Can secondary compounds of an invasive plant affect. larval amphibians? J. C. MAERZ, C. J. BROWN, C. T. CHAPIN * and B. BLOSSEY

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1 Functional Ecology 2005 Can secondary compounds of an invasive plant affect Blackwell Publishing, Ltd. larval amphibians? J. C. MAERZ, C. J. BROWN, C. T. CHAPIN * and B. BLOSSEY Ecology and Management of Invasive Plants Program, Department of Natural Resources, Cornell University, Ithaca, NY , USA, and USDA/ARS/PPRU Federal Nutrition Laboratory, Tower Road, Ithaca, NY 14853, USA Summary 1. There is significant concern over the impacts of plant invasions on habitat quality for native fauna. Recent research suggests that non-native Purple Loosestrife (Lythrum salicaria) invasions may negatively affect the performance of larval American Toad tadpoles (Bufo americanus), and that compounds leached from L. salicaria leaves play a direct or indirect role in this effect. 2. We raised individual B. americanus and Gray Treefrog (Hyla versicolor) tadpoles on high-quality diets in aqueous extracts of senescent leaves from L. salicaria, native Broad-Leaf Cattail (Typha latifolia), and control water to determine whether loosestrife extracts directly affect anuran tadpole performance. 3. Even at high artificial food levels, B. americanus survival was significantly lower in L. salicaria extracts compared with T. latifolia extracts and a water control. Food level strongly affected B. americanus development, but tadpoles raised in L. salicaria extract were less developed compared with conspecifics raised in cattail extract or water. Unlike B. americanus, Hyla performance was not affected by exposure to any plant extract compared with the water control. 4. Our study implicates secondary plant compounds as a mechanism underlying the impact of an invasive plant on some but not all native fauna. We hypothesize that high tannin concentrations of L. salicaria leaves have the potential to create environments that are directly toxic to B. americanus tadpoles. We hypothesize that obligate gill breathers such as B. americanus tadpoles are highly sensitive to gill damage caused by high concentrations of phenolics. Other anuran species such as H. versicolor that develop well-functioning lungs early may be less affected by high tannin concentrations. Key-words: Amphibian larvae, Bufo americanus, detritus, Hyla versicolor, invasive plants, phenolics Functional Ecology (2005) doi: /j x Ecological Society Introduction The increasing spread of non-native plant species raises concerns about the impacts of plant invasions on habitat quality for native fauna. Generally, we expect that invasive plants will affect animal communities by altering resource availability (trophic effects) or habitat structure. However, some invaders may have particular traits that contribute to their effects on native fauna in unexpected ways. For example, Purple Loosestrife (Lythrum salicaria) is a highly invasive plant in wetlands throughout North America. L. salicaria invasions are Author to whom correspondence should be address. jmaerz@forestry.uga.edu *Current address: National Park Service, Great Lakes Network Office, 2800 Lakeshore Drive E, Suite D, Ashland, WI linked to declines in habitat quality for breeding birds, mammals and turtles (reviewed by Blossey 1999). Recent evidence shows that L. salicaria invasions may also reduce growth and survival of American Toad tadpoles (Bufo americanus) (Brown et al. 2005). Brown et al. (2005) present evidence of changes in tadpole diets associated with L. salicaria invasions (a trophic effect), which might be the underlying cause of reduced performance in L. salicaria habitats. They also demonstrated raising B. americanus tadpoles in aqueous extracts of L. salicaria leaves could produce similar levels of mortality, raising the possibility that soluble compounds in L. salicaria leaves affect the productivity or composition of food resources or act directly on developing tadpoles. Alteration of the chemical environment is known for a number of invasive plant species (Blossey 1999; Caraco & Cole 2002; Ehrenfeld 2003), but little is 970

2 971 Invasive plant compounds and larval amphibians known about the effects of L. salicaria invasion on the chemistry of wetland habitats. Templer, Findlay & Wigand (1998) document changes in sediment chemistry associated with L. salicaria invasion. Rauha et al. (2001) found that L. salicaria leaves have particularly high concentrations of phenolics (tannins), but the fate of those compounds and their potential effects on host communities or ecosystems has not been addressed. We recently collected water samples from L. salicariainvaded habitats in 13 wetlands in New York, Rhode Island, and Minnesota, and found reactive phenolic concentrations during the spring when anurans were breeding as low as 1 mg l 1 and as high as 11 mg l 1 (B. Blossey and J. C. Maerz, unpublished data). These results show that while not ubiquitous, habitats invaded by L. salicaria can have high tannin concentrations that might affect aquatic fauna. High tannin concentrations can have direct and indirect effects on aquatic fauna. A growing body of literature shows that phenolics are retained at high concentrations in plant leaves, and through effects on decomposition rates (Rier et al. 2002; Tuchman et al. 2003b; Cornelissen et al. 2004; Parsons et al. 2004; Schweitzer et al. 2004; Rier, Tuchman & Wetzel 2005) and allelopathy (Ervin & Wetzel 2003), inhibit bacteria, phytoplankton and periphyton growth, which consequently affects the performance of organisms at higher trophic levels (Tuchman et al. 2002, 2003a). In addition to trophic effects, aquatic organisms in wetland habitats are literally bathing in leached plant compounds that they may ingest while feeding or that pass over gills as the animals respire; yet there has been little attention to the direct effects of tannins or other secondary plant compounds on aquatic fauna. Applebaum & Birk (1979) note that saponins leaching from aquatic plants poison fish by lysing blood cells, a process that has been recognized and exploited by fisherman since ancient times. Temmink et al. (1989) found that tannins leached into water from tree bark had lethal and subacute effects on fish at high and low concentrations, respectively. Lethal effects were related to gill lesions caused by phenolic reactions with sensitive tissues, and subacute effects may have been related to a combination of gill damage and phenolics reducing the nutritional quality of food or interfering with digestive enzymes. Though the results of Brown et al. (2005) suggests that compounds, most likely high concentrations of tannins, leached from L. salicaria may contribute to the plant s effect on B. americanus tadpole performance, the design of that study cannot distinguish between the indirect trophic effects of L. salicaria extracts on algal growth or direct toxicity as the mechanism. The purpose of this study was to determine whether compounds leached from L. salicaria leaves directly affect two species of larval anuran. Using high-quality artificial food supplements to reduce indirect trophic effects, we compared survival and development of tadpoles raised in extracts of L. salicaria, T. latifolia and control water. To follow up on previous research, we tested B. americanus tadpoles (hereafter referred to as Bufo). We tested a second amphibian species because studies of larval amphibian responses to pesticides (Relyea 2003) and different plant substrates (Skelly, Freidenburg & Kiesecker 2002) show that species can differ in their sensitivity similar compounds. We used Gray Treefrog tadpoles (Hyla versicolor, hereafter referred to as Hyla), which is another widely distributed anuran species that was available during our study. Both species will breed in the same wetlands and are common constituents of anuran communities in our region. We hypothesized that tadpoles exposed to aqueous L. salicaria extracts would show lower survival and development compared to conspecifics raised in T. latifolia extracts or control water. Materials and methods GENERATING AQUEOUS LEAF EXTRACTS We harvested senescing T. latifolia and L. salicaria at the Northern Montezuma Wetlands Complex, in upstate New York. Plants were taken to the lab, dried immediately, and stored dry in opaque paper bags until use. For all experiments, we used a concentration of 0 5 g dry leaf/l water, which was similar to the concentration used by Brown et al. (2005). Extracts were initially prepared by collecting dried leaves from 15 to 20 randomly selected plants, and leaching those leaves for 48 h in filtered reverse osmosis water at a concentration of 5 g l 1. Extracts were filtered through cheesecloth to remove solid material, and stored in opaque containers at 4 C. For a control, we also aged, filtered and stored reverse osmosis water in a manner identical to plant extracts. To measure phenolic concentration of extracts, we filtered 10 ml of stock extract through a 0 45 µm filter, used Folin phenol reagent (Sigma- Aldrich, St Louis, MO, USA) to reduce the active phenolics, and used a pre-made Folin-Ciocalteu reagent to determine sample concentration against a phenol standard (Clesceri & Eaton 1998). METHODS FOR EXPOSING TADPOLES TO PLANT EXTRACTS We exposed tadpoles to extracts in 1-l plastic containers floating in artificial outdoor ponds. We added 0 1 l stock control water or plant extract to 0 9 l treated tap water. For each treatment, we used 30 replicated cups. For statistical purposes, we randomly assigned 5 cups to one of six groups, creating six replicate groups composed of five independent replicates for each treatment. This design allowed us to measure a mean and variance for percentage survival within each treatment rather than a single measure of percent survival with no measure of variance. We floated cups in Styrofoam racks in outdoor ponds to provide natural photoperiod and temperature regimes, and we used a stratified random design to assign cups to locations within and among ponds.

3 972 J. C. Maerz et al. Fig. 1. Mean (± 2 SE) percentage survival of Bufo americanus tadpoles raised on low (white symbols), medium (grey symbols) and high (black symbols) supplemental food levels while exposed to aqueous extracts of senescent T. latifolia (circles) or L. salicaria (triangles) leaves. Different letters indicate significant differences between treatments at 21 days based on Tukey s Honest Significant Difference test. Experiment 1: Bufo responses to plant extracts and food levels We collected Gosner (1960) stage 25 B. americanus tadpoles from multiple artificial ponds on the grounds of the Cornell Resource Ecology and Management Facility (REM), Ithaca, NY, and added 1 tadpole to 180 cups containing either T. latifolia or L. salicaria extract and one of three artificial food supplement levels. Food levels were created by adding a 0 01 g, 0 1 g or 0 75 g Wardley s turtle food stick (Zoomed Laboratories, San Louis, CA, USA) as low, medium or high food treatments, respectively. Pilot studies showed that these artificial food supplements have no effect on 21- day survival of tadpoles, but generate slow, intermediate and maximal development rates, respectively (J. C. Maerz, unpublished data). We checked all cups after 1, 7, 14 and 21 days of exposure to determine whether tadpoles were still alive, and we harvested all tadpoles after 21 days. We determined the Gosner stage and wet mass of tadpoles that survived to the end of the experiment, and group means were used for all analyses. We used a two-way, repeated measure ANOVA to compare Bufo survival rates among treatments. We also used a two-way ANOVA and Tukey s post hoc tests to compare percentage survival at 21 days survival among treatments. Experiment 2: Comparison of Bufo and Hyla responses to plant extracts and a water control We used an experimental design similar to Experiment 1 to compare the performance of Bufo and Hyla raised in different plant extracts and a water control. Because these species breed at different times, tests on both species were not conducted concurrently (Bufo were tested in May and Hyla in July. The Bufo trial was run concurrently with Experiment 1, so we used the same extracts. We made fresh extracts from randomly selected plants from the same collection in the Hyla trial. For both species, we collected Gosner stage 25 tadpoles from the same REM ponds, and added 1 tadpole to each of 90 cups containing a water control, T. latifolia extract, or L. salicaria extract. We added a 0 5-g Wardley Reptile Stick, which pilot studies show was sufficient to allow maximum survival and rapid development of both species (J. C. Maerz, unpublished data). We harvested all surviving tadpoles after 21 days and determined their Gosner stage. We used one-way MANOVA to determine whether extract treatments affected tadpole survival and mean Gosner stage, and Tukey s Honest Significant Difference tests to compare survival or mean Gosner stage between treatments. Separate analyses were conducted for each species. Results EXPERIMENT 1: BUFO RESPONSES TO PLANT EXTRACTS AND FOOD LEVELS Reactive phenolic concentrations for T. latifolia and L. salicaria treatments in Bufo trials were 0 62 and mg l 1, respectively. Plant extract exposure and food level had significant interactive effects on Bufo survival rates (Extract Food Date interaction: F 6,90 = 4 655, P < 0 001; Fig. 1) and final percentage survival (Extract Food interaction: F 1,20 = 10 00, P = 0 005; Fig. 1). At all supplemental food levels, Bufo survival was lower in L. salicaria treatments than T. latifolia treatments. Within T. latifolia treatments, Bufo survival was high and did not differ among supplemental food treatments (Fig. 1); however, in L. salicaria treatments, Bufo survival was increased slightly by the high supplemental food treatment (Fig. 1). In all L. salicaria treatments, most mortality occurred within the first 7 days of exposure. Like survival, Bufo development was affected by extract treatment (F 1,16 = 15 42, P = 0 001) and food level (F 1,16 = 17 84, P < 0 001); however, there was no interaction between those factors (F 1,16 = 0 015, P = 0 905) and food level had a much greater effect on development than extract treatment did (Fig. 2). For high and medium food levels, exposure to L. salicaria extracts significantly reduced tadpole development (Fig. 2). EXPERIMENT 2: COMPARISON OF BUFO AND HYLA RESPONSES TO PLANT EXTRACTS AND A WATER CONTROL For the Bufo trial, phenolic concentrations in the control, T. latifolia and L. salicaria extracts were 0 01, 0 62 and mg l 1, respectively, and for the Hyla trials, phenolic concentrations in the control, T. latifolia and L. salicaria extracts were 0 01, 0 68 and mg l 1, respectively. Bufo performance differed significantly among treatments (Wilks λ = 0 129, F 4,28 = ,

4 973 Invasive plant compounds and larval amphibians Fig. 2. Mean (± 2 SE) development stage (Gosner 1960) of Bufo americanus tadpoles raised on low, medium and high food levels while exposed to aqueous extracts of senescent T. latifolia or L. salicaria leaves. Different letters indicate significant differences between treatments based on Tukey s Honest Significant Difference test. P < 0 001; Fig. 3). Percentage survival and mean Gosner stage were significantly lower in the L. salicaria treatment compared with the water control and T. latifolia treatments, and did not differ between T. latifolia and water control treatments (Fig. 3). Hyla performance did not vary among treatments (Wilks λ = 0 702, F 4,28 = 1 400, P < 0 274; Fig. 3). Mean percentage survival was greater than 90% and did not vary significantly among treatments (F 2,15 = 0 238, P = 0 791; Fig. 3). Tadpoles achieved a mean Gosner stage of 40 that also did not vary significantly among treatments (F 2,15 = 2 4, P = 0 124; Fig. 3). Discussion This study shows that natural compounds leached from decomposing L. salicaria leaves can directly affect Bufo tadpole performance. Bufo tadpoles showed dramatic and rapid mortality when exposed to L. salicaria extracts compared with conspecifics exposed to T. latifolia extracts and a water control. This early mortality cannot be explained by food stress, as Bufo showed high survival while on low food in water control and T. latifolia extract treatments and increasing food levels had only a minor positive effect on Bufo survival in L. salicaria treatments. These results indicate that L. salicaria extract acted directly on Bufo tadpoles. Given the high concentration of reactive phenolics (tannins) in L. salicaria extracts, we hypothesize that it is these compounds that may be acting upon tadpoles. Damage to gill tissues from exposure to tannins causes high and rapid mortality in fish (Temmink et al. 1989), and similar to fish, Bufo tadpoles are essentially obligate gill breathers (Ultsch, Bradford & Freda 1999). Bufo have rudimentary lungs through most of their development, and therefore, a limited ability to compensate for gill damage (Ultsch et al. 1999). Fig. 3. Mean (± 2 SE) percent survival and development stage (Gosner 1960) of Bufo americanus and Hyla versicolor tadpoles exposed to water control or aqueous extracts of senescent plant leaves. Though presented in a single figure, the two species were tested separately, so different letters indicate significant differences between treatments (Tukey s Honest Significant Difference test) within but not across species. We must acknowledge that our extract experiments cannot separate the effects of phenolics from other potential chemical differences between L. salicaria and T. latifolia. L. salicaria leaves are known to also contain anthocyanosides, flavonoid C-glycosides and alkaloids, though none of these compounds occurs in remarkable amounts compared with phenolics (Rauha et al. 2001). Because we did not measure other factors such as dissolved oxygen or ph, we also cannot rule out other confounding effects at this time. In follow-up studies, we have found that ph in cups with L. salicaria extract remained relatively neutral (7 8) and well within the physiological limits of tadpoles; however, we did find that dissolved oxygen levels in cups with loosestrife extract were lower than in water controls, and dropped between 25% and 50% h into experiments before returning to 50% saturation after 48 h (J. C. Maerz and B. Blossey, unpublished data). We do not know the tolerance of Bufo tadpoles to low dissolved oxygen, though B. terrestris tadpoles were rarely found in microhabitats within a pond where dissolved oxygen was below 50% (Ultsch et al. 1999). It is possible that reduced oxygen levels combined with damage to gills from exposure to tannins explains the high mortality of B. americanus tadpoles raised in L. salicaria extracts.

5 974 J. C. Maerz et al. Another important finding of our study was that tadpoles of two sympatric anuran species responded differently when exposed to L. salicaria extracts. Unlike Bufo, Hyla survival and development were high in loosestrife extract and indistinguishable from rates observed in cattail and control water treatments. Anuran tadpoles are known to show species-specific response to pesticides (Relyea 2003) and plant substrates (Skelly et al. 2002). We hypothesize that differences in the respiratory capacity between Bufo and Hyla explain the differential sensitivity of these species to L. salicaria extract. Unlike Bufo sp., Hyla sp. (as well as Rana sp.) have well-developed lungs early in larval development, and could compensate for damage to gills by breathing air at the water surface (Ultsch et al. 1999). Additional experiments are required to assess whether tannins, alone or in combination with other compounds or factors, in L. salicaria leaves are responsible for the effects we observed on tadpoles in the field. Field measurements of phenolic concentrations in L. salicariainvaded habitats were found to reach 11 mg l 1 ; but the range of phenolic levels across habitats or sites was very high (range 1 11 mg l 1 ; B. Blossey and J. C. Maerz, unpublished data). Water samples from cattail habitats at four sites ranged from 0 3 to 2 mg l 1 (B. Blossey and J. C. Maerz, unpublished data). We do not know the cause of variation in tannin levels among L. salicaria habitats, but likely factors include the amount of L. salicaria biomass production, length of invasion, hydrology and the presence of herbivorous biological control insects defoliating the plant (Blossey, Skinner & Taylor 2001). Our data indicate that L. salicaria invasions have the potential to result in high tannin concentrations in aquatic habitats, and where tannin levels are high, these compounds can contribute to the impact of the plant on wetland fauna. Direct acute effects of high tannin levels such as those we demonstrate for B. americanus, may be limited to particular species such as those that primarily respire with gills. It is also important to note that exposure to lower concentrations of tannins may have non-lethal affects on some aquatic fauna (Temmink et al. 1989), and that the negative effects of elevated phenolic levels on bacterial, algal and periphyton productivity (Tuchman et al. 2002, 2003a; Ervin & Wetzel 2003) may negatively affect a broader range of species. Our study draws attention to two neglected aspects of larval amphibian ecology. First, is the general affects of plants communities on larval amphibian ecology. Though research is limited, the effects of plant communities on amphibian habitats are shown to be important in understanding current changes in the distribution and abundance of amphibians (Skelly, Werner & Cortwright 1999, Skelly et al. 2002), and predicting the potential effects of future changes (Rubbo & Kiesecker 2004). Most tadpoles occupy the littoral habitats of ponds and lakes where emergent and aquatic macrophytes are abundant (Alford 1999). The structure and abundance of plants may affect physical conditions important to larval amphibian performance (e.g. temperature or hydrology, Skelly 1996; Halverson et al. 2003), and plants almost certainly provide important refuge from predators. Most importantly, detritus from the surrounding and emergent plant community accounts for > 90% of the energy and materials supporting aquatic food webs (Wetzel 1995), and variation in detritus chemistry, which can occur temporally within plant species or as a result of changes plant community composition, will affect decomposition processes, water chemistry and the availability of algal, periphyton and zooplankton resources upon which larval amphibians feed (Tuchman et al. 2002, 2003a; Ervin & Wetzel 2003). Second, concern over the worldwide decline of amphibian populations has lead to the realization that declines may often result from a suite of local interacting factors including invasions by non-native species (Beebee & Griffiths 2005). However, attention to invasive species has focused almost exclusively on the impacts of introduced predators, competitors and disease (Kiesecker 2003; Beebee & Griffiths 2005). There has been relatively little attention to the potential contribution of plant invasions to reduced habitat quality and population declines, despite the fact that invasive plants are a widespread and common constituent of amphibian habitats. Limited recent evidence indicates that plant invasions can degrade the larval and postmetamorphic habitats of amphibians (Brown et al. 2005; Maerz, Blossey & Nuzzo 2005), but far more research addressing a broader suite of invasive plant species, different amphibian habitats and population-level effects is needed. Studies of plant invasions offer an opportunity not only to understand the impacts of plant invasions on amphibians, but to better understanding the influence of plant communities on amphibian ecology in general. Acknowledgements We thank P. Baxter, W. Baxter, R. Farr, M. Miller, K. O Donnell, J. Peñafiel, D. Stratton and E. Wilhelm for their assistance. This research was supported, in part, through Cornell Hatch grant 3305 and the Ecology and Management of Invasive Plants Program. This work complied with the current state and federal laws, and the collection and use of amphibian species was approved by the New York State Department of Environmental Conservation (LCP03-236) and the Cornell University IACUC (protocol 03 24). References Alford, R.A. (1999) Ecology: resource use, competition, and predation. Tadpole: the Biology of Anuran Larvae (eds R.W. McDiarmid & R. Altig), pp University of Chicago Press, Chicago, IL. Applebaum, S.W. & Birk, Y. (1979) Saponins. Herbivores: Their Interaction with Secondary Plant Metabolites (eds G.A. Rosenthal & D.H. Janzen), pp Academic Press, Inc., London.

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