. 2007) because elevated nutrients favor exotic plants that possess traits that include rapid growth rate, low tissue production cost, and high phenotypic plasticity compared to resident native plants (Daehler 2003). However, our knowledge of nutrient enrichment’s direct and indirect effects on higher trophic levels, including exotic herbivores, is limited (Alonso and Camargo 2003). To better predict and understand exotic herbivore invasions, we need to increase our understanding of how anthropogenic factors such as nutrient enrichment directly and indirectly via plants affect exotic herbivore survival and growth. Nutrient enrichment’s direct effects on exotic herbivores are rarely investigated (Ebeling et al. 2013), but indirect effects via plants have been examined for native herbivores and on herbivores used as biocontrol agents. Nutrient enrichment may directly and indirectly affect exotic herbivore survival, growth, or reproduction, but our understanding of some of these effects is limited. For instance, an experimental study showed that elevated nutrients increased native aquatic snail mortality due to higher toxicity levels with elevated nutrient levels (Alonso and Camargo 2003). Similarly, studies on the indirect effects of nutrient enrichment on exotic herbivores via exotic plants are limited. Studies 44 evaluating nutrient enrichment indirect effects on herbivores often focus on herbivores that have been introduced to control and manage troublesome exotic plants, biocontrol agents. For example, nutrient enrichment increased foliar nitrogen in exotic plant water hyacinth (Eichhornia crassipes) which resulted in a shift of water hyacinth grasshopper (Cornops aquaticum), E. crassipes’ biocontrol agent, sex ratio towards females and increased female fecundity and body mass (Bownes et al. 2013a). Similarly, nutrient enrichment effects on E. crassipes increased the population growth and reproduction of weevils Neochetina eichhorniae and N. bruchi, biocontrols of E. crassipes (Center and Dray 2010). Together these studies emphasize that our understanding of the direct and indirect effects of nutrient enrichment on exotic herbivores is limited. Exotic herbivores may influence exotic plant invasions via several mechanisms, including higher consumptive effects on co-occurring native plants (Nuñez et al. 2010) and via non-consumptive effects on exotic plants. For instance, exotic red deer (Cervus elaphus) and fallow deer (Dama dama) enhanced the invasion of exotic Douglas fir (Pseudotsuga menziesii) by preferentially consuming native evergreen conifer (Austrocedrus chilensis) (Relva et al. 2009). In addition, non-consumptive damage by exotic crayfish (Orconectes rusticus) on exotic Eurasian watermilfoil (Myriophyllum spicatum) increased the probability of plant dispersal via plant fragmentation allowing plants to colonize and invade surrounding habitats (Maezo et al. 2010). Exotic plants may also influence exotic herbivore performance. For example, exotic plants increased larval survival and pupal weight and reduced development time of the 45 exotic generalist herbivore Epiphyas postvittana when compared to native plants (Engelkes and Mills 2013). Together these studies show that exotic herbivores may influence plant invasions via different mechanisms and that exotic plants may influence the performance of exotic herbivores. Studies have shown that the effect of biocontrol agents, special cases of introduced herbivores, on exotic plants can vary with nutrient availability (Coetzee et al. 2007; Bownes et al. 2013b). Elevated nutrients increased E. crassipes growth and reproduction and increased the population growth of biocontrol mirid (Eccritotarsus catarinensis) (Coetzee et al. 2007). In addition, elevated nutrients also increased E. catarinensis damage on E. crassipes but the effect of E. catarinensis on E. crassipes’ performance did not vary with nutrient levels (Coetzee et al. 2007). Nutrient enrichment and exotic herbivores interactions may alter their individual effects on exotic plants. For instance, exotic herbivore effects may be greater at low nutrient levels because exotic plants do not have the resources needed to recover quickly form herbivore damage. For example, exotic E. crassipes’ performance was lower at low nutrients with biocontrol C. aquaticum herbivory (Bownes et al. 2013b). In addition, exotic herbivore effects on exotic plants may be less severe with high resource availability because plant may have the resources needed to recover more quickly from herbivore damage. These studies showed that elevated nutrients alone increased exotic plant mass, biocontrol herbivores alone reduced exotic plant mass, but in combination they altered each other’s effects. These studies guide our predictions on the effects of exotic herbivores on exotic plants, but 46 studies on the effect of exotic herbivore and nutrient enrichment interactions on exotic plants are needed to predict exotic plant and exotic herbivore performance, probability of invasion success, and their impacts on native species in invaded ecosystems. Here, we conducted two greenhouse mesocosm experiments to investigate: 1) direct and indirect effects of nutrient enrichment on exotic aquatic snail survival and growth with living and non-living plants and 2a) nutrient enrichment effects on snail growth with single native or exotic aquatic plant species and 2b) effects of nutrient enrichment and exotic aquatic snail interactions on native and exotic plants from freshwater wetland ecosystems. 3.4. Methods 3.4.1. Study system In southeast Texas, exotic aquatic plants and snails heavily invade local freshwater wetlands. Exotic species that co-occur in this region include the exotic plants Alternanthera philoxeroides (alligator weed - Amaranthaceae), Eichhornia crassipes (water hyacinth - Pontederiaceae), and the exotic snail Pomacea maculata (island apple snail - Ampullariidae; synonym P. insularum; (Hayes et al. 2012). These exotic plants and snails are sympatric in their native (South America) and introduced ranges (USA). These exotic species co-occur with native aquatic plants that include Hydrocotyle umbellata (water pennywort Araliaceae) and Limnobium spongia (frog's bit - Hydrocharitaceae) in southeast Texas. Native H. umbellata and exotic A. philoxeroides were included because 47 they are both emergent plants that can grow in terrestrial and aquatic habitats and also co-occur in wetland ecosystems. Native L. spongia and exotic E. crassipes were included in this study because they are both free-floating aquatic plants with clonal reproduction and similar plant structure with leaves above the water surface. Alternanthera philoxeroides is native to the Parana River region of South America (Julien et al. 1995) and was first recorded in Mobile, Alabama, USA in 1897 and invaded coastal states from North Carolina to Texas by 1963 (Spencer and Coulson 1976). In aquatic habitats, A. philoxeroides anchors to banks and reproduces clonally, but does not produce viable seeds in its introduced ranges (Spencer and Coulson 1976; He et al. 2014). It has hallow stems that can root at the nodes when fragmented resulting in large interwoven mats or free-floating monocultures (Spencer and Coulson 1976; Sainty et al. 1997). Eichhornia crassipes is considered one of the worst aquatic weeds in the world with high socio-economic and ecological impacts (Lowe et al. 2004). It was introduced from South America to the USA in 1884, invaded southern USA states by the 1900’s, and its current distribution includes more than 50 countries on five continents (Lowe et al. 2004). This perennial herb is characterized by free-floating rosettes with clonal reproduction. Newly formed rosettes break easily, allowing E. crassipes to disperse and colonize neighboring habitats. Pomacea maculata’s native range expands north to the Amazon basin of Brazil and occurs in Uruguay, Paraguay, and Argentina (Hayes et al. 2008; Karatayev et al. 2009). It was discovered in 1989 in southeast Texas with source 48 populations likely from the lower Parana River Basin in La Plata region of Argentina (Hayes et al. 2012). Pomacea maculata populations have been reported along the Gulf Coast from Texas to Florida and along the Atlantic coast to South Carolina (Rawlings et al. 2007; Byers et al. 2013). It has been shown to have negative effects on several native freshwater wetland plants (Meza-Lopez and Siemann 2015). 3.4.2. Experimental set-up We conducted two mesocosm experiments in a temperature-controlled greenhouse at Rice University, Texas. We collected exotic plants and P. maculata egg clutches from Armand Bayou (Pasadena, TX). To simulate natural P. maculata hatching conditions in lab, we placed egg clutches on a mesh on top of a container that was partially filled with water; snails fell into the water as they hatched. They were reared on commercially purchased romaine lettuce until enough juvenile snails were available for each experiment (Conner et al. 2008). Native plants in the second experiment were commercially purchased. 3.4.3. Exp.1: Nutrients’ direct and indirect effects on P. maculata mortality and shell length Experiment 1 was conducted in fall 2009 with forty-five 19L containers filled with ~11.4L of dechlorinated municipal water. Fifteen juvenile P. maculata (~6-7mm initial foot length) were added to each mesocosm. Mesocosms were randomly assigned to: 1) nutrients: control (ambient nutrients in municipal water), low (0.5mg/L Nitrogen and 1mg/L of Phosphorus), or high (5mg/L Nitrogen and 49 10mg/L Phosphorus) and 2) plants: detached non-living lettuce leaves without roots or a single living exotic plant with roots (A. philoxeroides or E. crassipes) with five replicates per treatment in a factorial design. Nitrogen treatments were selected based on nutrient levels in aquatic ecosystems in southeast Texas. Phosphorus levels selected were higher than levels in Texas during non-drought conditions, but are typical of phosphorus levels during drought conditions. Including higher phosphorus levels would increase our understanding of the effects of nutrient enrichment under varying environmental conditions. Exotic plant treatments received 25g of a living exotic plant a single time and lettuce treatments received cut lettuce biweekly to refresh and replenish lettuce consumed by snails. Cut lettuce was used to investigate direct effects of nutrient enrichment on P. maculata because cut lettuce without roots is unlikely to absorb significant amounts of water nutrients before snails consume it compared to exotic living plants that have roots. Pomacea maculata hatchlings may feed on periphyton but switch to plants with increasing size. We replaced ~7.57L of water and replenished nutrients weekly to reduce snail waste accumulation and to maintain nutrient levels constant throughout the study. After nine weeks, we counted and measured snail shell length from apex to aperture end (Conner et al. 2008) and weighed exotic plants. 3.4.4. Exp. 2: Nutrient enrichment effects on P. maculata, plants, and plant-herbivore interactions Experiment 2 was conducted at the end of summer 2010 with ninety-six 19L containers filled with ~11.4L of dechlorinated municipal water. Mesocosms were 50 randomly assigned to: 1) plant treatment: a single native (L. spongia or H. umbellata) or exotic (E. crassipes or A. philoxeroides) plant; 2) nutrient treatment: control (ambient nutrients in municipal water), low (3mg/L N and 1mg/L of P), or high (6mg/L N and 2mg/L P); and 3) snail treatment: absence or presence (10 juvenile P. maculata) in a factorial design with four replicates per treatment. Mesocosms received the following plant mass depending on plant treatments: L. spongia = 20.60±0.06g, H. umbellata = 20.35±0.06g, E. crassipes = 20.47±0.07g, or A. philoxeroides = 20.50±0.05g a single time. Nutrient levels selected differed from experiment 1 to further assess the effects of nitrogen and phosphorus levels that are typical in southeast Texas on exotic species performance. Water was changed weekly as described in experiment 1. After six weeks, we measured snail operculum length and weighed native and exotic plant mass. Snail operculum length was used instead of shell length to quantify snail growth in experiment 2 because snail operculum length was recommended as a reliable measurements for snail growth and is widely used by researchers (Youens and Burks 2007). 3.4.5. Data analyses For experiment 1 data, we conducted 2-way ANOVAs to test the dependence of snail shell length and percent mortality on water nutrients, plants, and their interaction. Residuals for snail mortality were non-normal, primarily due to one tank with very high mortality (12 of 15 dead) compared to other replicates for that treatment (0 or 1 dead). To determine if it would be appropriate to exclude or keep that replicated tank, we conducted a 2-way ANOVA with and without that 51 replicate. Exclusion of that point made the main effects non-significant but the interaction term significant. We report results with all points included despite the departure from normality. For experiment 2 data, we conducted a 2-way ANOVA to test the dependence of snail operculum length on nutrients, plants, and their interactions. Average snail shell length and operculum length per tank were used and dead snails were excluded in snail size ANOVAs in experiment 1 and 2. We conducted a 3-way ANOVA to test the dependence of wet plant mass on snails, nutrients, plants, and their interactions. In addition, we conducted 2-way ANOVAs to test the dependence of individual plant species wet mass on nutrients, snails, and their interactions. Data met the assumptions of ANOVA (with the exception of snail mortality data). All analyses were conducted in SAS (v 9.4). 3.5. Results 3.5.1. Exp. 1: Nutrient enrichment’s direct and indirect effects on P. maculata mortality and shell length Pomacea maculata’s shell length varied with nutrients, plants, and their interaction (Table 3-1, Appendix Figure 3-1, Figure 3-1a). Snail mortality varied with plants (Table 3-1, Figure 3-1b). Nutrients or its interaction with plants did not alter snail mortality (Table 3-1). 52 3.5.2. Exp. 2: Nutrient enrichment effects on P. maculata, plants, and plant-herbivore interactions Pomacea maculata operculum length increased with elevated nutrients and varied with plant identity (Table 3-1, Figure 3-2). The combined effect of nutrient and plant treatments did not alter snail operculum length (Table 3-1). Wet plant mass was lower with snails and varied with plant treatments (Table 3-1, Figure 3-3). Nutrients and other factor interactions had no effect on wet plant mass (Table 3-1). Snails reduced H. umbellata, A. philoxeroides, and E. crassipes wet mass with no effect on L. spongia wet mass (Appendix Figure 3-1), Appendix Figure 3-2). Nutrients and its interaction with snails had no effect on wet plant species masses (Appendix Table 3-1). 3.6. Discussion Nutrient enrichment increased P. maculata operculum length with no effect on shell length or native and exotic plant mass. Nutrient enrichment did not alter snail shell length when snails were fed lettuce, but varied when they were fed exotic plants in experiment 1, indicating that P. maculata responded to nutrients indirectly via plants. In addition, nutrient enrichment increased snail operculum length in experiment 2, suggesting indirect nutrient enrichment effects. Varying snail shell and operculum length may have been a result of changes in plant nutritional value or varying plant physical defenses with elevated nutrients, but our study did not quantify either of these changes. However, studies conducted on biocontrol herbivores have shown that elevated nutrients increased exotic 53 E. crassipes tissue quality indirectly increasing the performance of biocontrol grasshopper (Bownes et al. 2013a), a mirid bug (Coetzee et al. 2007), and weevils (Center and Dray 2010). Furthermore, elevated nutrients may also change the physical defenses of plants which can then alter the survival, growth, and reproduction of herbivores. For example, nutrient enriched brown alga Fucus vesiculosus increased the growth rate, consumption, and reproduction of herbivorous isopod Idotea baltica by increasing insoluble sugars, reducing total carbon content, and reducing the physical toughness of the thallus (Hemmi and Jormalainen 2002), suggesting that nutrient enrichment can alter the nutritional value and physical defenses of plants. In experiment 1, P. maculata shell length was greater when they were fed lettuce than when they were fed either E. crassipes or A. philoxeroides. Similarly, Lach et al. (2000) found that P. canaliculata grew faster when fed cut lettuce than when fed E. crassipes or P. stratiotes. In our study, differences in snail shell length with plant identity may have been a result of varying plant physical defenses. The leaves and stems of E. crassipes and A. philoxeroides were physically tougher compared to lettuce leaves, but these differences in plant physical defenses were not quantified. Because our study did not quantity plant changes with nutrient enrichment and the length of our studies was not long enough to assess P. maculata’s population growth or reproduction, longer field studies on several native and exotic plants that quantify plant changes with nutrient enrichment and that assess the effect of plant changes on exotic herbivore growth and reproduction are needed. 54 Nutrients did not affect P. maculata mortality but mortality varied with plant identity in experiment 1. In contrast, survival of native aquatic snail Potamopyrgus antipodarum varied with nitrogen compound types with ammonia (96 hour LC50 = 2.02mg/L) being more toxic than nitrite (535mg/L) or nitrate (1042mg/L) (Alonso and Camargo 2003). However, ammonia levels in our high nutrient treatments (~0.05mg/L) were lower than those that caused snail mortality within 96 hours in their study (LC.01 = 0.16mg/L) (Alonso and Camargo 2003). In addition, a study in the Philippines showed that P. maculata mortality increased after nitrogen fertilizer was applied (Stuart et al. 2014), but ammonia concentrations in their study were equivalent to 80 to 400mg/L. These studies show that nutrient enrichment can have direct negative effects on native and exotic aquatic snails, but our results suggest that an increase in exotic snail operculum length with elevated nutrients via native and exotic plants in experiment 2 may be more important than the direct negative effects of nutrient levels in eutrophic southeast US waters. Pomacea maculata reduced overall wet plant mass and wet plant mass varied with plant species, but plant mass for both exotic plant species was higher compared to both native species. Another study showed that exotic African snail (Achatica fulica) and Cuban brown slug (Veronicella cubensis) had varying effects on native and exotic plants with high damage to some native and some exotic woody plants, but little damage to other native or exotic species (Shiels et al. 2014). In addition, a meta-analysis showed that exotic invertebrate herbivores can reduce native and exotic plant performance, but suggested that this 55 reduction in exotic plant performance would not influence exotic plant invasion success (Oduor et al. 2010). Higher exotic plant mass independent of snail presence compared to native plants suggests that exotic plants may survive and grow under varying biotic environmental conditions, but plant interactions under varying environmental conditions would need to be assessed to increase our understanding of plant invasion success. Our results on a limited number of native and exotic plants suggest caution about drawing inferences about aquatic plant invasion success based on changes in individual plant species mass with snails and in no-choice experiments with single plant species because herbivore feeding preferences and their effects on plant interactions can be complex and influence exotic plant performance and the their invasion success. Elevated nutrient enrichment did not alter wet plant mass or individual plant species masses. In contrast, other studies have shown significant effects of elevated nutrients on plant performance. For instance, nutrient enrichment affected native and exotic grasses in a fertilization experiment in western North American grasslands but its effect did not depend on plant origin (Seabloom et al. 2011). There is evidence that elevated nutrients increase plant mass with a general pattern of higher exotic plant productivity compared to native plants in deserts (Brooks 2003), grasslands (Huenneke et al. 1990), salt marshes (Tyler et al. 2007), and marine habitats (Gennaro and Piazzi 2014). A model also predicted that elevated nutrients can increase E. crassipes growth ten-fold (Wilson et al. 2005), suggesting that this may be one exotic aquatic plant species that may invade more readily with elevated nutrients. These studies show that 56 elevated nutrients increase plant productivity with greater effects on exotic plants in many ecosystems, but our study does not support those findings. We might not have been able to detect an increase in native and exotic plant mass with elevated nutrients with greater effects on exotic plants for several reasons that include the length of study, numbers of plant species included (single vs. many), type of study (greenhouse vs. field), time of the year when study was conducted, or nutrients may not have been limiting under greenhouse conditions. To increase our understanding of nutrient enrichment effects on native and exotic plants and their effects on exotic herbivores, longer field studies that include several native and exotic plants individually and in combination are needed. In conclusion, these studies suggest that freshwater wetland ecosystems may become readily invaded by exotic herbivores and multiple exotic plants. Nutrient enrichment may increase the probability of P. maculata invasions in aquatic ecosystems by increasing snail growth, potentially reducing time to reach reproductive maturity as has been suggested for P. canaliculata (Lach et al. 2000) that could increase their reproduction, growth rate, and spread to neighboring eutrophic habitats. Snails reduced exotic plants mass, but exotic plant mass was greater than native plants independent of snail presence, suggesting that these plants may survive and grow in habitats with varying biotic environmental conditions, but studies evaluating both plant and plant-herbivore interactions are needed. Together these results suggest that nutrient enrichment may increase the probability of multiple plant and herbivore invasions in freshwater wetland ecosystems. 57 3.7. Acknowledgments We thank Erika Soltero for her assistance in the lab and Romi Burks, Jenn Rudgers, Amy Dunham, Volker Rudolf, and Anna Armitage for helpful discussions. This work was supported by an AGEP-Rice University Fellowship (to MML) and a Ford Foundation Predoctoral Fellowship (to MML). Table 3-1 The dependence of exotic P. maculata snail shell length and percent mortality in experiment 1 and P. maculata operculum length and wet plant mass in experiment 2 on nutrients, plants, snails, and their interactions in ANOVAs. Significant results are shown in bold Experiment 1 Snail shell length Factor df Fx,36 P Experiment 2 Snail % mortality Fx,36 P Snail operculum length df Fx,34 P Wet plant mass df Fx,72 P Nutrients 2 15.2 <0.0001 0.1 0.9430 2 71.5 < 0.0001 2 0.9 0.3939 Plants 2 1,361.4 <0.0001 3.4 0.0449 3 3.0 0.0432 3 79.2 < 0.0001 Nutrients*plants 4 11.5 <0.0001 1.3 0.2724 6 2.0 0.0954 6 1.1 0.4019 Snails 1 30.0 <0.0001 Snails*nutrients 2 1.0 0.3819 Snails*plants 3 2.1 0.1041 Snails*nutrients*plants 6 1.0 0.4170 58 59 25 a a a Nutrients Control Low High a 20 15 cd 10 b b bc d e 5 0 20 P. maculata % mortality P. maculata shell length (mm) 30 b a 15 10 ab 5 b 0 Lettuce E. c. A. p. Plants Lettuce E. c. A. p. Plants Figure 3-1 The dependence of exotic P. maculata (a) shell length on nutrients and plants and (b) percent mortality on plant species in experiment 1. Means+1SE. Means with the same letter were not significantly different in post-hoc tests. E. c. = E. crassipes and A. p. = A. philoxeroides P. maculata operculum length (mm) 12 a b a b 8 c 4 0 Control Low Nutrients High L. s. H. u. A. p. E. c. Plants Figure 3-2 The dependence of exotic P. maculata operculum length on (a) nutrients and (b) plants in experiment 2. Means +1SE. Means with the same letters were not significantly different in post-hoc tests. L. s. = L. spongia, H. u. = H. umbellata, A. p. = A. philoxeroides, E. c. = E. crassipes 60 a b a Wet plant mass (g) 40 30 a b 20 b c 10 d 0 No snails Snails Snails L. s. H. u. A. p. E. c. Plants Figure 3-3 The dependence of wet plant mass on (a) snails and (b) plants in experiment 2. Means +1SE. Means with the same letters were not significantly different in post-hoc tests. L. s. = L. spongia, H. u. = H. umbellata, A. p. = A. philoxeroides, E. c. = E. crassipes 61 Chapter 4 Nutrient enrichment effects on invasions depend on climate warming: an experimental test with freshwater wetland plant communities 4.1. Abstract Anthropogenic environmental change can increase species invasions and reduce native biodiversity. Nutrient enrichment may favor exotic plant species that typically have higher growth rates. Warming may increase the performance of pre-adapted exotic species from warmer native ranges and/or decrease the performance of locally adapted native species. However, the community level impacts of nutrient enrichment and warming will depend on their combined effects on individual species and their interactions. We conducted an 11-month field experiment with freshwater wetland plant communities in 474L tanks in 62 63 Houston, TX that manipulated 1) plant community composition: native plants [4 species], exotic plants [3 species from warm, nutrient rich habitats], or both native and exotic plants [7 species]; 2) nutrients: ambient or high (+6mg/L Nitrogen and +1.7mg/L Phosphorus); and 3) temperature: ambient, +1°C, or +2°C in a factorial design. Elevated nutrients increased plant mass, favored exotic plants, and increased exotic plant diversity. Exotic plants reduced native plant diversity and reduced native plant mass in elevated nutrients. Elevated nutrients increased (2 exotic species) or decreased (1 exotic species, 3 native species) species’ relative abundance but for the majority of species the magnitude of these changes depended on warming. These results suggest that nutrient enrichment may increase invasions by some exotic plants but warming will modify the magnitude of these effects. In addition, warming will influence the particular exotic plant species that dominate invasions with nutrient enrichment and the native species that will be most heavily impacted in freshwater wetland ecosystems. 4.2. Keywords warming, nutrient enrichment, Alternanthera philoxeroides, Eichhornia crassipes, Pistia stratiotes, freshwater mesocosms 4.3. Introduction Anthropogenic factors can contribute to species invasions and reduce native biodiversity (Stevens et al. 2004; Walther 2010; Cockrell and Sorte 2013) and 64 generate novel communities (Hobbs et al. 2006; Williams and Jackson 2007). Researchers have emphasized the need to investigate the interactive effect of multiple anthropogenic factors on native and exotic species performance (Didham et al. 2007; Crain et al. 2008; Bradley et al. 2010; Dukes et al. 2011) and species interactions (Tylianakis et al. 2008; van der Putten et al. 2010; Gennaro and Piazzi 2011; Verlinden et al. 2014). Many studies have investigated the effect of individual anthropogenic factors on species performance (Tyler et al. 2007; Henry-Silva et al. 2008; Cockrell and Sorte 2013; Gennaro and Piazzi 2014) but only a few studies have investigated the effects of multiple anthropogenic factors on exotic species (Crain et al. 2008; Dukes et al. 2011; Griffiths et al. 2014; You et al. 2014). Nutrient enrichment typically increases plant growth, but often has larger positive effects on exotic plant performance compared to native plants, increasing exotic plant dominance and success in invaded habitats (Rickey and Anderson 2004; Tyler et al. 2007). Nutrient enrichment may favor exotic plants because some of the exotic plants possess characteristics that include rapid growth rates and high phenotypic plasticity which allows them to quickly take advantage of available resources. For instance, elevated nutrients increased native riparian species and Arundo donax mass, but favored A. donax (Quinn et al. 2007). In addition, these characteristics make exotic plants suitable candidates for wastewater remediation because they can efficiently remove nutrients from enriched water. Some of the exotic plants used in remediation include Eichhornia crassipes, Pistia stratiotes, and Alternanthera philoxeroides 65 (Boyd 1970; Jayaweera and Kasturiarachchi 2004; Rodríguez-Gallego et al. 2004; Lu et al. 2010). These subtropical exotic plants might also be used for wastewater remediation because they originate from aquatic ecosystems with high water nutrient levels (Roberto et al. 2009). Warming effects on exotic species have been investigated using various methods including climate based models (e.g. CLIMEX) that assess exotic species habitat suitability and potential species distributions in the introduced range (Ramsfield et al. 2007; Kriticos et al. 2007; Potter et al. 2009) and experiments that evaluate the effect of warming on exotic species performance (Dukes et al. 2011). Climate based models predict how warming may influence exotic species distribution by comparing the climate in the native and the introduced range of exotic species or by quantifying changes in the distribution of exotic species at a particular location over time. Climate based models that compare climatic conditions in the native and introduced range of exotic species are limited because they primarily focus on the range of tolerance of exotic species. Furthermore, climate based models often predict wider species distributions than what is observed because they do not consider the effect of dispersal, species interactions, or the interaction of warming with other anthropogenic factors (Davis et al. 1998a; Wharton and Kriticos 2004). Warming effects via changes in maximum, minimum, and average temperatures may alter the distribution of exotic species by altering the length of the growing season which may affect exotic species survival, growth, and reproduction. Furthermore, warming may alter the outcome of native and exotic 66 species interactions, altering community composition patterns if native and exotic plant responses to warming vary in direction or magnitude (Davis et al. 1998b; Dukes and Mooney 1999; Rahel and Olden 2008; Walther et al. 2009). For example, experimental warming increased exotic yellow star thistle’s growth and abundance with no effect on native plants, suggesting that warming may favor those exotic plants (Dukes et al. 2011). In contrast, experimental warming with temperatures above 30°C reduced the growth rate of native Azolla filiculoides and Lemna minor and exotic Salvia molesta, suggesting that warming can have a negative effect on both native and exotic plants (van der Heide et al. 2006b). However, warming may increase the performance of exotic subtropical plants such as E. crassipes, P. stratiotes, and A. philoxeroides that are sensitive to cold temperatures, hard freezes, and ice cover (Owens and Madsen 1995; Li et al. 2012). These studies show varying effects of warming on native and exotic plants, suggesting that additional studies are needed to increase our understanding of warming effects on exotic species and their effect on native species. Eichhornia crassipes (water hyacinth - Pontederiaceae) is considered one of the worst aquatic weeds in the world with high socio-economic and ecological impacts (Lowe et al. 2004). It was intentionally introduced from South America to the USA in 1884, invaded southern USA states by the 1900’s, and it currently invades more than 50 countries on five continents (Lowe et al. 2004). The optimum temperature for E. crassipes growth is 28-30°C. A CLIMEX model showed that southern United States is suitable habitat for E. crassipes (Brunel 67 2012). In addition, a study showed that low temperatures ranging from 5-8°C reduced the regrowth potential of E. crassipes, changing its distribution (Owens and Madsen 1995). Furthermore, winter temperatures in the Great Lakes reduced E. crassipes winter survival (Rixon et al. 2005), influencing population growth and infestation during the growing season. However, warming may allow E. crassipes to overwinter in the Great Lakes. Eichhornia crassipes has been observed in various locations with air and water temperatures between 8-10°C and 10-11°C (Adebayo et al. 2011). In addition, experimental warming in the winter increased E. crassipes’ survival and clonal reproduction (You et al. 2013). Pistia stratiotes (water lettuce - Araceae) is on the list of the World’s Worst Weeds (Evans 2013). It was introduced from South America to the United States, likely accidentally, and was first reported in Florida in 1765 (Stuckey and Les 1984). It is currently found in Europe, Africa, and Asia (Neuenschwander et al. 2009). The optimum temperature for P. stratiotes growth is 22-30°C. A study showed that freezing temperatures in South Florida increased P. stratiotes’ winter mortality, suggesting that P. stratiotes is sensitive to low temperatures (Dewald and Lounibos 1990). Warming may increase the distribution of P. stratiotes by increasing temperature during winter. Pistia stratiotes has been able to overwinter in a natural thermal stream in Topla in Slovania that has water temperature above 17°C all year (Šajna et al. 2007) and in ponds with warm water discharges in the winter (Kadono 2004; Tamada et al. 2015). Alternanthera philoxeroides (alligator weed- Amaranthaceae) was accidentally introduced from the Parana River region of South America (Julien et 68 al. 1995). In the United States, it was first recorded in Mobile, Alabama in 1897 and invaded coastal states from North Carolina to Texas by 1963 (Spencer and Coulson 1976). The optimum temperature for A. philoxeroides growth is 15-20°C. Shorter growing seasons and a higher number of frost events at higher latitudes have been shown to kill the top growth of A. philoxeroides, reducing its mass accumulation during the growing season (Julien et al. 1992). A climate based model using distribution data from the native range of A. philoxeroides showed that the southern United States is suitable habitat for A. philoxeroides (Julien et al. 1995). However, an experimental study showed that warming effects on A. philoxeroides depend on the plant’s physical characteristics. Warming reduced the growth of A. philoxeroides fragments that had connected stolons but increased growth of fragments with severed stolons (Li et al. 2012). Warming in Houston may benefit these exotic plants by reducing frost events that may reduce winter mortality and increase mass accumulation during the growing season. Species interactions may alter the effect of warming on species with other anthropogenic factors influencing the effects of warming. For instance, warming increased exotic Solidago gigantea dominance when it was planted with native Plantago lanceolata, but reduced the dominance of S. gigantea when planted with native Epilobium hirsutum (Verlinden et al. 2014). In addition, nitrate reduced exotic Centaurea solstitialis establishment in ambient temperature, but increased its establishment with warming (Dukes et al. 2011). Furthermore, nutrient enrichment together with warming increased the growth and clonal 69 propagation of E. crassipes in China (You et al. 2014). Studies investigating the interactive effect of multiple anthropogenic factors on exotic species are increasing, but our understanding of how plant interactions alter the effect of anthropogenic factors on species invasions and their impact on native species remain limited (Didham et al. 2007). Here, we investigated the individual and interactive effects of nutrient enrichment and warming on plant invasions in native freshwater wetland plant communities and their effect on native plants with a field mesocosm experiment. 4.4. Methods 4.4.1. Study system Alternanthera philoxeroides, E. crassipes, and P. stratiotes heavily invade wetlands in southeast Texas. These plants are sympatric in their native range (South America) and co-occur in the introduced range with native wetland plants that include Hydrocotyle umbellata (water pennywort - Araliaceae), Limnobium spongia (frog's bit - Hydrocharitaceae), Pontederia cordata (pickerelweed Pontederiaceae), Typha latifolia (cattail - Typhaceae), and native snails Physa acuta (bladder snail - Physidae) and Planorbella trivolvis (marsh ramshorn snail Planorbidae). We selected plants in this study based on their functional characteristics. Native H. umbellata, T. latifolia, P. cordata and exotic A. philoxeroides are emergent plants. Hydrocotyle umbellata and A. philoxeroides can grow in both terrestrial and aquatic habitats. Exotic E. crassipes, P. stratiotes, and native L. spongia are free-floating aquatic plants 70 with clonal reproduction. Eichhornia crassipes and L. spongia have similar plant structure. Furthermore, P. cordata and E. crassipes belong to the same family. Alternanthera philoxeroides in aquatic habitats anchors to banks and reproduces clonally with no viable seeds in its introduced range (Spencer and Coulson 1976; He et al. 2014). It can form large interwoven mats or free-floating monocultures from stem fragments because the hollow stems can float and root at the nodes (Spencer and Coulson 1976; Sainty et al. 1997). Eichhornia crassipes reproduces clonally and has free-floating rosettes. Newly formed rosettes can break easily, allowing E. crassipes to disperse and colonize neighboring habitats. Similarly, P. stratiotes is characterized by free-floating rosettes that can exist on their own or as interconnected clones forming free-floating mats. 4.4.2. Experimental setup To investigate the individual and interactive effect of nutrient enrichment and warming on native and exotic plants and their interactions, we conducted a 3×2×3 factorial mesocosm experiment that manipulated 1) plant composition: native plants only (H. umbellata, L. spongia, P. cordata, and T. latifolia), 2) exotic plants only (A. philoxeroides, E. crassipes, and P. stratiotes) or both (four native and three exotic plant species), 2) nutrients: control (ambient) or high (+6mg/L Nitrogen and +1.7mg/L Phosphorus), and 3) temperature: ambient, +1°C, or +2°C with four replicates per treatment. In May 2012, we set up 72 mesocosms by adding 15cm of topsoil/sand mixture and water to 454L tanks at the Rice University South Campus Field 71 Station. We added an individual of each of the four native plant species to tanks assigned to native plant only communities. We added an individual of each of three exotic plant species to tanks assigned to exotic plant only communities. We added approximately 160g of fresh plant mass to each of these tanks. We added an individual of each of the four native and an individual of each of the three exotic plant species to the tanks with both groups (~ 320g of fresh plant mass). We replaced plants that did not survive at least two weeks after the initial planting with a new individual of equivalent mass. After two weeks, we added 40 native P. acuta and P. trivolvis snails to all mesocosms to reduce snail variation among plant communities (because they are often accidentally introduced to mesocosms). We selected nutrient levels based on nutrient levels in southeast Texas wetlands. To deliver the randomly assigned nutrient and temperature water treatments to each community, we setup a flow-through watering system with drip emitters in each tank that resulted in a four-day hydraulic residence time. We mixed a soluble fertilizer (28-8-18) with municipal water in a holding tank that was then added into the water lines for the elevated nutrient treatments with a fertilizer injector (Dosatron) and then delivered to communities. To achieve temperature treatments, we passed water through zero (ambient), two (+1°C), or four (+ 2°C) solar water heaters (each 1.2 x 6 m, Specialty Pool Products, East Windsor CT, USA) before being delivered to communities. We supplemented solar water heaters with electric water heaters (four 1800 watt heaters, SR-15L, Chronomite, City of Industry CA, USA) during the winter months due to low solar radiation. We conducted monthly visual plant surveys to estimate plant cover by 72 species. After 11 months, we harvested aboveground plant mass that was sorted by species, oven-dried, and weighed. 4.4.3. Data analyses We used ANOVAs to test the dependence of plant mass and diversity (Shannon Diversity Index) on plant composition, nutrients, temperature, and their interactions. We conducted repeated measures ANOVAs to test the dependence of percent cover on community composition, nutrients, temperature, and their interactions. We excluded native plant only communities from exotic plant analyses and exotic plant only communities from native plant analyses. We conducted repeated measures ANOVAs on species’ proportional cover in communities with both native and exotic plants to test their dependence on nutrients, temperature, and their interactions. Data met the assumptions of ANOVA and repeated measures ANOVA. We conducted post-hoc tests to determine the differences between treatment levels for significant variables with more than two levels. We also conducted PERMANOVAs to examine community variation in native plant only, exotic plant only, and communities with both native and exotic plants in relation to nutrients, temperature, and their interaction using plant mass. We performed all ANOVAs using SAS (v 9.4) and PERMANOVAs using R (v 3.2.0). We conducted NMS ordinations using mass by species to examine variation in native plant only, exotic plant only, and communities with both native and exotic plants in relation to nutrients, temperature, and their interaction (PCord4, 500 iterations, Sorensen [Bray-Curtis] distance, 2 initial axes, 0.01 step-down threshold, 0.005 stability criterion). In addition, we 73 predicted exotic to native plant mass ratios from native plant only and exotic plant only communities and compared it to the observed exotic to native plant mass ratio in plant communities with both native and exotic plants. The predicted exotic to native plant mass ratio was obtained by randomly pairing exotic and native plant masses from native plant only and exotic plant only communities (5000 times, without replacement). 4.5. Results Native plant only, exotic plant only, and communities with both native and exotic plants varied in relation to nutrients but were not influenced by temperature or their interaction (Table 4-1, Figure 4-1). Elevated nutrients increased native plant mass and native plant mass was higher in native plant only communities (Table 4-2, “nutrients”, “composition*nutrients”, Figure 4-1b,1d, Figure 4-3a). Other factors and their interactions did not affect native plant mass (Table 4-2). Native plant diversity depended on community composition and on nutrients (Table 4-2, Figure 4-3a). Temperature and factor interactions did not affect native plant diversity (Table 4-2). Exotic plant mass depended on community composition, nutrients, and their interaction (Table 4-2, “nutrients”, “composition*nutrients”, Figure 4-1a,1c, Figure 4-3c). Temperature and other factor interactions did not affect exotic plant mass. Elevated nutrients increased exotic plant diversity independent of community composition, temperature, and their interactions (Table 4-2, “nutrients”, Figure 4-3c-d). 74 Native plant percent cover varied with community composition, nutrients, and their interaction (Appendix Table 4-1). Temperature and other factor interactions did not affect native plant percent cover (Appendix Table 4-1). Elevated nutrients increased exotic plant percent cover independent of community composition, temperature, and their interaction (Appendix Table 4-1, “nutrients”, Figure 4-2). In most cases, the expected exotic to native plant mass ratio was significantly higher than in communities with both native and exotic plants with the exception of ambient nutrients and in +1°C treatments (Table 4-3). Elevated nutrients increased native P. cordata and T. latifolia mass with no effect on H. umbellata (Appendix Figure 4-1g,n,k; respectively). Limnobium spongia’s mass varied with community composition, nutrients, and their interaction (Appendix Figure 4-1i-j). Elevated nutrients increased L. spongia’s mass in native plant only communities (Appendix Figure 4-1i, Appendix Figure 4-2a). Temperature and other factor interactions did not affect the mass of native species (Appendix Figure 4-1g-n). Eichhornia crassipes’ mass varied with community composition, nutrients, and their interaction (Appendix Figure 4-1). Elevated nutrients increased E. crassipes’ mass in exotic plant only communities (Appendix Figure 4-2b). Elevated nutrients increased exotic A. philoxeroides’ mass but had no effect on P. stratiotes’ mass (Appendix Figure 4-1c,e). Temperature and other factor interactions did not affect E. crassipes, A. philoxeroides, and P. stratiotes’ mass (Appendix Figure 4-1a-f). Elevated nutrients increased proportional plant cover of exotic P. stratiotes and E. crassipes and reduced proportional plant cover of exotic A. philoxeroides and native P. cordata, L. spongia, and T. latifolia (Table 4-4). Elevated nutrients had 75 no effect on H. umbellata and warming reduced P. cordata’s proportional plant cover (Table 4-4). Furthermore, warming reduced the effect of elevated nutrients on E. crassipes’ proportional plant cover (Table 4-4). Warming with elevated nutrients increased H. umbellata’s proportional plant cover (Table 4-4). Warming increased L. spongia’s proportional plant cover in ambient temperature treatments (Table 4-4). 4.6. Discussion Nutrient enrichment increased exotic plant invasions but warming influenced the magnitude of nutrient enrichment effects on exotic species. Variation in species’ responses to nutrient enrichment and warming suggest that these effects are species specific results of plant characteristics including growth rate. For instance, exotic E. crassipes and native L. spongia had similar responses to nutrient enrichment and warming possibly because both have similar phenotypes and growth forms. They also grow rapidly with elevated nutrients, allowing them to take advantage of available resources possibly outcompete other species in the community. A study conducted in China also found that the growth, clonal reproduction, and shoot/root ratio of E. crassipes increased with nutrient enrichment and with warming but without an interactive effect (You et al. 2014). Nutrient enrichment favored exotic plants increasing plant invasion success but these effects were greater for exotic plants in exotic plant only communities than for exotic plants in communities with both native and exotic plants. Other studies have shown that competition with native plants can reduce the positive 76 effects of nutrient enrichment on exotic plants. For instance, elevated nutrients increased exotic Centaurea solstitialis mass in monoculture but had no effect on C. solstitialis mass when grown with native Avena barbata (Dukes et al. 2011). In addition, exotic Acacia saligna outperformed native Protea repens in elevated nutrient conditions, but their performance was equivalent when grown together in low nutrient conditions (Witkowski 1991). In our study, native T. latifolia and exotic E. crassipes were the dominant species in communities with both native and exotic plants. These species could have reduced each other’s performance and that of other species because both have rapid growth rates that allow them to take advantage of available resources. Even though native T. latifolia had higher mass than L. spongia in our study, it is possible that L. spongia had a greater effect on E. crassipes’s invasion compared to T. latifolia because L. spongia and E. crassipes have similar phenotypes and growth rates. Both have low stature in ambient or low nutrient conditions and elongated petioles and broader leaves in elevated nutrient conditions. They are also often mistaken for one another in the field because they look very similar to each other especially in high nutrient water habitats. Furthermore, higher predicted exotic to native plant mass ratio from native plant only or exotic plant only communities than in communities in which they co-occurred suggests that it is necessary to investigate effects of multiple anthropogenic factors at the community level to understand their effects on plant invasions and their combined impact on native species. 77 Nutrient enrichment increased plant invasion success overall but not all exotic plants benefitted from elevated nutrients. Althernanthera philoxeroides was the most abundant exotic plant in ambient nutrient conditions, suggesting that it may be a more problematic invader in low resource habitats. Another study, showed that E. crassipes may survive in ambient nutrient conditions but requires abundant nitrogen, phosphorus, and potassium to grow (Rodríguez-Gallego et al. 2004). Only some species had positive responses to nutrients, perhaps due to variation in their characteristics. Our results show that exotic plant mass varied with nutrient enrichment and suggest that exotic species’ mass is not always higher than native species’ mass under the same environmental conditions in similar conditions. Exotic species performance may be higher, lower, or equivalent to the performance of native species depending on species identity and environmental conditions (Daehler 2003). Furthermore, we expected elevated nutrients to reduce exotic plant diversity because some of these exotic plants have higher growth rates that may result in an increase in competitive ability, potentially excluding slow growing plants. In our study, elevated nutrients increased exotic plant diversity because more species were abundant with elevated than with ambient nutrients. It is possible that fewer exotic plants persisted in ambient nutrient conditions because these exotic plants are preadapted to nutrient rich water in their native range (Roberto et al. 2009). Our results suggest that nutrient poor habitats may be less susceptible to plant invasions and that nutrient rich habitats will increase the probability of multiple plant invasions in aquatic ecosystems. 78 We expected warming to increase plant invasions but instead it had no effect on plant invasions. We expected exotic plants to be closer to their optimum climatic conditions during the growing season with warming and to have lower winter mortality and higher exotic plant regrowth and mass accumulation during the growing season with an increase in winter temperatures. However, we might not have detected an effect of warming in Houston because winter temperatures were mild, suggesting that it may not have had an effect on winter plant mortality. In addition, climate based models predict that areas in Houston where E. crassipes and A. philoxeroides occur are already suitable habitats for these species (Julien et al. 1995), suggesting that an increase of 1°C to 2°C in Houston will not affect plant invasions but may influence the effect of nutrient enrichment on some exotic species. Furthermore, the length of the study may not have been long enough to quantify warming effects on exotic plant regrowth and mass accumulation during the growing season. Other studies have shown that warming may have positive and negative effects on plant invasions (Bradley et al. 2010). For instance, De Sassi et al. (2012) showed that warming reduced native species richness and increased exotic plant abundance. Warming may increase plant invasions by increasing exotic species growth rates in areas that are already suitable for the species or by reducing winter mortality closer to their northern range of distribution as predicted by climate based models. In contrast, warming reduced the growth of exotic plants in an Australian temperate grassland relative to native plant growth (Williams et al. 2007). Warming may also reduce exotic plant performance when its maximum temperature tolerance 79 is reached. These studies show that warming may have opposing effects on plant invasions; therefore additional field studies that use information from climate based models are necessary to increase our understanding of the mechanisms by which warming may affect plant invasions and exotic species performance throughout their predicted distribution range and their effect on native communities. In conclusion, nutrient enrichment will likely increase plant invasions in these ecosystems but the magnitude of this effect on exotic species likely depends on warming. Elevated nutrients increased exotic plant mass and diversity, increasing the probability of multiple plant invasions in invaded habitats. However, nutrient enrichment effects on plant invasions based on exotic plant only communities overestimated the actual effects in communities with both native and exotic plants. In addition, warming had no effect on plant invasions possibly because these exotic species already occur in habitats with suitable climatic conditions as predicted by climate based models. However, it did increase the relative abundance of some exotic species. Therefore, additional experimental studies that use information from climate based models are necessary to increase our understanding of the mechanism by which nutrient enrichment and warming affect plant invasions and their combined effect on native communities. 4.7. Acknowledgements We thank: Candice Tiu, Qiang Yang, Peter Sylvers, Jordan Bunch, Daniel McDermott, Abby Lamb, Cassandra Corrales, Dana Galvan, Michelle Shapiro, 80 Sarah Margolis, Ariel Nixon, Ling Zhang, and Gang Liu for their assistance in the field; Romi Burks, Amy Dunham, Volker Rudolf, and Anna Armitage for helpful discussions. This work was supported by a Ford Foundation Predoctoral Fellowship (to MML), an Environmental Protection Agency Science to Achieve Results Fellowship for Graduate Environmental Study (to MML), a Rice University Shell Center for Sustainability research grant (to MML), and a Rice centennial award to support undergraduate research (to MML). 81 Table 4-1 The dependence of community composition using plant mass in native plant only, exotic plant only, and communities with both native and exotic plants on nutrients, temperature, and their interaction in PERMANOVAs. Significant results are shown in bold Native only Factor Nutrients Temperature Nutrients*temperature Exotic only Native + Exotic DF Pseudo-Fx,18 P(perm) Pseudo-Fx,18 P(perm) Pseudo-Fx,18 P(perm) 1 2 2 36.0 2.0 2.2 0.0002 0.1152 0.0872 57.6 0.7 0.6 0.0002 0.2482 0.3202 22.5 0.7 0.6 0.0002 0.6164 0.6914 Table 4-2 The dependence of native and exotic plant mass and diversity (Shannon Diversity Index) on plant composition, nutrients, temperature, and their interactions in ANOVAs. Significant results are shown in bold Native mass Factor DF Fx,36 2.6 P Native diversity F x,36 P Exotic mass F x,36 Composition 1 0.1152 9.5 0.0039 6.5 Nutrients 1 90.4 <0.0001 18.0 0.0001 Temperature 2 1.7 0.1903 1.2 0.3297 2.4 Composition*nutrients 1 5.2 0.0280 2.0 0.1616 Composition*temperature 2 0.1 0.8904 2.5 Nutrients*temperature 2 0.8 0.4711 Composition*nutrients*temperature 2 1.0 0.3711 P Exotic diversity F x,36 P 0.0153 3.4 0.0730 90.8 <0.0001 18.8 0.0001 0.1079 0.5 0.6140 6.5 0.0153 2.9 0.0978 0.0957 0.5 0.6131 1.2 0.3051 1.7 0.1950 2.3 0.1128 0.1 0.9479 0.5 0.6169 0.5 0.5965 0.1 0.5835 82 83 Table 4-3 The dependence of exotic to native plant mass ratio on nutrients, temperature and their interaction, in contrasts of expected ratio from native only and exotic only plant communities and the observed ratio from communities with both native and exotic plants. Significant results are shown in bold Contrast Expected Observed P All communities 0.37 0.28 0.0080 Ambient nutrients High nutrients +0°C +1°C +2°C Ambient nutrients, +0°C High nutrients, +0°C Ambient nutrients, +1°C High nutrients, +1°C Ambient nutrients, +2°C High nutrients, +2°C 0.07 0.45 0.50 0.34 0.29 0.06 0.36 0.36 0.35 0.16 0.1500 <0.0001 <0.0500 0.1200 <0.0001 0.09 0.06 0.0400 0.56 0.48 0.0400 0.05 0.08 0.0400 0.45 0.44 0.0400 0.06 0.04 0.0400 0.34 0.20 0.0400 84 Table 4-4 The dependence of species cover on nutrients, temperature, and their interactions in repeated measures ANOVAs using proportional cover for each species in communities with both native and exotic plants. Significant results are shown in bold Repeated measures Species Nutrients F1,18 Exotic A. alligator 79.7 philoxeroidesweed water E. crassipes 236.4 hyacinth P. stratiotes water lettuce 17.8 Native T. latifolia cattail 29.4 L. spongia frog’s bit 96.7 water H. umbellata 2.9 pennywort P. cordata pickerelweed 29.5 Temperature Nutrients *temperature F2,18 P P F2,18 P <0.0001 0.2 0.8014 0.9 0.4094 <0.0001 4.3 0.0301 3.9 0.0404 0.0005 1.9 0.1746 2.8 0.0873 <0.0001 <0.0001 2.9 4.1 0.0827 0.0338 1.4 6.3 0.2636 0.0085 0.1034 6.7 0.0066 6.4 0.0080 <0.0001 3.7 0.0467 2.3 0.1255 85 25 a. exotics only b. natives only 0.6 20 0.3 Axis 2 Rank 15 10 +0C - control +1C - control +2C - control +0C + NP +1C + NP +2C + NP 5 0 -0.3 1 kg 2 kg 4 kg -0.6 1.0 1.0 c. native + exotic [exotic mass] 0.5 d. native + exotic [native mass] 0.5 Axis 2 Axis 2 0.0 0.0 -0.5 0.0 -0.5 -1.0 -1.0 -1 0 Axis 1 1 2 -1 0 1 2 Axis 1 Figure 4-1 The dependence of plant mass in communities with (a) exotic plants only or (b) native plants only ,and (c) exotic plant mass and (d) native plant mass in communities with both native and exotic plants. 86 Percent cover (average over 11 months) 120 100 80 60 Native T. latifolia L. spongia H. umbellata P. cordata Exotic A. philoxeroides P. stratiotes E. crassipes 40 20 0 ol ol ol i gh i gh i gh contr contr contr C-h C-h C-h 0 1 2 C C + + + C 2 + +0 +1 Temperature - nutrients Figure 4-2 The dependence of percent cover for each native and exotic plant species on temperature and nutrients in communities with both native and exotic plants 87 (a) (b) 0.8 a 4 b 3 2 c c a Native diversity Native mass (kg) 5 Nutrients control high b 0.6 0.4 0.2 1 0.0 0 np np+ ep (c) np + ep ep np + ep 4 a 3 2 b Exotic diversity Nutrients control high 5 Exotic mass (kg) np (d) 0.8 0.6 0.4 0.2 1 c c 0.0 0 ep np + ep Community composition Community composition Figure 4-3 The dependence of (a-b) native and (c-d) exotic plant mass and diversity on nutrients and community composition. Means +1SE. Means with the same letters were not significantly different in post-hoc tests. Np = native plant, ep = exotic plant, np + ep = native and exotic plants in community 88 Chapter 5 Warming increased exotic snail reproduction independent of nutrient enrichment and plant invasions in wetland communities 5.1. Abstract Anthropogenic factors can contribute to multiple plant and animal invasions in invaded ecosystems. Climate warming and nutrient enrichment individually and in combination may affect plant and herbivore invasions directly or impact invasions by changing plant-herbivore interactions. To investigate warming and nutrient enrichment effects on plant and snail invasions and their effect on native plant and snail communities, we established forty freshwater wetland communities with native plants and snails that were then invaded by an exotic snail and assigned to three treatments in a factorial design: 1) native or exotic 89 plant addition, 2) control (no addition) or elevated nutrients [+6mg/L Nitrogen and +2mg/L Phosphorus], and 3) ambient or +2°C temperature. Warming did not affect plants but increased exotic snail reproduction four-fold. Nutrient enrichment and warming increased exotic snail mass resulting in an increase in snail reproduction and population growth rates by reducing the time needed for snails to reach reproductive maturity. Elevated nutrients also increased plant mass and native snail abundance. However, exotic plant mass was higher than native plant mass, suggesting that nutrient enrichment will increase the likelihood of plant invasions. In addition, exotic plants reduced native plant mass while elevated nutrients and warming had no effect on native plant mass. Our results suggest that warming with elevated nutrients could increase the likelihood of aquatic herbivore invasions and that nutrient enrichment could enhance the effect of exotic plants on native plants in freshwater wetland communities. 5.2. Keywords warming, nutrient enrichment, Pomacea maculata, Alternanthera philoxeroides, Eichhornia crassipes, Pistia stratiotes, wetland communities, mesocosms 5.3. Introduction Many anthropogenic factors are known to contribute to species invasions that decrease native biodiversity (Stevens et al. 2004; Walther 2010; Cockrell and Sorte 2013) and generate novel communities (Hobbs et al. 2006; Williams and Jackson 2007). Researchers have repeatedly emphasized the need to 90 investigate the interactive effect of multiple anthropogenic factors on exotic species and species interactions (Gritti et al. 2006; Scherer-Lorenzen et al. 2007; Didham et al. 2007; Tylianakis et al. 2008; Crain et al. 2008; van der Putten et al. 2010; Griffiths et al. 2015) to better predict species invasions and increase our understanding of the effects of multiple anthropogenic factors on exotic species and their effect on native ecosystems. Anthropogenic factors such as warming and nutrient enrichment may increase the probability of plant and consumer invasions based on studies that have investigated their individual effects (Wilson et al. 2005; Quinn et al. 2007). However, predictions of the interactive effects of multiple anthropogenic factors on species based on their individual effects may under or overestimate these effects because their interactive effect can be complex (Dukes et al. 2011; Hoover et al. 2012). Experimental studies in terrestrial (Hoover et al. 2012) and aquatic ecosystems (Doyle et al. 2005) have shown that the interactive effect of multiple anthropogenic factors may be larger (synergistic) or smaller (antagonistic) than their expected additive effects (Folt et al. 1999; Darling and Côté 2008). Studies investigating interactive anthropogenic factor effects on plant and consumer invasions are increasing, but our understanding of these interactive effects on exotic plants and consumers at the community level remains limited (Dukes and Mooney 1999; Vilà et al. 2007; Didham et al. 2007; Tylianakis et al. 2008). Warming can increase the survival, growth, and reproduction of exotic aquatic plants (You et al. 2014) and aquatic animals (Sorte et al. 2010) by increasing the length of the growing season and reducing winter mortality (Davis et al. 1998b; 91 Dukes and Mooney 1999; Rahel and Olden 2008; Walther et al. 2009). Studies show that warming can enhance (Dukes and Mooney 1999; Sorte et al. 2010; Chuine et al. 2012) or inhibit species invasions (Williams et al. 2007) and also influence species interactions (Verlinden et al. 2014). For instance, warming increased exotic Solidago gigantea dominance depending on the plant species with which it was grown, suggesting that warming influences plant interactions (Verlinden et al. 2014). Warming also increased exotic consumer herbivory (Llorens et al. 2004; Rahel and Olden 2008), but herbivore effects on plants can depend on warming effects on plants, suggesting that warming can alter plantherbivore interactions (van der Heide et al. 2006a). These studies show that our understanding of the effects of warming on exotic plants, consumers, and plant-herbivore interactions at the community level is limited. Studies have investigated nutrient enrichment effects individually and in combination with other anthropogenic environmental factors on terrestrial (Dukes et al. 2011) and on aquatic plants (Wersal and Madsen 2011; You et al. 2014). Elevated nutrients increase native and exotic plant growth, but frequently favor exotic plants, increasing their dominance in invaded habitats (Daehler 2003; Rickey and Anderson 2004; Scherer-Lorenzen et al. 2007; Tyler et al. 2007). The dominance and performance of exotic plants often increases with available resources because exotic plants often have characteristics that include rapid growth rate and high phenotypic plasticity that allows them to quickly take advantage of available resources, outcompeting native plants that do not possess these characteristics (Kennedy et al. 2009). Similarly, elevated nutrients 92 may alter exotic consumer performance, but their effect on herbivores is not as clear as for exotic plants because nutrient enrichment effects on exotic consumers vary by species (Wikström and Hillebrand 2011). However, these studies suggest that nutrient enrichment may increase plant and herbivore invasions possibly altering native community structure (Kennedy et al. 2009). Here we investigated the individual and interactive effects of warming and nutrient enrichment on exotic plants and snails, on plant-herbivore interactions, and their effect on native plants and snails in freshwater wetland communities with a field mesocosm experiment. Specifically, we assessed the effects of 1) elevated nutrients, increased temperature, and their interaction on native and exotic snails and plants and 2) plant invasion effects on native plants and on native and exotic snails in wetland communities. 5.4. Methods 5.4.1. Study system The exotic aquatic plants Alternanthera philoxeroides (alligator weed Amaranthaceae), Eichhornia crassipes (water hyacinth-Pontederiaceae), Pistia stratiotes (water lettuce-Araceae), and the exotic snail Pomacea maculata (island apple snail–Ampullariidae; synonym P. insularum) (Hayes et al. 2012) are sympatric in their native range (South America) and co-occur in heavily invaded local wetlands in southeast Texas. These exotic species invade habitats characterized by the native wetland plants Hydrocotyle umbellata (water pennywort–Araliaceae), Limnobium spongia (frog's bit–Hydrocharitaceae), 93 Pontederia cordata (pickerelweed-Pontederiaceae), Typha latifolia (cattail Typhaceae), and the native snails Physella acuta (bladder snail- Physidae) and Planorbella trivolvis (marsh ramshorn snail - Planorbidae) in the introduced range. The plant species were selected in part based on their growth forms in addition to being common. Native H. umbellata, P. cordata, T. latifolia and exotic A. philoxeroides are emergent plants. In addition, H. umbellata and A. philoxeroides can grow in both terrestrial and aquatic habitats. Exotic P. stratiotes and E. crassipes and native L. spongia are free-floating aquatic plants that reproduce clonally. Eichhornia crassipes and L. spongia have similar phenotypes in varying environmental conditions. Moreover, native P. cordata and E. crassipes are from the same family. Alternanthera philoxeroides was introduced from the Parana River region of South America (Julien et al. 1995). It was first recorded in Mobile, Alabama in 1897 and invaded coastal states from North Carolina to Texas by 1963 (Spencer and Coulson 1976). In aquatic habitats, it anchors to banks and reproduces clonally but does not produce viable seeds in the introduced range (Spencer and Coulson 1976; He et al. 2014). Alternanthera philoxeroides has hollow stems that root at the nodes, resulting in large interwoven mats or free-floating monocultures when fragmented (Spencer and Coulson 1976; Sainty et al. 1997). Eichhornia crassipes is considered one of the worst aquatic weeds in the world with high socio-economic and ecological impacts (Lowe et al. 2004). It was introduced from South America to the USA in 1884, invaded southern USA states by the 1900’s, and its current exotic distribution includes more than 50 countries 94 on five continents (Lowe et al. 2004). This perennial herb is characterized by free-floating rosettes with clonal reproduction; newly formed rosettes can break easily, allowing them to disperse and colonize neighboring habitats. Pistia stratiotes is on the list of the world’s worst weeds (Evans 2013). It was introduced from South America to the United States where it was first reported in Florida in 1765 (Stuckey and Les 1984). It is currently found in Europe, Africa, and Asia (Neuenschwander et al. 2009). Pistia stratiotes may disperse and colonize neighboring habitats by forming mats of free-floating rosettes that can exist on their own or as interconnected clones. Pomacea maculata’s native range extends north to the Amazon basin of Brazil and also occurs in Uruguay and Paraguay (Karatayev et al. 2009). It was discovered in 1989 in SE Texas with source populations likely from the lower Parana River Basin in La Plata region of Argentina (Hayes et al. 2012). Pomacea maculata populations have been reported along the Gulf Coast from Texas to Florida and along the Atlantic coast to South Carolina (Rawlings et al. 2007; Byers et al. 2013). These snails are voracious generalist plant consumers (Rawlings et al. 2007) with high reproductive rates of an average of 2000 eggs per clutch (Barnes et al. 2008). Pomacea maculata’s ecological impact on wetland plant communities is similar to another apple snail P. canaliculata’s impact on Asian wetlands (Carlsson et al. 2004) because it can significantly reduce native plant survival, growth, and diversity by preferentially consuming native plants than exotic plants in wetland communities (Meza-Lopez and Siemann 2015). 95 5.4.2. Experimental setup To investigate the individual and interactive effects of warming and nutrient enrichment on exotic plants and snails, on plant-herbivore interactions, and the effects on native plant and snail communities, we conducted a 2×2×2 factorial field experiment that manipulated plant addition, nutrients, and temperature in established freshwater wetland communities at Rice University South Campus Field Station. We established native wetland communities in fall 2011 by adding 15cm topsoil/sand mixture and municipal water to 473L tanks. We then added an individual of each native plant: L. spongia, H. umbellata, and P. cordata, and 40 of each native snail P. acuta and P. trivolvis to mesocosms. We added native T. latifolia to all mesocosms and allowed it to establish for five months, while other native plants got established for nine months. We replaced native plants that did not survive after two weeks of the initial planting with a new individual to ensure that all native species were represented in established plant communities. In summer 2012, three juvenile P. maculata snails were introduced to previously established native wetland plant communities randomly assigned to: 1) plant addition: native (L. spongia, H. umbellata, and P. cordata) or exotic (A. philoxeroides, E. crassipes, and P. stratiotes), 2) nutrients: control (no addition) or high (+6mg/L Nitrogen and +2mg/L Phosphorus), and 3) temperature: ambient or +2°C with five replicates per treatment. We added a total of 160g of plants that were either native (3 species) or exotic (3 species) to each established native plant community to only manipulate plant origin (native 96 vs. exotic) in plant addition treatments. Nutrient treatments were based on nutrient levels in southeast Texas waters. Nutrients were mixed with municipal water before being added to wetland communities using a Dosatron and a flowthrough watering system that resulted in a four-day hydraulic residence time for water in communities. In addition, we used four solar water heaters (each 1.2 x 6m, Specialty Pool Products, East Windsor CT, USA) to increase the water temperature before delivering it to each community. For the duration of the study, we conducted weekly P. maculata egg mass surveys and monthly visual plant surveys. After 19 weeks, we harvested, sorted, dried, and weighed native and exotic aboveground plant mass and collected and quantified native and exotic snail abundance and size. 5.4.3. Data analyses We used three-way ANOVAs to test the dependence of total, native, and native plant species masses, native snail P. acuta and P. trivolvis abundances, and exotic P. maculata size (operculum length) and mass on nutrients, temperature, plant addition, and their interactions. We also tested the dependence of exotic plant mass and exotic plant species’ masses on nutrients, temperature, and their interaction with two-way ANOVAs. In addition, we conducted repeated measures ANOVAs to test the dependence of P. maculata egg clutch number and egg clutch mass on nutrients, temperature, plant addition, and their interactions. Data met the assumptions of ANOVAs and repeated measures ANOVAs. Post-hoc tests were performed to determine differences between treatment levels for 97 significant variables with more than two levels. We performed all statistical analyses using SAS (v 9.4). 5.5. Results 5.5.1. Snails Warming increased P. maculata egg clutch number with no effect of nutrients, plant addition, or their interactions (Table 5-1). Pomacea maculata egg clutch number changed over time and depended on the interaction of plant addition, nutrients, and temperature over time (Table 5-1, “time”, “plant addition*nutrients* temperature*time”, Appendix Figure 5-1a, Appendix Figure 5-4). Pomacea maculata egg mass increased four-fold with warming while nutrients, plant addition, and their interactions had no effect on egg mass (Table 5-1, Figure 5-2). Pomacea maculata egg mass changed over time and was influenced by the interaction of plant addition, nutrients, and temperature over time (Table 5-1, “time”, “plant addition*nutrients* temperature*time”, Appendix Figure 5-1b). Nutrients, temperature, and plant addition treatments did not affect P. maculata mass (Table 5-1). However, elevated nutrients combined with warming increased P. maculata mass (Table 5-1, “nutrients*temperature”, Figure 5-2 ). Other factor interactions did not affect P. maculata mass (Table 5-1, Appendix Figure 5-2). Elevated nutrients increased native P. acuta abundance with no effect of temperature, plant addition, and their interactions (Table 5-1, Appendix 98 Figure 5-3). Nutrients, temperature, plant addition and their interactions had no effect on native P. trivolvis abundance (all P > 0.05, Appendix Figure 5-3b). 5.5.2. Plants Elevated nutrients increased total and native plant mass (Table 5-2). Warming had no effect on total or native plant mass (Table 5-2). Nutrient and plant addition interactions did not affect total plant mass (Table 5-2, “plant addition*nutrients”, Figure 5-4a). Temperature together with plant addition treatments influenced total and native plant mass (Table 5-2, “plant addition*temperature”, Figure 5-4b,d). Other factor interactions did not affect total plant mass (Table 5-2, Appendix Table 5-1). In addition, exotic plants on their own and in combination with nutrients reduced native plant mass (Table 5-2, “plant addition*nutrients”, Figure 5-4c). Additional factor interactions did not influenced native plant mass (Table 5-2, Appendix Table 5-1). Elevated nutrients increased native L. spongia, P. cordata, T. latifolia, and H. umbellata mass (Appendix Table 5-2). Exotic plants reduced L. spongia, P. cordata, and T. latifolia mass but had no effect on H. umbellata mass (Appendix Table 5-2). Elevated nutrients and plant addition treatments increased native plants L. spongia, P. cordata, and T. latifolia mass but had no effect on H. umbellata (Appendix Table 5-2, Appendix Figure 5-4). Temperature and other factor interactions did not affect native plant L. spongia, P. cordata, T. latifolia, or H. umbellata mass (Appendix Table 5-1, Appendix Table 5-2). Exotic plant mass increased with elevated nutrients while warming had no effect on exotic plant mass (Table 5-2, Figure 5-3 e-f). Nutrients and warming 99 together had no effect on exotic plant mass (Table 5-2, Appendix Table 5-1, “nutrients*temperature”). Elevated nutrients increased exotic plants E. crassipes and A. philoxeroides mass while P. stratiotes was the most abundant exotic plant in ambient nutrient treatments (Appendix Table 5-3, Appendix Figure 5-5). Temperature and factor interactions had no effect on E. crassipes, A. philoxeroides, or P. stratiotes mass (Appendix Table 5-1, Appendix Table 5-3). 5.6. Discussion Warming increased exotic snail invasions with no nutrient enrichment effect, but nutrient enrichment together with warming increased snail performance. Warming increased P. maculata reproduction (egg clutch number and egg clutch mass) and warming with elevated nutrients increased P. maculata adult mass, suggesting that warming will increase P. maculata invasions. Elevated nutrients increased native snail P. acuta abundance but had no effect on P. maculata. In addition, warming had no effect on native snails. These results suggest that nutrient enrichment and warming effects on snails may depend on the origin or identity of snails. Similarly, warming effects on plants depended on plant origin and plant identity. Warming reduced native plant mass and had no effect on exotic plant mass, suggesting that warming may contribute to an increase in exotic plant dominance. Furthermore, elevated nutrients increased total plant mass but had greater effects on exotic plants compared to native plants, suggesting that nutrient enrichment will increase the likelihood of plant invasions and their dominance in invaded habitats. Together this suggests that warming 100 and nutrient enrichment may contribute to multiple plant and snail invasions in these ecosystems. Studies have shown that warming often increases invader success in terrestrial (Dukes and Mooney 1999) and marine communities (Sorte et al. 2010; Dijkstra et al. 2011). Warming may increase the likelihood of invader success by altering an invader’s performance (growth and reproduction) and increasing the length of the growing season (Cockrell and Sorte 2013) for invaders, specifically invaders that originate from a warmer native range because warming would make environmental conditions more similar to the conditions that invaders are exposed to in their native range. For instance, warming increased the population size of exotic tropical flies Drosophila simulans and D. melanogaster compared to non-tropical D. obscura (Davis et al. 1998b). These results agree with our findings and suggest that warming may increase exotic P. maculata reproduction with an increase in temperatures by providing optimal conditions for reproduction. Warming could also increase the length of the growing season in the introduced range, resulting in an increase in P. maculata’s lifetime reproductive output but we did not test this given the design and duration of our study. Others have found that warming in combination with elevated nutrients increased the performance of consumers similar to the observed increase in P. maculata size in our study. For instance, warming and elevated nutrients together increased native grassland herbivore abundance (de Sassi et al. 2012) and native zooplankton density (O’Connor et al. 2009). De Sassi et al. (2012) suggested that an increase in herbivore abundance may be a result of elevated 101 nutrient effects on exotic plants. Additional studies have shown that elevated nutrient effects via changes in exotic plants increase the performance of exotic grasshopper (Bownes et al. 2013a), a mirid bug (Coetzee et al. 2007), and a weevil (Center and Dray 2010). In our study, elevated nutrients had greater effects on exotic plants than native plants, suggesting that nutrient enrichment can influence the performance of P. maculata. However, it is unlikely that elevated nutrient effects via changes in exotic plants increased P. maculata size because P. maculata has been shown to preferentially consume native plants (Burlakova et al. 2009; Meza-Lopez and Siemann 2015). Even though P. maculata can grow and survive when fed exotic plants (Burks et al. 2011), their growth rate is higher when they consume lettuce compared to exotic plants (Lach et al. 2000). These studies indicate that warming and elevated nutrients increase exotic insect performance primarily via nutrient enrichment effects on exotic plants but the mechanism by which warming and nutrient enrichment influence P. maculata performance is unclear. Elevated nutrients have been shown to increase the performance of herbivores by increasing their growth (Rosemond et al. 1993; de Sassi et al. 2012). In our study, elevated nutrients had no effect on adult P. maculata snails but increased native P. acuta snail abundance, suggesting that nutrient enrichment may more strongly increase native snail population growth rates. Similarly, phosphorus enriched water increased grazing caddisfly Leucotrichia pictipes and Psychomyia flavida larval mass, developmental rates, and population densities, and increased periphyton biomass. It was suggested that 102 an increase in the performance of aquatic herbivores with elevated nutrients could be a result of increased periphyton quality and quantity with elevated nutrients (Hart and Robinson 1990). In our study, elevated nutrients may have increased P. acuta abundance by increasing periphyton mass, but our study did not assess nutrient enrichment effects on periphyton performance. In addition, our study was unlikely to detect nutrient enrichment effects on adult P. maculata via periphyton because the diet of P. acuta and P. maculata vary depending on their size. Physella acuta consumes periphyton (Wikström and Hillebrand 2011) and adult P. maculata are voracious plant consumers (Rawlings et al. 2007). However, P. maculata consumes periphyton as hatchlings and juveniles when they are similar in size to juvenile and adult native snails and then switch from periphyton to plants as they grow. These results suggest that nutrient enrichment may increase the performance of native snails, but it is unlikely that it influences P. maculata invasions via an increase in periphyton. Warming had no effect on plant invasions but may reduce native plant mass. It has been suggested that warming can reduce plant performance by increasing heat or water stress (White et al. 2000). In our study, it is probable that warming reduced native plant mass by increasing heat stress compared to water stress because water levels in wetland communities remained constant throughout the study. Furthermore, exotic plants reduced native plant mass, suggesting that exotic plants may increase warming effects on native plants. This suggests that warming in combination with plant invasions probably reduce native plant mass, increasing plant invasion success. 103 Higher exotic plant mass than native plant mass with elevated nutrients, suggests that elevated nutrients will magnify the effect of exotic plants on native plants and increase exotic plant dominance. Elevated nutrients have been shown to favor exotic plants and increase the dominance of exotic plants in terrestrial (Huenneke et al. 1990; Brooks 2003; Mahdavi-Arab et al. 2014) and aquatic ecosystems (Tyler et al. 2007; Gennaro and Piazzi 2014). In addition, nutrient enrichment is known to increase exotic plant mass and competitive ability in many ecosystems (Wedin and Tilman 1996; Brooks 2003; Tyler et al. 2007; Mahdavi-Arab et al. 2014). In our study, E. crassipes had higher mass than A. philoxeroides or P. stratiotes with elevated nutrients. In addition, P. stratiotes only survived in low nutrient conditions, suggesting that nutrient enrichment can also have negative effects on exotic aquatic plants depending on plant interactions. Exotic plants may also outcompete other exotic plants in high resource habitats. This may have been the case with P. stratiotes and E. crassipes because E. crassipes can completely cover the surface of water potentially crowding out native and other exotic plants. Furthermore, a model predicted that elevated nutrients may increase exotic plant E. crassipes growth ten-fold (Wilson et al. 2005). Exotic plants that have increased performance with nutrient enrichment often have characteristics that include high growth rates and high phenotypic plasticity that allows them to quickly take advantage of available resources. However, native plant performance in some cases may be higher or equivalent to exotic plants in habitats with low resource availability (Daehler 2003; Bradley et al. 2010). This suggests that environmental conditions can 104 significantly influence plant invasions and exotic plant impacts on native plants in invaded ecosystems. In addition, P. maculata may enhance the effect of exotic plants and nutrient enrichment on native plants because P. maculata preferentially consumes native plants (Burlakova et al. 2009; Meza-Lopez and Siemann 2015). However, we did not test P. maculata effects on native and exotic plants because P. maculata was present in all native wetland communities in our study. These results suggest that elevated nutrients will favor exotic plants and nutrient enrichment in combination with exotic plants will further reduce native plant performance, contributing to multiple plant invasions and an increase in exotic plant dominance in nutrient enriched aquatic ecosystems. Even though we did not detect significant positive or negative effects of warming on exotic plants, warming may increase the likelihood of plant invasion success because it reduces native plant performance. In addition, warming may increase the performance of exotic plants in the introduced range by creating a novel environment that closely resembles the environment in the exotic plant’s native range especially for exotic species from warmer native ranges (Walther et al. 2002). Warming may reduce winter frost events and increase the length of the growing season, reducing annual winter exotic plant mortality and increase growth and reproduction for species from warmer environments. For instance, with warmer winters the exotic palm Trachycarpus fortune survived outdoors all year without protection (Walther et al. 2007).Warming can also inhibit plant invasions if it does not increase resource availability in invaded habitats, reducing the probability that exotic plants become dominant and outcompete native plants 105 in invaded habitats (Bradley et al. 2010). These studies suggest that warming may increase the likelihood of plant invasions when resources are available but we did not observe this in our study. In conclusion, our results indicate that warming alone and in combination with nutrient enrichment will contribute to herbivore invasions and that nutrient enrichment will increase the likelihood of plant invasion success, reducing native plant mass in aquatic ecosystems. Furthermore, our results showed that the interactive effects of multiple anthropogenic factors on native and exotic species are complex and could not be predicted based on their individual effects. Effects of anthropogenic factors alone and in combination varied depending on snail and plant identity as well as their origin (native vs. exotic); therefore, additional studies investigating the interactive effects of multiple anthropogenic factors on multi-trophic invaders considering species interactions at the community level are needed. 5.7. Acknowledgements We thank: Steve Pennings for tanks; Chef Derrix at a Rice University Servery for providing lettuce for snail populations; Hannah Chen, Cassandra Corrales, Jordan Bunch, Dana Galvan, Josh Hwang, Hayden Ju, Daniel McDermott, Ariel Nixon, Peter Sylvers, Candice Tiu, Ling Zhang, Qiang Yang, and Boris Zhong for their assistance in the field and lab; Romi Burks, Jenn Rudgers, Amy Dunham, Volker Rudolf, and Anna Armitage for helpful discussions. This work was supported by a Ford Foundation Predoctoral Fellowship (to MML), an 106 Environmental Protection Agency Science to Achieve Results Fellowship for Graduate Environmental Study (to MML), a Rice University Shell Center for Sustainability research grant (to MML), and a Rice centennial award to support undergraduate research (to MML). 107 Table 5-1 The dependence of exotic snail P. maculata egg clutch number, egg clutch mass, adult mass, and native snail P. acuta abundance on nutrients, temperature, plant addition, and their interactions in ANOVAs or repeated measures ANOVAs. Significant results are shown in bold P. maculata clutch # P. maculata egg mass P. maculata mass P. acuta abundance Factor DF F P F P F P F P Nutrients Temperature Plant addition Nutrients*temperature Plant addition*nutrients Plant addition*temperature Plant addition*nutrients*temperature Error Time Nutrients*time Temperature*time Plant addition*time Nutrients*temperature*time Plant addition *nutrients*time Plant addition*temperature*time Plant addition*nutrients*temperature*time Repeated error 1 1 1 1 1 1 1 32 8 8 8 8 8 8 8 8 256 0.1 8.0 1.5 0.2 0.6 0.1 0.1 0.8193 0.0081 0.2354 0.6895 0.4595 0.8193 0.7319 0.5 9.9 1.0 0.8 0.9 0.1 0.0 0.4851 0.0036 0.3429 0.3785 0.3413 0.7228 0.9833 1.7 0.1 2.5 4.4 0.6 0.1 3.1 0.2036 0.8914 0.1254 0.0446 0.4434 0.9595 0.0868 8.5 0.2 0.1 1.9 0.1 0.3 0.3 0.0064 0.6429 0.9588 0.1813 0.7893 0.6164 0.6164 3.2 1.1 0.8 1.1 0.6 1.1 0.8 2.9 0.0018 0.3602 0.6496 0.3670 0.7539 0.3602 0.6496 0.0043 2.9 0.5 0.7 0.7 0.5 0.7 1.5 3.8 0.0043 0.8403 0.7256 0.6750 0.8756 0.6552 0.1424 0.0003 108 Table 5-2 The dependence of total, native, and exotic plant masses on nutrients, temperature, plant addition, and their interactions in ANOVAs. Significant results are shown in bold Total plant mass Native plant mass Factor F1,32 P F1,32 Nutrients 110.8 <0.0001 19.1 Temperature 1.0 0.3229 Nutrients*temperature 0.3 Plant addition P Exotic plant mass F1,16 P 0.0001 155.0 <0.0001 2.4 0.1345 0.5 0.5052 0.6118 0.4 0.5115 0.1 0.8845 0.8 0.3698 67.1 <0.0001 Nutrients*plant addition 0.3 0.5603 46.1 <0.0001 Temperature*plant addition 5.8 0.0222 5.3 0.0287 Nutrients*temperature*plant addition 0.1 0.7846 0.2 0.6934 109 (a) 8 (b) Nutrients Temperature control high (c) Plant addition ambient +2°C native exotic Egg mass (g) 6 4 2 0 2 4 6 8 10 Time (week) 2 4 6 Time (week) 8 10 2 4 6 8 10 Time (week) Figure 5-1 Exotic P. maculata snail egg clutch mass dependence on (a) nutrients, (b) temperature, and (c) plant addition treatments over time. Means ±1SE 110 Pomacea maculata mass (g) 60 Nutrients ab 45 control high ab b a 30 15 0 ambient +2°C Temperature Figure 5-2 Exotic P. maculata snail mass dependence on nutrient and temperature treatments. Means +1SE. Means with the same letters were not significantly different in post-hoc tests 111 Physella acuta abundance 80 a 60 40 b 20 0 control high Nutrients Figure 5-3 Native P. acuta snail abundance dependence on nutrient treatments. Means +1SE. Means with the same letters were not significantly different in post-hoc tests 112 Total plant mass (kg) (a) 4 Nutrients (b) control high 3 Temperature ambient +2°C a ab a b 2 1 0 Native plant mass (kg) (c) 4 (d) Nutrients a 3 Temperature ambient +2°C control high a b 2 b bc 1 c c c 0 Exotic plant mass (kg) (e) 4 3 (f) Nutrients control high a Temperature ambient +2°C 2 1 b 0 native exotic Plant addition native exotic Plant addition Figure 5-4 (a) Total plant mass dependence on nutrients together with plant addition and (b) on temperature together with plant addition treatments. (c) Native plant mass dependence on nutrients together with plant addition and (d) on temperature together with plant addition treatments. (e) Exotic plant mass dependence on nutrients and (f) on temperature. Means +1SE. Means with the same letters were not significantly different in post-hoc tests 113 References Abbott KL, Green PT (2007) Collapse of an ant–scale mutualism in a rainforest on Christmas Island. 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Means with the same letters were not significantly different in post-hoc tests 128 5 0.0 -0.4 0 1 -1 0 -0.8 0.0 -0.4 -0.8 -1.2 0 1 -1 Axis 1 -0.4 1 0.0 -0.4 0.0 -0.4 1 2 0 0.4 1 0.0 -0.4 2 0.4 0.0 1 2 j. native + exotic L. spongia ( =0.8kg) 0.8 0.4 0.0 -0.4 -1.2 -1 0 1 2 Axis 1 -0.4 0 -0.8 -2 m. native only T. latifolia ( =1.5kg) 0.8 -1 Axis 1 -1.2 -1 -2 i. native only L. spongia ( =0.8kg) Axis 1 Axis 2 0.4 0 -0.8 -2 2 l. native + exotic H. umbellata ( =0.3kg) 0.8 Axis 2 0.4 0.0 Axis 1 k. native only H. umbellata ( =0.3kg) 0.8 0 -1.2 -1 0.8 -1.2 -2 2 0.4 -0.4 Axis 1 h. native + exotic P. cordata ( =0.1kg) 0.8 0.0 -0.8 -2 1 0.4 -2 -1 0 1 2 Axis 1 n. native + exotic T. latifolia ( =1.5kg) 0.8 Axis 2 -1 0 -0.8 -1.2 -2 -0.4 Axis 1 Axis 2 0.4 0.4 -1.2 -1 2 g. native only P. cordata ( =0.1kg) 0.8 Axis 2 Axis 2 0.0 -0.4 Axis 2 1 0.0 e. exotic only P. stratiotes ( =0.1kg) 0.8 -0.8 Axis 2 f. native + exotic P. stratiotes ( =0.1kg) 0.4 10 Axis 1 Axis 1 0.8 0.4 15 0 -1.2 -1 d. native + exotic A. philoxeroides ( =0.4kg) 0.8 5 -0.8 0 c. exotic only A. philoxeroides ( =0.4kg) Axis 2 10 20 Axis 2 0.4 15 25 b. native + exotic E. crassipes ( =1kg) 0.8 Axis 2 a. exotic only E. crassipes ( =1kg) Axis 2 Rank 20 Rank 25 0.0 Temp - nutrients +0C - control +1C - control +2C - control +0C - high -0.4 +1C - high -0.8 -0.8 -0.8 -0.8 -1.2 -1.2 -1.2 -1.2 -2 -1 0 Axis 1 1 2 -2 -1 0 Axis 1 1 2 -2 -1 0 Axis 1 1 2 +2C - high -2 -1 0 1 2 Axis 1 Appendix Figure 4-1 The dependence of exotic (a-b) E. crassipes, (c-d) A. philoxeroides, and (e-f) P. stratiotes and native (g-h) P. cordata, (i-j) L. spongia, (k-l) H. umbellata, and (m-n) T. latifolia in relation to nutrients and temperature in exotic or native only communities and communities with both native and exotic plants. Symbol size represent species dry plant mass 129 Nutrients control high a 1.2 0.8 0.4 b 0.0 (b) 2.0 a E. crassipes mass (kg) L. spongia mass (kg) (a) 1.6 1.6 1.2 b 0.8 0.4 b b 0.0 native Nutrients control high c d native + exotic Community composition exotic native + exotic Community composition Appendix Figure 4-2 (a) Native L. spongia and (b) exotic E. crassipes mass dependence on community composition and nutrients. Bars represent +1SE. Means with the same letters were not significantly different in post-hoc tests. Scale is different for each species 130 a temperature*nutrients*plant addition +2°C, control, native +2°C, high, native +2°C, control, exotic +2°C, high, exotic 2.0 16 ambient, control, exotic ambient, high, exotic ambient, control, native ambient, high, native Egg mass (g) Egg clutch number 2.5 1.5 1.0 temperature*nutrients*plant addition b +2°C, control, native +2°C, high, native +2°C, control, exotic +2°C, high, exotic ambient, control, exotic ambient, high, exotic ambient, control, native ambient, high, native 12 8 4 0.5 0.0 0 2 4 6 Time (week) 8 10 2 4 6 8 Time (week) Appendix Figure 5-1 Exotic P. maculata snail (a) egg clutch number and (b) egg mass dependence on the interactive effect of temperature, nutrients, and plant addition treatments over time. Means ± 1SE 10 131 60 P. maculata mass (g) 50 Plant addition native exotic 40 30 20 10 0 control, ambient control, +2°C high, ambient high, +2°C Nutrient - temperature treatments Appendix Figure 5-2 Pomacea maculata snail mass dependence on plant addition, nutrients, and temperature treatments. Means +1SE 132 a 120 Plant addition native exotic b 100 80 P. trivolvis abundance P. acuta abundance 100 60 40 20 Plant addition native exotic 80 60 40 20 0 am , l ro nt o c nt bie l, tro n co °C +2 m ,a h g bi 0 t en hi h, hig °C +2 Nutrient - temperature treatments l, ro t n co am bie nt nt l tro n co C 2° + , hi m ,a h g bie C 2° + h, g i h Nutrient - temperature treatments Appendix Figure 5-3 (a) P. acuta and (b) P. trivolvis snail dependence on plant addition, nutrients, and temperature treatments. Means +1SE 133 (a) 0.16 (b) 2.4 Nutrients Plant mass (kg) Nutrients control high 0.12 1.8 L. spongia H. umbellata 0.08 control high a 1.2 b bc 0.04 0.6 c 0.00 0.0 (c) 0.4 b Nutrients (d) 0.9 Nutrients control high Plant mass (kg) control high a 0.3 P. cordata T. latifolia 0.6 0.2 0.3 a b b b a 0.1 a 0.0 0.0 native exotic Plant addition native exotic Plant addition Appendix Figure 5-4 Native (a) H. umbellata (no significant interactive result), (b) L. spongia, (c) P. cordata, and (d) T. latifolia mass dependence on nutrients and plant addition treatments. Means +1SE. Means with the same letters were not significantly different in post-hoc tests. Note that the y-axis scales differ among species 134 (a) 2.5 a E. crassipes (b) 0.6 (c) A. philoxeroides Plant mass (kg) a 0.009 P. stratiotes a 2.0 0.4 0.006 1.5 1.0 0.5 0.2 b 0.003 b 0.0 control high Nutrients b 0.000 0.0 control high Nutrients control high Nutrients Appendix Figure 5-5 Exotic (a) E. crassipes, (b) A. philoxeroides, and (c) P. stratiotes mass dependence on nutrient treatments. Means +1SE. Means with the same letters were not significantly different in post-hoc tests. Note that the y-axis scales differ among species 135 Appendix Table 3-1 The dependence of native L. spongia and H. umbellata and exotic A. philoxeroides and E. crassipes wet plant mass in experiment 2 on nutrients, snails, and their interactions in ANOVAs. Significant results are shown in bold L. spongia Factor Nutrients Snails Nutrients*snails df Fx,18 2 1 2 3.0 2.5 1.1 P 0.0744 0.1350 0.3623 H. umbellata Fx,18 1.8 4.7 1.2 P 0.1931 0.0431 0.3228 A. philoxeroides Fx,18 P 1.0 55.2 2.5 0.3963 <0.0001 0.1110 E. crassipes Fx,18 0.3 8.3 0.8 P 0.7316 0.0099 0.4658 136 Appendix Table 4-1 The dependence of percent cover on community composition, nutrients, temperature, and their interaction in repeated measures ANOVAs. Significant results are shown in bold Factor DF Composition 1 Nutrients Temperature 1 2 Composition*nutrients Composition*temperature Nutrients*temperature Composition*nutrients*temperature Error Time Composition*time Nutrients*time Temperature*time Composition*nutrients*time Composition*temperature*time Nutrients*temperature*time Composition*nutrients*temperature*time Repeated error 1 2 2 2 36 7 7 7 14 7 14 14 14 252 Native plant % cover Exotic plant % cover F P F 27.4 51.0 1.5 <0.0001 <0.0001 0.2346 39.6 1.2 1.0 0.9 <0.0001 0.3223 0.3632 0.3994 52.2 16.7 20.0 1.1 <0.0001 <0.0001 <0.0001 0.3447 20.8 1.4 1.7 1.5 <0.0001 0.1812 0.0630 0.1092 P 2.8 0.1041 286.5 0.1 1.4 0.6 0.1 0.5 <0.0001 0.8884 0.2475 0.5719 0.9023 0.6114 35.8 1.4 <0.0001 0.2092 56.0 1.7 0.9 1.2 1.7 1.2 <0.0001 0.0604 0.5383 0.3027 0.0680 0.2793 137 Appendix Table 4-2 The dependence of exotic plant masses on nutrients, temperature, and their interaction in ANOVAs. Significant results are shown in bold Factors Nutrients Temperature Nutrients*temperature A. philoxeroides E. crassipes mass mass DF Fx,16 P F x,16 P 1 1 1 55.4 2.8 2.5 <0.0001 0.1113 0.1340 72.6 <0.0001 0.9 0.3561 0.3 0.6256 P. stratiotes mass F x,16 P 19.1 0.0005 0.6 0.4468 0.6 0.4468 138 Appendix Table 5-1 Total and native plant mass and individual native species mass dependence on plant addition, nutrients, and temperature. Exotic plant mass and individual exotic species mass dependence on nutrients and temperature. Means±SE Treatment Exotic, control, ambient Exotic, control, +2°C Exotic, high, ambient Exotic, high, +2°C Native, control, ambient Native, control, +2°C Native, high, ambient Native, high, +2°C Total plant Native plant Exotic plant H. umbellata P. cordata T. latifolia 948.5±264 719.3±268 229.3±040 22.3±14 67.1±15 170.5±052 1338.8±377 1008.0±255 330.7±122 37.0±22 41.1±07 3071.8±282 504.9±116 2567.0±308 56.3±21 3180.9±220 456.3±120 2724.5±181 1369.2±377 L. spongia P. stratiotes A. philoxeroides E. crassipes 459.4±260 5.9±2 53.2±05 170.1±038 290.7±057 639.2±187 8.5±2 47.4±05 274.8±126 60.8±16 113.7±068 274.2±080 0 542.5±67 2024.5±370 29.5±21 78.7±58 233.1±080 115.1±069 0 365.5±84 2359.1±243 1369.1±377 28.3±15 89.5±18 251.5±121 999.8±289 803.6±083 803.6±083 15.1±09 109.0±29 140.7±036 538.8±107 3184.2±127 3184.2±127 146.6±44 349.7±45 749.3±160 1938.5±248 2534.1±270 2534.1±270 62.9±37 299.2±62 575.1±154 1596.9±159 139 Appendix Table 5-2 The dependence of native plant masses on nutrients, temperature, plant addition, and their interactions in ANOVAs. Significant results are shown in bold Factors H. umbellata L. spongia mass mass DF Fx,32 P F x,32 P Nutrients 1 6.8 0.0136 5.6 0.0243 Temperature 1 2.2 0.1488 2.1 0.1607 Plant addition 1 2.1 0.1535 Nutrients*temperature 1 2.3 0.1379 Plant addition*nutrients 1 3.6 0.0670 Plant addition*temperature 1 1.3 0.2581 2.3 Plant addition*nutrients*temperature 1 0.2 0.6950 0.7 43.4 <0.0001 0.2 P. cordata mass F x,32 P 20.7 <0.0001 0.1 T. latifolia mass F x,32 P 8.1 0.0078 0.7142 0.1 0.8756 32.1 <0.0001 10.0 0.0035 0.6897 0.1 0.8062 0.1 0.8252 24.7 <0.0001 15.7 0.0004 13.2 0.0010 0.1403 0.1 0.8298 3.3 0.0779 0.4062 1.2 0.2896 0.1 0.8298 140 Appendix Table 5-3 The dependence of exotic plant masses on nutrients, temperature, and their interaction in ANOVAs. Significant results are shown in bold Factors Nutrients Temperature Nutrients*temperature DF 1 1 1 A. philoxeroides mass Fx,16 P 55.4 2.8 2.5 <0.0001 0.1113 0.1340 E. crassipes mass F x,16 P 72.6 0.9 0.3 <0.0001 0.3561 0.6256 P. stratiotes mass F x,16 P 19.1 0.6 0.6 0.0005 0.4468 0.4468 141