Competitive outcomes between herbivorous consumers can be predicted from their stoichiometric demands

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1 Competitive outcomes between herbivorous consumers can be predicted from their stoichiometric demands TSUBASA IWABUCHI AND JOTARO URABE Graduate School of Life Sciences, Tohoku University, 6-3 Aramaki-Aza-Aoba, Sendai, Miyagi Japan Citation: Iwabuchi, T., and J. Urabe Competitive outcomes between herbivorous consumers can be predicted from their stoichiometric demands. Ecosphere 3(1):7. Abstract. Exploitative competition for food resources is one of the crucial biological interactions in nature. However, few studies have experimentally tested if competitive ability of consumer species changes depending on elemental contents of the food, although it has been suggested by the theories of resource ratio for competition and ecological stoichiometry. To verify if competitive superiority actually changes according to elemental contents of the food, competition experiments were conducted using high and low P algal food and three Daphnia species (D. galeata, D. pulicaria, and D. tanakai) with different threshold levels of carbon (TFC) and phosphorus (TFP) necessary for individual growth. Since the TFCs of D. tanakai and D. pulicaria were similar to each other but lower than that of D. galeata, we specifically predicted that when fed high P food (i.e., limited by C), D. tanakai and D. pulicaria would be competitively equal to each other but superior to D. galeata. We also predicted from their TFPs that when fed low P food, D. pulicaria would outcompete D. tanakai and D. galeata, while neither of the latter two would be competitively superior to the other. The results showed that when one of the two competitors was predicted to be competitively superior, the food level under competition was similar to the TFC (or TFP) of the superior species but lower than that of the other inferior species, and that the biomass of the former was much less affected by the competition. Also, when the two competing species were predicted to be competitively equal, the food levels under competition were generally similar to the TFC (or TFP) of both species and their biomasses decreased by the same magnitude. The results thus accorded well with the predictions from the TFC and TFP, and indicate that competitive superiority between the same two Daphnia species changes depending on P:C ratio of algal food. This study provides firm evidence for the first time that resource ratio theory can be applied to competition between animal consumers for essential substances packaged within a single food resource. Key words: algae; competition; Daphnia; P:C stoichiometry; resource ratio theory; threshold food level; zooplankton. Received 25 August 2011; revised 12 December 2011; accepted 16 December 2011; final version received 9 January 2012; published 24 January Corresponding Editor: D. P. C. Peters. Copyright: Ó 2012 Iwabuchi and Urabe. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits restricted use, distribution, and reproduction in any medium, provided the original author and sources are credited. iwa283@gmail.com INTRODUCTION Since the pioneering work by Tilman (1982), the resource ratio theory of competition has been one of the most successful and attractive theories in ecology (Miller et al. 2005). In this theory, an outcome of competition can be predicted from a minimum resource level (R*) required to maintain an equilibrium population at any mortality rates due to, for example, predation (Chase et al. 2002). When there are two or more limiting resources, the theory states that competitive superiority between the same two species can change depending on a supply ratio of these resources if R* for each resource differs between the competing species. The theory has been v 1 January 2012 v Volume 3(1) v Article 7

2 successfully substantiated in chemostat studies with phytoplankton (Tilman 1982, Sommer 1989, Grover 1997). Grover (1997) and Miller et al. (2005) pointed out that although the theory had been tested even on higher plants and microorganisms, it has not been examined for animals except in the study by Rothhaupt (1988), who showed that the competitive outcomes between two rotifer species were predictably determined by supply rates and ratios of two algal food. In the latter study, however, the supplied food resources were substitutable for each other and cannot be treated separately as in the case of nutrient elements for plants. Thus, surprisingly, despite the well-established classical theories in ecology, applicability of the resource ratio theory has not yet been examined for competition between animal consumers for essential resources that are not substitutable for each other. For consumers, the quantity of a suitable food has often been a prime matter for determining their growth rate. However, a growing body of evidence has shown that contents of essential substances in a food resource such as nutritional elements can also be a factor in determining the growth rates of consumers (Hessen 1992, Urabe and Watanabe 1992, Sterner and Elser 2002, Raubenheimer et al. 2009) because animals require a suitable amount of macro- and trace elements (Robbins 1983). This fact implies that if food is considered as a package of various essential elements such as carbon (C), nitrogen (N) and phosphorus (P), a single food may generate an additional niche space called stoichiometric niche (Elser and Hessen 2005) because a requirement for each element likely differs among animal species. This niche space can theoretically predict changes in competitively superior species depending on an elemental composition of food (Hall 2004, Loladze et al. 2004), but few studies have experimentally tested if competitive ability of consumer species actually changes depending on the elemental contents of food. It is well known that algae, the major primary producer in aquatic systems, greatly alter P and N contents relative to C according to rate and ratio of nutrient and light supplies (Harrison et al. 1990, Urabe and Sterner 1996, Sterner et al. 1997). Since it is often in short supply in aquatic ecosystems, and since it is an important element constituting ribosomal RNA where protein synthesis takes place (Sterner and Elser 2002), the supply of P relative to energy or C is crucial for determining growth rates of not only plants but also animals. A number of studies have shown that a low P food slows down growth rates of key freshwater zooplankton (Urabe et al. 1997, DeMott 1998, Sterner et al. 1998, Urabe et al. 2002, Seidendorf et al. 2010), but the effect of P- deficiency differs among taxa (DeMott and Pape 2005, Ferrão-Filho et al. 2007, Iwabuchi and Urabe, submitted ). Some studies have reported that the dominant cladoceran species in nature seasonally changes due to changes in abundance and composition of algae, which were presumably different in nutrient content and digestibility (DeMott 1983, Hoenicke and Goldman 1987, Hu and Tessier 1995). Others have shown a variety of results in competition experiments even when the same two cladoceran species were used (Neill 1975, Lynch 1978, Goulden et al. 1982, Smith and Cooper 1982, Hall et al. 2004). Among these, Hall et al. (2004) showed that supply rates of light relative to P changed dominant cladoceran species in enclosure experiments. However, it is not yet clear if these changes in relative abundance of cladoceran species can be attributed to changes in competitive superiority accompanied by changes of nutrients that limit growth as described by the resource ratio theory, although some theoretical studies have suggested such a possibility (Hall 2004, Loladze et al. 2004). In their classical works, Lampert (1977) and Lampert and Schober (1980) suggested that competitive superiority among Daphnia species can be predicted from threshold food levels in terms of carbon (C), at which the growth rate of the individual is zero, because these are equivalent to minimum food levels sustaining the population under negligible mortality rates. In our previous study (Iwabuchi and Urabe, submitted ), we estimated the threshold food levels of several Daphnia species in terms of not only C but also phosphorus (P) (hereafter, TFC and TFP). Because species with lower TFC did not necessarily have lower TFP, it is likely that competition superiority among Daphnia species may change depending on P contents relative to C contents in the food. However, it is not clear if these differences in threshold food levels indeed change outcomes of competition among Daphnia v 2 January 2012 v Volume 3(1) v Article 7

3 species. In this study, therefore, we examined (1) if outcomes of competition among consumer species change according to the elemental composition of food in a predictable way from the TFC and TFP, and thus (2) if the resource ratio theory for essential elements can be applied to consumer animals. For these objectives, we first predicted the outcomes of competition among Daphnia species from their known TFC and TFP. Then, the predictions were tested with a series of experiments using the Daphnia species. Predictions of competitive outcomes between Daphnia species We have previously shown that TFC and TFP were not the same among eight Daphnia species (Iwabuchi and Urabe, submitted ). Three of these eight Daphnia species, i.e., D. tanakai, D. galeata, and D. pulicaria, were used in this study because these species showed the lowest and highest values for either TFC or TFP. Fig. 1 shows zero net growth isoclines (ZNGIs) of the three species for food C and P as we assume that R*s for C and P are respectively equivalent to TFC and TFP. In this figure, the slope of the line drawn from the origin represents the food P:C ratio, while x- and y-values on this line represent the amounts of C and P in the food, respectively. Assuming that consumers do not instantly affect the P:C ratio of food, the food moves toward the origin along the resource ratio line as the food is consumed and the population increases. When fed a high P:C food, animals are C-limited and the food first reaches a threshold level for C (TFC). As D. galeata has the highest TFC, it will first cease to grow, while the other two species can still grow. Thus, D. galeata will decrease in abundance and eventually be excluded when competing against D. tanakai or D. pulicaria with high P:C food. Because the latter two species have no significant difference in TFC, they may coexist for a long time when competing for the high P:C food. When fed a low P:C food, animals suffer from P- deficiency. Because D. pulicaria has a lower TFP than the other species, they will dominate when competing against either of the other two by reducing the food abundance lower than their TFP. Because TFP was similar between D. tanakai and D. galeata, these two can coexist for a long time when competing for low P:C food. These predictions were tested with a series of experiments using algal food with the high and low P:C ratios by examining dynamics of the abundances of algal food and Daphnia species in the flow-through reaction vessels. METHODS Algal food and animals Scenedesmus obliquus (Turpin) Kürzing was used as algal food for the three Daphnia species. Algal cells with high and low P contents (HIP and LOP algae, hereafter) were cultured in chemostats containing COMBO (Kilham et al. 1998), an artificial inorganic medium. The chemostats were kept at 208C with a light intensity of 200 le m 2 s 1. The P content of these two algae was manipulated by the P concentration in the medium (100 lm K 2 HPO 4 for HIP and 25 lm K 2 HPO 4 for LOP) and the dilution rate (0.5 per day for HIP and 0.2 per day for LOP). The N concentrations were the same between HIP and LOP media. After they had attained an equilibrium cell density, the algae were harvested, washed with distilled water and used as food. Atomic P:C ratios of HIP and LOP algae were measured by the method described in Iwabuchi and Urabe (submitted ), and were and , respectively. D. tanakai (Ishida et al. 2006) was collected from a marsh in Mt. Zao, Miyagi, Japan. D. galeata was collected from an irrigation pond in Yamaguchi, Japan in D. pulicaria was collected from Lake Biwa (Urabe et al. 2003) and has been kept in the lab since then. All the species were reared at 208C with sufficient food (.1.0 mg C/L) for several months before the experiments. Both in stock cultures and experiments, Daphnia individuals were reared in 40% strength of N- and P-free COMBO, which was made by dilution with distilled water. Before use, we adjusted the ph of the basal COMBO to Ten to twenty individuals of each species were reared for several generations in 1 L bottles with 1.0 mg C/L of HIP algae. The medium and food were renewed every other day. In these stock cultures, neonates from the third clutch of each generation were used to establish the next generation in order to ensure constant condition of the neonates. Furthermore, we renewed the medium and algal food every day for the parents of the neonates used in the experiments. v 3 January 2012 v Volume 3(1) v Article 7

4 Fig. 1. Graphical presentation showing competitive interactions between three Daphnia species when fed high and low P:C algae. The horizontal and vertical lines represent the threshold food levels in terms of C (TFC) and P (TFP) estimated in our previous study (Iwabuchi and Urabe, submitted ), respectively. The gray areas represent 1SD. The two diagonal lines with arrows represent high and low P:C food lines, respectively. An x-value on those lines represents a food quantity, and their slope represents the P:C ratio of algal food. Arrows represent consumption vectors, which move toward the origin along the food lines as grazers consume the food. Culture system For experiments, we used a flow-through system similar to the one used by Kreutzer and Lampert (1999) that consisted of reservoir tanks and reaction vessels. The reservoirs contained either 0.15 mg C/L of HIP algae or 1.83 lg P/L (0.60 mg C/L) of LOP algae, both of which were above the TFC and TFP of all three Daphnia species (Iwabuchi and Urabe, submitted ). The algal carbon abundance in the reservoirs was calculated as the product of daily microscopic count (cells/l) and the algal carbon cell quota (lg C/L), which was estimated several times during the experiment. Fresh food and medium were renewed in the reservoir tanks everyday. A 100- lm plankton net was installed at the bottom of the reaction vessels to allow the medium and unfed algae but not Daphnia to pass through it. The volume of food suspension above the net, where Daphnia were kept, was 150 ml. Following Kreutzer and Lampert (1999), the inflow rate from the reservoirs to the vessels was set at 20 ml/h by a peristalic pump and the outflow was made by gravity, establishing a turnover rate at 3.5 per day in the vessels. All experiments were conducted at 208C under a dim light condition. The reaction vessels were cleaned every two to four days to remove detritus, algae, and exuviae and carcasses of Daphnia on the wall or mesh net at the bottom. While cleaning the reaction vessels, all animals were temporarily transferred to small beakers, which took less than five min per vessel. According to Kreutzer and Lampert (1999), algal abundance reached stable levels within nine days in this experimental setting. When algal abundance reached such a stable level, we considered it to be the minimum food level sustaining Daphnia population (F*). Ideally, the food level when consumer biomass is at equilib- v 4 January 2012 v Volume 3(1) v Article 7

5 rium (R*) should be measured to estimate competition superiority (Tilman 1982). However, in Daphnia populations, biomass can fluctuate due to a time lag between food uptake and reproduction (e.g., McCauley et al. 1988) making it difficult to measure R* precisely. In this study, therefore, we used F* as a gauge for competitive superiority together with difference in abundance between competing Daphnia species as in Kreutzer and Lampert (1999). Single species experiment To examine if the threshold level at the individual level (TFC and TFP) estimated in our previous study (Iwabuchi and Urabe, submitted ) are retained at the population level (F* for C and P) in each Daphnia species, we first conducted experimental runs with a single Daphnia species. To initiate an experiment, three 11-day-old adults and five 1-day-old neonates were placed into each of six reaction vessels for each species. The algal abundances in the flow-through reaction vessels were determined every two to four days during the experiments. To determine an algal abundance, we used a particulate counter (CASY cell counter Model TT, Innovatis AG, Reutlingen, Germany) as in Kreutzer and Lampert (1999). We set the size range of the particles to be counted to lm so that both HIP and LOP cells would be covered. In the measurement procedure, we withdrew a 2 ml sample of algal suspension from the reaction vessel by a micropipette, diluted the sample to 10 ml with CASYton (Innovatis), an isotonic particle-free (.0.2 lm) solution, and averaged five measurements of 400 ll from each sample. In parallel with the measurements of algae in the reaction vessels, we also measured the volumetric abundance of algal cells in the reservoir tanks at a known algal carbon biomass and estimated conversion factors for carbon biomass from the volumetric abundance. Using these conversion factors, we estimated algal C biomass in the reaction vessels. For LOP algae, food abundance in terms of P was calculated from the C cell quota and P:C ratios of the LOP cells. After day 14, the number of individuals in each reaction vessel was also counted when algal abundance was determined. The number of individuals was not counted before that date to minimize any damage to animals during the growing phase. The experimental run lasted for 25 days. On day 20, individuals in three randomly selected vessels were sacrificed to determine Daphnia biomass, which was measured after drying at 608C for a day. On the last day (day 25), Daphnia in the remaining three vessels were sacrificed in the same way as those on day 20. The same procedures were performed for experiments with HIP and LOP algae. Competition experiment We performed experiments by inoculating one of three combinations in species pairs (D. tanakai vs. D. galeata, D. tanakai vs. D. pulicaria, and D. pulicaria vs. D. galeata) into reaction vessels receiving either HIP or LOP algae. In any competition experiments, three 11-day-old adults and five 1-day-old neonates of each species were placed into each vessel to initiate an experiment. We performed competition experiments with the same procedures as those of the single species experiments, except that routine counts of Daphnia individuals after day 14 were done under a microscope to distinguish the species. Damage due to the microscopic observation was negligible. Data analysis The stable phase of algal biomass for the single species experiments was determined by the linear regression analysis. A linear regression was first estimated for algal biomass in terms of C against time using all the data between day 4 and 25. Then a data point on the earliest day of the data set was successively removed, until the slope no longer differed from zero ( p. 0.05) (Kreutzer and Lampert 1999). According to this analysis, the algal biomass became stable after day 14 in all our experiments. Therefore, we averaged algal data obtained after day 14 and defined the averaged algal biomass as the minimum food level for population (F*) according to Kreutzer and Lampert (1999). T-tests were used to test if there was a significant difference between the F* for C (F C *) in the experiment with HIP algae and the TFC estimated in our previous study (Iwabuchi and Urabe, submitted ) for each Daphnia species. Similarly, t-tests were also employed to determine if there was a significant difference between the F* for P (F P *) in the experiment with LOP algae and the TFP estimatv 5 January 2012 v Volume 3(1) v Article 7

6 ed in our previous study (Iwabuchi and Urabe, submitted ) was tested for each species. To examine differences in F C * and F P * among the three species fed the same algal food, we used generalized linear mixed models (GLMMs) with the model selection procedure using Akaike s information criteria (AIC) (Akaike 1973). We used this method because the experiments were conducted with repeated measures and the number of replication vessels differed with experimental date (six until day 20 and three after that). Each model assumed a Gaussian distribution and included a dummy variable representing one of five combinations in multiple comparisons for differences in F* between the three species. The dummy variable was treated as a fixed effect, while replication and observation date were incorporated as random effects. A model with the lowest AIC value and models with AIC not greater than 2 from the lowest AIC (i.e., DAIC, 2) were selected as the best models from the five models (Burnham and Anderson 2002). We also used GLMMs with the model selection procedure described in the previous paragraph to examine differences in the F* between competition experiments and the corresponding single species cultures. Models for each competition experiment included a dummy variable representing one of five combinations in multiple comparisons for differences in F* between the competition experiment and the corresponding two single species experiments. In this model, the dummy variables were treated as a fixed effect. To examine the magnitude of the competition effect on competing species, we calculated the ratio of biomass in the competition experiment to that in the single species experiment for each Daphnia species. In this estimation we used the average biomass among replications of the single species experiment. Because the number of observed data was limited, it was not possible to identify exact data distribution of the population. Therefore, the ratios were transformed by the Box-Cox transformation with the estimated transformation parameters (k ¼ and for HIP and LOP treatments, respectively) to stabilize the variance (Sokal and Rohlf 1981). The GLMMs with Gaussian distributions were then constructed and a model selection procedure was performed to examine if the competition effect was different between the two competing species. In this modeling, we used species as a fixed effect and the observation date as a random effect. All statistical analyses were done using R (R Development Core Team 2009). RESULTS Single species experiments The algal food abundances in the reaction vessels with Daphnia individuals are shown in Fig. 2a and e. The F*forC(F C *) in the experiment using D. tanakai fed HIP algae was significantly higher than the TFC estimated in our previous study (t ¼ 3.88, p, 0.05 with the Bonferroni correction). However, no such significant differences were detected between F C * and TFC in the case of D. galeata and D. pulicaria (t ¼ 1.45, p and t ¼ 1.71, p. 0.05, respectively). When fed LOP algae, the F* for P (F P *) was not significantly different from the TFP estimated in our previous study for any of the species (t ¼ 2.29, p. 0.05; t ¼ 1.92, p. 0.05; and t ¼ 0.15, p for D. tanakai, D. galeata, and D. pulicaria, respectively). In experiments with HIP algae, the F C *was higher for D. galeata than for D. tanakai and D. pulicaria, while those for the latter two were similar to each other (Fig. 2a). The best two GLMMs (DAIC, 2) included the fixed effects representing difference of D. galeata from D. tanakai and D. pulicaria (Table 1). These results imply that D. tanakai and D. pulicaria reduced the food abundance to a level lower than that reduced by D. galeata. Thus, rank order of the F C * among these three species was consistent with that of TFC estimated in our previous study (Iwabuchi and Urabe, submitted ). In experiments with LOP algae, the best GLMM for F P * indicated that D. pulicaria reduced the food abundance to a level lower than those by D. tanakai and D. galeata did, and the latter two showed similar F P * (Fig. 2e, Table 1). Thus, the rank order of F P * among the three species was again consistent with that of TFP estimated in our previous study (Iwabuchi and Urabe, submitted ). In any experiment, the number of individuals of Daphnia increased for the first 15 days and continually increased more or less thereafter (Fig. 3a, e), except for D. galeata fed LOP algae. However, their biomass did not significantly v 6 January 2012 v Volume 3(1) v Article 7

7 Fig. 2. Food abundances in the single species and competition experiments with (a d) high-p (HIP) and (e h) low-p (LOP) algae: (a, e) single species experiments for three Daphnia species, (b, f ) competition experiment for D. tanakai and D. galeata, (c, g) competition experiment for D. tanakai and D. pulicaria, (d, h) competition experiment for D. pulicaria and D. galeata. The results of the single species experiments of the corresponding species are also shown in the results of the competition experiments for comparison. Note that abundances of HIP algae are expressed in terms of C concentration (mg C/L), and that of LOP algae were expressed in terms of P concentration (lg P/L). The error bars show 61 SD. v 7 January 2012 v Volume 3(1) v Article 7

8 Table 1. Results of GLMMs for stable food abundances (F*) in the single species and competition experiments. Single species T vs. G T vs. P G vs. P Fixed effect DAIC Fixed effect DAIC Fixed effect DAIC Fixed effect DAIC HIP algae TGP 0.0 TGC 0.0 TPC 0.0 GPC 0.0 TGP 0.4 TGC 1.3 T P C (null) 0.9 G PC 0.6 T G P 9.9 T G C 9.1 T P C 2.0 GPC 10.6 T G P (null) 14.1 TGC(null) 11.7 TPC 2.1 G P C (null) 16.2 T G P 15.3 T G C 12.5 T P C 2.3 G P C 17.5 LOP algae TGP 0.0 TGC 0.0 T PC 0.0 G PC 0.0 TGP 1.5 TGC 1.7 T P C 2.0 G P C 0.6 TGP 26.1 TGC 4.1 T P C 17.2 G P C 30.7 T G P 28.9 T G C (null) 5.1 TPC 17.7 GPC 35.8 T G P (null) 30.1 T G C 6.5 T P C (null) 18.7 G P C (null) 36.4 Notes: Models are represented by their fixed effects and DAIC. The letters T, G, and P represent D. tanakai, D. galeata, D. pulicaria in the single species experiments, respectively. The letter C represents the competition experiment for given pairs of the species. In each fixed effect, the underlined letters represent that they are not different in effect on F* between them. For each experiment, models with DAICs, 2 are selected as the best models and denoted in bold letters. HIP, high P content; LOP, low P content. differ between day 20 and 25 for all three species in both HIP and LOP experiments (t ¼ 1.21, p, 0.05; t ¼ 0.37, p, 0.05; t ¼ 1.98, p, 0.05 for D. galeata, D. tanakai, and D. pulicaria in HIP experiments, respectively, and t ¼ 0.74, p, 0.05; t ¼ 0.12, p, 0.05; t ¼ 0.26, p, 0.05 for D. galeata, D. tanakai, and D. pulicaria in LOP experiments, respectively). When fed LOP algae, D. galeata did not show an increase in number of individuals or biomass (Fig. 3e). In contrast, D. pulicaria achieved a higher number of individuals and biomass when fed LOP algae than when fed HIP algae, probably due to the higher supply of algal food. Competition experiments D. tanakai vs. D. galeata. In the experiments with HIP algae, the best GLMMs for F C * included the fixed effect representing the difference of the competition experiment from the single species experiment for D. galeata but not from that for D. tanakai (Table 1). The result implies that F C * in the competition experiment was at least lower than that in the single species experiment for D. galeata (Fig. 2b). In both species, the number of individuals was lower in the competition experiment than in the single species experiments (Fig. 3b). However, the biomass of D. tanakai in the competition experiment was similar to that in the single species experiments (Fig. 4b). The best GLMM (Table 2) indicated that the biomass ratio between the competition experiment and single species experiments was higher in D. tanakai than in D. galeata (Fig. 5a). When LOP algal food was used, the best GLMM indicated that F P * in the competition experiment was lower than those in the single species experiments (Fig. 2f, Table 1), which were similar to each other as described in Results: single species experiments. The number of individuals and biomasses of both D. tanakai and D. galeata in the competition experiment decreased compared with those in the single species experiments (Figs. 3f and 4f ). For the biomass ratio between the competition experiment and single species experiments, however, AIC did not greatly differ between the model with the fixed effect representing difference between the two species and the null model (DAIC, 2; Table 2). The result suggests that magnitude of the biomass decrease due to the competition did not necessarily differ between these species (Fig. 5d). D. tanakai vs. D. pulicaria. When fed HIP algae, the best GLMM of algal food indicated that F C * was similar between the competition experiment and the single species experiments for both species (Fig. 2c, Table 1). In the competition experiment, both species showed lower number of individuals and biomass compared to the single species experiments (Figs. 3c and 4c). According to the GLMM of the biomass ratio between the competition experiment and single species experiments, magnitude of the v 8 January 2012 v Volume 3(1) v Article 7

9 Fig. 3. Number of Daphnia individuals in the single species and competition experiments with (a d) high-p (HIP) and (e h) low-p (LOP) algae: (a, e) single species experiments for three Daphnia species, (b, f ) competition experiments for D. tanakai and D. galeata, (c, g) competition experiment for D. tanakai and D. pulicaria, (d, h) competition experiment for D. pulicaria and D. galeata. The results of the single species experiments of the corresponding species are inserted in the panels of competition experiments for comparison. The error bars show 61 SD. v 9 January 2012 v Volume 3(1) v Article 7

10 Fig. 4. Daphnia biomass in the single species and competition experiments with (a d) high-p (HIP) and (e h) low-p (LOP) algae: (a, e) single species experiments, (b, f ) competition experiments for D. tanakai and D. galeata, (c, g) competition experiments for D. tanakai and D. pulicaria, (d, h) competition experiments for D. pulicaria and D. galeata. The error bars show 61 SD. biomass decrease by the competition did not necessarily differ between these species (Fig. 5b, Table 2). When we used LOP algae as food, the best GLMM of algal food indicated that F P * in the competition experiment was similar to that of the v 10 January 2012 v Volume 3(1) v Article 7

11 Table 2. Results of GLMMs for biomass ratios between the single species and competition experiments of Daphnia species fed high-p (HIP) and low-p (LOP) algae. T vs. G T vs. P G vs. P Fixed effect DAIC Fixed effect DAIC Fixed effect DAIC HIP algae TG 0.0 TP 0.0 GP 0.0 T G (null) 14.2 T P (null) 1.3 G P (null) 19.8 LOP algae T G (null) 0.0 TP 0.0 GP 0.0 TG 1.4 T P (null) 14.2 G P (null) 22.6 Notes: Models are represented by their fixed effect and DAIC. The letters T, G, and P represents D. tanakai, D. galeata, and D. pulicaria, respectively. In each fixed effect, the underlined letters represent that the paired species have the same effect on biomass ratios. For each experiment, DAICs less than 2 are denoted in bold letters. single species experiment for D. pulicaria, but lower than that of the single species experiment for D. tanakai (Fig. 2g, Table 1). Both species showed lower number of individuals and biomass compared to the corresponding single species experiments (Figs. 3g and 4g). However, the biomass ratio between the competition experiment and single species experiments was higher for D. pulicaria than for D. tanakai (Fig. 5e) as evidenced by the GLMMs (Table 2). D. pulicaria vs. D. galeata. The best GLMM for HIP algal food indicated that F C * in the competition experiment was similar to that in the single species experiment for D. pulicaria, but lower than that in the single species experiment for D. galeata (Fig. 2d, h, Table 1). The same result was obtained for F P * in experiments with LOP algae. In the competition experiments, D. pulicaria maintained higher individual number (Fig. 3d, h) and biomass (Fig. 4d, h) than D. galeata did regardless of P content of the algal food. According to the best GLMM for the biomass ratio (Table 2), magnitude of the biomass decrease due to the competition was much greater for D. galeata than for D. pulicaria (Fig. 5c, f ). DISCUSSION In general, a species can be judged as competitively superior to others sharing the same resource if the former excludes the latter from the exploitative competition arena. However, it is often very laborious to identify competitively superior species by observing the long-term population dynamics of different consumer species. Indeed, it takes a long time for a species to competitively eradicate an inferior species even in experiments using zooplankton with short life spans (DeMott 1989, McCauley et al. 1999, Urabe et al. 2002). However Lampert (1977) and Lampert and Schober (1980) suggest that competitive superiority among Daphnia species can be assessed from species-specific differences in minimum food requirement to sustain individual growth. Therefore, we first predicted competitive superiority between Daphnia species from the threshold food levels of C and P for individual growth (TFC and TFP). Then the predictions were examined experimentally by measuring the minimum food level needed to sustain the population (F*) using a specifically designed flow-through system as in Kreutzer and Lampert (1999). The experiments showed that the rank orders of F* forc(f C *) and for P (F P *) among the three Daphnia species examined were the same as those of TFC and TFP, respectively. Moreover, no significant difference was found between F C * and TFC except in D. tanakai, nor between F P * and TFP in any of the Daphnia species. The results suggest that minimum food concentration in terms of not only C but also P was at almost the same level for individual and population growth rates, and thus that competitive superiority among Daphnia species can be predicted from minimum food level sustaining individual growth if mortality is negligible (see below). According to the resource ratio theory, the supply rate and ratio of two resources determine competitive outcomes, which can be predicted by the threshold level of each resource necessary to maintain the population of the competitors (Tilman 1982). On the one hand, we predicted from v 11 January 2012 v Volume 3(1) v Article 7

12 Fig. 5. Competition effect represented by Box-Cox transformed biomass ratio between the single species and competition experiments with (a c) high-p (HIP) and (d f ) low-p (LOP) algae. The horizontal dashed lines represent zero in the transformed biomass ratio, representing no change in biomass between single species and competition experiments. The error bars show 61 SD. the TFC that when fed high P food (HIP algae), D. tanakai and D. pulicaria would be competitively equal to each other but superior to D. galeata. On the other hand, according to their TFP, it was predicted that when fed low P food (LOP algae), D. pulicaria would outcompete D. tanakai and D. galeata, while neither of the latter two would be competitively superior to the other. In accordance with these predictions, when fed HIP algae, D. tanakai and D. pulicaria were competitively equal and reduced food abundance to a level lower than TFC of D. galeata. Consequently, the biomass v 12 January 2012 v Volume 3(1) v Article 7

13 of Daphnia species with higher TFC was reduced when competing for HIP algae with the species with lower TFC. When fed LOP algae, D. pulicaria reduced the food abundance to a level as low as its own TFP, which was lower than those of D. tanakai and D. galeata. As a result, the biomass of Daphnia species with higher TFP was reduced when this species competed for LOP algae with the other species with lower TFP. These results confirmed the predictability of competitive outcomes from threshold food levels that are necessary for individual growth. As in other herbivorous animals (Moran and Whitham 1990, Chase et al. 2002, Kaplan and Denno 2007, Preisser and Elkinton 2008), a number of studies have examined competitive interactions among herbivorous zooplankton and shown a variety of experimental results even when the same two species were used (Neill 1975, Lynch 1978, Goulden et al. 1982, Smith and Cooper 1982). The inconsistent outcomes of competition between the same two species could be because relative elemental contents of the food may have been different between experiments in these studies. However, few studies have tested if competitive superiority between consumers changes depending on the relative element contents of food, although these changes have long been suggested by the resource ratio theory (Tilman 1982, Grover 1997) and theories of ecological stoichiometry (Sterner and Elser 2002, Hall 2004, Loladze et al. 2004). This study showed for the first time that competitive superiority between consumers could change depending on the relative element contents of food. Note that, among the three species we used, D. pulicaria was never inferior to the other two species both in experiments with HIP and LOP algae. Thus, when only competition for algal food matters, this species can dominate any habitat over the other two species. However, D. pulicaria and D. galeata sometimes occur in the same pond or lake (Leibold and Tessier 1991, Cáceres 1998, Urabe et al. 2003). A possible drawback of D. pulicaria may be its vulnerability to predation. Since D. pulicaria is the largest in body size and thus more vulnerable to fish predation, coexistence of this species with D. galeata relies on habitat segregation by depth, as D. pulicaria inhabits deeper water, where predation is less intense (Cáceres 1998, Urabe et al. 2003). Although it was inferior to D. pulicaria at a low P:C food, D. tanakai was not inferior to D. galeata regardless of P content of the algal food. The results suggest that D. tanakai could be dominant over D. galeata in nature. The distribution of D. tanakai is, however, restricted only to high mountain areas (Ishida et al. 2006). Because D. tanakai is similar in body size to D. galeata, itis unlikely that the former is more susceptible to size-selective predation by fish than the latter. Other than body size, agility is also an important trait for avoiding predation by not only fish but also invertebrates such as Chaoborus and Cyclops (Browman et al. 1989, Chang and Hanazato 2003). Since D. tanakai is laterally stocky in body shape compared to D. galeata, this species seems to be poorer in agility than D. galeata. Indeed, the former individuals were much easier to pick up by a pipette during a procedure of culture maintenance (T. Iwabuchi, personal observation). The fact suggests that there is a trade-off between the competitive superiority for food resources and vulnerability to predation in herbivorous consumers. Due to such a trade-off, the distribution of D. tanakai may be restricted in mountain lakes and small pools in moors, where few predators are found. Other than herbivorous zooplankton, recent studies have shown that in a variety of animals including snails, ants, caterpillars and true bugs, the individual growth rate is highly sensitive to changes in elemental or chemical contents of the food resources (Sterner and Elser 2002, Fink and Von Elert 2006, Apple et al. 2009, Zehnder and Hunter 2009, Wilder et al. 2011). Thus, if minimum food level sustaining positive rate of the individual growth is related with that maintaining the population as seen for Daphnia, and if demands of elements for the individual growth differ among competitors, competition outcomes would change depending on elemental contents of the food. It is known that elemental contents of algae and foliage change with a number of environmental variables such as nutrient supply, light and CO 2 levels, and temperature (Sterner and Elser 2002, Valkama et al. 2007, Urabe and Waki 2009, Makino et al. 2011). These environmental variables can thus affect herbivore communities through change in competitive superiority among herbivores. v 13 January 2012 v Volume 3(1) v Article 7

14 To summarize, our experiments showed that competitive superiority among herbivorous consumers changed according to elemental composition of the food. Thus, the effects of elemental composition of the food resource on competition between consumers are basically similar to those of supply ratios of multiple nutrients on competition between algae. Tilman (1982) showed that two algal species can stably coexist depending on the supply rates and ratio of two resources when each species consumes a greater amount of the resource that most limits its own growth than its competitors. In the case of this study, as long as TFC and/or TFP are not the same, consumer species would not exhibit stable coexistence because the relative contents of C and P in the food were fixed. However, stable coexistence of these species may be possible if nutrient recycling driven by consumers greatly influences the relative content of C and P in algae and if the recycling efficiency of each element differs between species, as suggested in Hall (2004) and Loladze et al. (2004). Further experimental studies are necessary to test this possibility. ACKNOWLEDGMENTS We deeply appreciate James M. Hood and Maren Striebel for their valuable comments and suggestions on the early version of our manuscript. We also thank Wataru Makino for his valuable help to experiments and data analyses. We also appreciate the members of the Community and Ecosystem Ecology Laboratory at Tohoku University for discussion and suggestions. This research was financially supported by a grant-inaid for scientific research A (No ) from MEXT Japan, Environmental Research & Technology Development Fund (D-1002) from Ministry of the Environment of Japan and the Global COE program J03 of MEXT Japan. LITERATURE CITED Akaike, H Information theory and an extension of the maximum likelihood principle. Pages in B. N. Petrov and F. Czáki, editors. Second International Symposium on Information Theory. Akademiai Kiadó, Budapest, Hungary. Apple, J. L., M. Wink, S. E. Wills, and J. G. Bishop Successional change in phosphorus stoichiometry explains the inverse relationship between herbivory and lupin density on Mount St. Helens. PLoS ONE 4:e7807. Browman, H. I., S. Kruse, and W. J. O brien Foraging behavior of the predaceous cladoceran, Leptodora kindti, and escape responses of their prey. Journal of Plankton Research 11: Burnham, K. and D. Anderson Model selection and multi-model inference. Springer, New York, New York, USA. Cáceres, C. E Seasonal dynamics and interspecific competition in Oneida Lake Daphnia. Oecologia 115: Chang, K. H. and T. Hanazato Vulnerability of cladoceran species to predation by the copepod Mesocyclops leuckarti: laboratory observations on the behavioural interactions between predator and prey. Freshwater Biology 48: Chase, J. M., P. A. Abrams, J. P. Grover, S. Diehl, P. Chesson, R. D. Holt, S. A. Richards, R. M. Nisbet, and T. J. Case The interaction between predation and competition: a review and synthesis. Ecology Letters 5: DeMott, W. R Seasonal succession in a natural Daphnia assemblage. Ecological Monographs 53: DeMott, W. R The role of competition in zooplankton succession. Pages in U. Sommer, editor. Plankton ecology: succession in plankton communities. Springer-Verlag, Berlin, Germany. DeMott, W. R Utilization of a cyanobacterium and a phosphorus-deficient green alga as complementary resources by daphnids. Ecology 79: DeMott, W. R. and B. J. Pape Stoichiometry in an ecological context: testing for links between Daphnia P-content, growth rate and habitat preference. Oecologia 142: Elser, J. J. and D. Hessen Biosimplicity via stoichiometry: the evolution of food-web structure and processes. Pages 7 18 in A. Belagrano, D. Scharler, and U. Ulanowicz, editors. Aquatic food webs: an ecosystem approach. Oxford University Press, Oxford, UK. Ferrão-Filho, A. D., A. J. Tessier, and W. R. DeMott Sensitivity of herbivorous zooplankton to phosphorus-deficient diets: Testing stoichiometric theory and the growth rate hypothesis. Limnology and Oceanography 52: Fink, P. and E. Von Elert Physiological responses to stoichiometric constraints: nutrient limitation and compensatory feeding in a freshwater snail. Oikos 115: Goulden, C. E., L. L. Henry, and A. J. Tessier Body size, energy reserves, and competitive ability in three species of cladocera. Ecology 63: Grover, J Resource competition. Chapman & Hall, New York, New York, USA. Hall, S. R Stoichiometrically explicit competition between grazers: species replacement, coexistence, v 14 January 2012 v Volume 3(1) v Article 7

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