Freshwater Biology (2008) 53, 1291–1302 doi:10.1111/j.1365-2427.2008.01962.x

Stoichiometric relationships in vernal pond plankton communities

CARLA E. CA´ CERES*,ALANJ.TESSIER†, ANDRI ANDREOU* AND MEGHAN A. DUFFY‡ *School of Integrative Biology, University of Illinois at Urbana-Champaign, Urbana, IL, U.S.A. †Division of Environmental Biology, National Science Foundation, Arlington, VA, U.S.A. ‡School of Biology, Georgia Institute of Technology, Atlanta, GA, U.S.A.

SUMMARY 1. The light-nutrient hypothesis (LNH) predicts that changes in light supply can alter the balance of nutrient and energy limitation in primary producers. We tested this prediction by examining temporal changes in vernal forest ponds, which are highly dynamic systems with respect to seasonal change in light and nutrient supply. In three vernal ponds that differ in productivity, we measured changes in light, total and seston nitrogen and phosphorus, and seston carbon and chlorophyll during the spring, before and after tree leaf-out. We also quantified changes in the population dynamics of the major zooplankton grazers in these systems. 2. In each pond, nutrient levels increased and light levels declined, creating a temporal shift in light-nutrient supply to the plankton. Results generally supported predictions of stoichiometric theory and the LNH, but there were notable exceptions. 3. Seston C : N : P ratios rapidly changed in response to dramatic increases in N and P supply rates. However, seston N : P was typically lower than values for total N : P in the water. Furthermore, as predicted, we observed a decline in seston C : P as the light : nutrient ratio declined, but seston C : N simultaneously increased. These results suggest an unexpected shift towards potential nitrogen limitation. Alternatively, this change in nutrient ratios may be driven by a seasonal change in phytoplankton composition or nutritional mode. 4. Seston carbon concentrations remained stable despite seasonal changes in grazing intensity associated with the phenology of large-bodied Daphnia grazers. However, chlorophyll concentrations declined dramatically as the season progressed, resulting in a simultaneous decline in the C : Chlorophyll ratio of seston. Both pond shading and increased grazing probably contributed to the decline in chlorophyll.

Keywords: carbon, Daphnia, light-nutrient hypothesis, nitrogen, phosphorus

questions of energy versus nutrient limitation can be Introduction addressed at multiple trophic levels (Hassett et al., Ecological stoichiometry, the balance of nutrients and 1997; Elser et al., 2000; Hall et al., 2006). The elemental energy in ecological interactions, provides a valuable composition of primary producers fluctuates in framework for investigating trophic interactions response to external changes in nutrient supply (Sterner & Elser, 2002). By tracking supply rates of whereas that of grazers is more constant (Andersen elements such as carbon, nitrogen and phosphorus & Hessen, 1991; Hessen & Faafeng, 2000). Hence, through the primary producers and into grazers, stoichiometric theory predicts thresholds of change where, depending upon the nutrient supply ratios, it Correspondence: Carla E. Ca´ceres, 515 Morrill Hall, Urbana, IL is expected that grazers can become limited by 61801, U.S.A. Email: [email protected] nutrients (food quality) rather than energy (food

2008 The Authors, Journal compilation 2008 Blackwell Publishing Ltd 1291 1292 C. E. Ca´ceres et al. quantity). Such transitions impact energy flow and reasons. First, these systems tend to exhibit dramatic nutrient recycling within ecosystems, can shift com- temporal changes in light and nutrient supply levels petitive relationships among species, and shape the (Colburn, 2004). In forested ponds, tree leaves pre- evolution of organism physiology and life history dictably open out over the course of a few weeks, (Andersen, 1997; Sterner & Elser, 2002). decreasing light levels dramatically. In addition, The stoichiometric framework considers more than vernal ponds form from nutrient-dilute snowmelt just the supply of elemental nutrients. Light is also and rain, but should experience a large increase in important, as is the interaction between light and nutrient supply due to decomposition from forest nutrients. In their light-nutrient hypothesis (LNH), floor litter (Wilbur, 1997). Second, temporary ponds Sterner et al. (1997) make several predictions regard- are often dominated by mixotrophic algae (Colburn, ing how nutrient use efficiency should vary across 2004). As the season progresses, this ability to change systems experiencing different light to nutrient ratios. nutritional mode as light and nutrient supply ratios The general concept is that as light becomes more are altered, coupled with overall changes in species limiting to primary producers, their elemental com- composition, may contribute to variance in the stoi- position should become enriched in nutrients such as chometric ratios of the seston. Third, the influence of nitrogen and phosphorus relative to carbon, resulting top-down factors on seston stoichiometry can fluctu- in increased food quality for grazers and higher ate just as dramatically as the bottom-up factors of trophic transfer efficiency. Of course, low light condi- light and nutrients (DeMott & Tessier, 2002). Hall tions will also reduce overall primary productivity, et al. (2007) showed evidence that grazers may play a decreasing food quantity for grazers. However, the key role in explaining field patterns of variation in evidence available from lakes suggests that limitation seston stoichiometry. In particular, top-down effects via food quality (including stoichiometry, phyto- of grazers increase turnover rate of the producers and plankton assemblage, fatty acid composition, etc.) is recycling rates of elements, both of which are often more important than is limitation by food expected to influence the stoichiometry of producers. quantity (Sterner & Schulz, 1998). Large-bodied daphniid species, which are a hallmark Several predictions of the LNH have been con- of temporary ponds (Schneider & Frost, 1996; firmed by focusing on comparisons among lakes that Colburn, 2004), hatch from diapausing eggs and can differ in mixing depth (mean light conditions for the quickly attain high biomass. Just as quickly, these phytoplankton), transparency and productivity either populations can disappear from the water entirely, as by using short-term manipulations such as mesocosm the result of producing diapausing rather than imme- experiments or broad-scale comparative studies (e.g. diately hatching eggs and increased predation. This Urabe & Sterner, 1996; Sterner et al., 1997; Hessen, creates a highly dynamic system with respect to Færøvig & Andersen, 2002; Diehl, Berger & Wohrl, grazer-imposed mortality rates on the primary pro- 2005; Schade et al., 2005; Dickman, Vanni & Horgan, ducers. 2006). Hall et al. (2007) extended the LNH to include In this study, we ask how temporal changes in light ponds that differ in light caused by tree shading, but and nutrient supply coupled with seasonal changes in still considered only spatial variation among commu- grazer abundance relate to seston stoichiometry in nities. However, light limitation has long been viewed three temporary ponds located in southwestern MI, as central in explaining temporal variation in primary U.S.A. Specifically, we addressed the following ques- productivity in most temperate ecosystems, and is tions (1) Are the patterns of temporal changes in pivotal to our understanding of seasonal succession in seston stoichiometry similar across ponds that differ the plankton (Sommer et al., 1986). The LNH predicts in trophic state (nutrient supply)? (2) Are these that an annual reduction in light level may cause a temporal changes related to the reduction in light shift in the balance of nutrient and light limitation in supply caused by tree leaf-out? In addition, we the phytoplankton. However, this prediction has discuss how seasonal changes in grazer abundance received limited testing (Chrzanowski & Grover, may influence, or be influenced by, seston stoichiom- 2001). etry. Our study was motivated by the premise that Vernal ponds are intriguing systems in which to temporal shifts in elemental ratios, including light, address these stoichiometric concepts for a number of may be particularly important in forested temporary

2008 The Authors, Journal compilation 2008 Blackwell Publishing Ltd, Freshwater Biology, 53, 1291–1302 Stoichiometry in temporary ponds 1293 ponds, where canopy closure, temporal variation in <15 lm and <3 lm) were also filtered onto GF ⁄F glass- supply rates and variable grazing pressure can create fibre filters, extracted in cold 95% ethanol and mea- substantial seasonal changes. sured by use of narrow band fluorometry (Welsch- meyer, 1994). To sample the zooplankton assemblage, we col- Methods lected three replicate zooplankton samples (12–21 L We studied three natural, vernal forest ponds that each) on each date by pooling 4–7 (depending on differ in productivity as reflected by nutrient concen- pond size) grab samples, taken with a 3-L pitcher. trations of nitrogen and phosphorus (Roughwood, The grab samples were taken while walking a Woodfrog and West Gull). The ponds, located in transect through the middle of each pond, taking Kalamazoo County, MI, U.S.A., fill with snowmelt care not to resample previously disturbed areas and rainwater in early spring (March–early April), at (Dudycha, 2004). Two of the samples were passed which time they achieve their greatest volume. This is through a 170 lm sieve to concentrate the zooplank- also the time of peak sunlight to these ponds. ton and were individually preserved in >70% ethanol Deciduous trees in and around the ponds form a and processed under a dissecting microscope. All forest canopy that shades the ponds after leaf-out, individuals were identified and counted to species which is typically in May. Hence, each pond experi- (Daphnia and Daphniopsis), genus (all other cladocer- ences a temporal gradient in light over the course of ans) or order (copepods). Daphnia adults and juve- only 2 months. The ponds typically dry in June, but niles were counted separately, and for Daphnia pulex Woodfrog has a higher groundwater input than the Leydig which was the most common species, a other two ponds and can remain wet in some years random sample of adults was measured for body (S. Hamilton, pers. comm). length and fecundity. These body length measure- To quantify temporal changes in light, nutrient ments were used to estimate grazing rate, based on supply rate, seston stoichiometry and grazer dynam- the equation given in Knoechel & Holtby (1986). The ics, we began sampling the ponds immediately after third zooplankton sample was used to estimate total ice out on 29 March 2001 for Roughwood and grazer biomass. That sample was returned to the Woodfrog Ponds, and on 6 April 2001 for West Gull laboratory on ice and filtered live onto a pre-weighed Pond. We sampled weekly until 20 June, after which glass fibre filter, dried overnight at 55 C, and time shallow water precluded sampling. We measured weighed using an analytical balance. light levels at the pond surface on each sampling date We measured pond surface dimensions and depth in April and May using a LI-COR Quantum meter and on each sampling date. Using these morphometric PAR sensor (LI-COR Biosciences, Lincoln, NE, U.S.A.). parameters, we estimated pond volume on each These values were expressed as a percent of full sun date, so that zooplankton abundances could be values by making simultaneous light measurements in expressed in terms of total pond volume. Analyzing ) open areas adjacent to each pond. We collected grab zooplankton values as number L 1 can confound samples of water for nutrient analysis from the middle temporal changes in population dynamics with a of the water column, making certain to avoid inclusion concentration of individuals as the pond dries of sediment. Water samples were frozen for later (Hairston, 1988). analysis of total phosphorus (molybdate-ascorbic acid To assess the possibility of temporal changes in method; APHA, 1980; Prepas & Rigler, 1982) and total food availability to the grazers, we used the egg ratio ) nitrogen (Bachmann & Canfield, 1996). We also mea- method to calculate birth rates (day 1) for D. sured the C, P and N content of the seston by passing pulex (Edmondson, 1960; Paloheimo, 1974): b= the water through a 62 lm sieve and then filtering it ((ln(E ⁄N)+1)) ⁄D. This equation is based on the num- onto pre-combusted, GF ⁄F glass fibre filters (What- ber of eggs per individual (E ⁄N) and the temperature- man, Clifton, NJ, U.S.A.). Particulate nitrogen and dependent development time D. Development times carbon were measured using a Carlo-Erba C : H : N were calculated based on the equation given in analyzer (Carlo-Erba, Milan, Italy) and particulate Bottrell et al. (1976). The temperature we used in phosphorus was measured by the molybdate-ascorbic calculating egg development times was the mean of acid method. Samples for chlorophyll a (total, <62 lm, the minimum and maximum temperatures that

2008 The Authors, Journal compilation 2008 Blackwell Publishing Ltd, Freshwater Biology, 53, 1291–1302 1294 C. E. Ca´ceres et al. occurred between each sampling date, as recorded Results from a max–min thermometer left in each pond in a ) shaded location. Population growth rates (r, day 1) The three ponds differed in size and trophic status, as were estimated from the logarithms of the water- measured by TP, chlorophyll and particulate carbon column abundance (N) on two successive sampling concentrations in early April (Table 1, Fig. 1). ) dates (t2 and t1): r = (ln N2 ln N1) ⁄(t2-t1) (e.g. Hall, Roughwood is oligotrophic with chlorophyll of only ) ) 1964). Death rates (d, day 1) were then estimated by 2 lgL 1, Woodfrog is typical of mesotrophic condi- ) taking the difference between birth and realized tions, with chlorophyll values of 4–6 lgL 1, and West growth rates. Gull is eutrophic with chlorophyll values exceeding ) Seasonal changes in the three ponds were statisti- 20 lgL 1. Similarly, particulate carbon is low ) cally modelled using a repeated measures analysis of (400 lgL 1) in Roughwood, but nearly double that variation (SAS, 2002, ver 9.1, Cary, NC, U.S.A.), in Woodfrog, and nearly double that again in West following log transformation (except for light). For Gull (Fig. 1b). most variables, we were interested in changes that Light levels dramatically declined from April to occurred in response to shading of the ponds by leaf- May following leaf-out of the canopy (RM ANOVA – out, i.e. the contrast of the first 4 weeks of sampling time F1,8 = 20.39, P = 0.002, Fig. 1a). By early May, the (open) to the second 4 weeks of sampling (shaded). To average amount of light reaching the surface of the compare the responses of the three ponds, for each three ponds was only 22% of that in open areas pond, we treated samples collected in the first (Table 1). Of course, total hours of daylight increased 4 weeks as replicates for the open time period and from 12 h, 43 min in early April to 15 h, 5 min on 31 samples collected in the second 4 weeks as replicates May. However, the additional hours of daylight for the shaded period. represent <20% increase, as compared to the 84% In testing stoichiometric predictions, we treated our reduction of light reaching the ponds surface due to estimates of TN and TP as proxies of nutrient supply leaf-out. We represented light levels as percentage of rates (Hall et al., 2005, 2007). Similarly, we used our the open area to avoid confounding temporal estimates of per cent of full sun as a relative measure decreases caused by leaf-out with temporal variance of light resource to create ratios with TN or TP in in cloud cover or angle of sun. Nevertheless, the testing the light : nutrient ratio hypothesis. Temporal amount of PAR we measured at the pond surface in variation in seston elemental composition (C : N : P) May was considerably lower than April values. was related to these resource supply ratios, and to The results of the cholorphyll size fractionation grazer biomass (log transformed), using ANCOVA, confirmed that the phytoplankton assemblage was with pond treated as a categorical factor (SYSTAT, composed primarily of small cells in all three ponds 2000 ver.10; San Jose, CA, U.S.A.). For each ANCOVA (Table 1). Hence, the <62 lm fraction, which con- model, we first confirmed the homogeneity of slopes tained >90% of the total chlorophyll, represented the (all pond*factor interactions P > 0.16). bulk of the seston. Not surprisingly, the reduction in

Table 1 The three ponds differed in size and trophic status, as measured by total phosphorus, chlorophyll and particulate carbon concentrations in early April

Max April TP April May %Chl-a %Chl-a %Chl-a April May ) Pond volume (m3) (lgL 1) light (%) light (%) <3 lm <15 lm <62 lm C : Chl C : Chl

Roughwood 760 14.3 39 9 24 94 99 205 423 Woodfrog 230 59.4 69 37 26 72 91 61 782 West Gull 2160 212.8 54 20 24 89 94 168 838

Values for April and May light levels are the percentage of full sun values reaching the pond surface, estimated by making simul- taneous light measurements in the pond and at open areas adjacent to each pond. The size structure (% of total Chl-a represented by the <3 lm, <15 lm, <62 lm fraction) of the phytoplankton was relatively constant through time. Hence, the average percentage of chlorophyll in each size class (<3 lm, <15 lm, <62 lm) are averages for the entire sampling period. April and May cabon : chlorophyll ratios (C : Chl) are for the <62 lm size fraction.

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(a) (a) 100 1000 N

80 ) –1

60 P 100 Light (%)

40 N or P (µg L

20

10 0 (b) (b) 1000 100 Carbon ) C : P –1 100 Molar ratio 10

carbon (µg L 10 Chla C : N Chlorophyll or particulate

1 21 Mar 10 Apr 30 Apr 20 May 9 Jun

21 Mar 10 Apr 30 Apr 20 May 9 Jun Fig. 2 (a) Concentrations of total nitrogen (TN) and total phos- phorus (TP) through time. Although both TN and TP increase Fig. 1 (a) Light levels in each pond expressed as the percentage through time, the rate of increase in TP is much faster than that of ambient light reaching the pond surface. The lines represent a of TN. (b) Molar ratio of carbon : phosphorus and car- LOWESS smoother with a tension of 0.6. (b) Temporal changes bon : nitrogen of the seston (<62 lm). In both panels, triangles in concentrations of particulate carbon and chlorophyll a. Both are West Gull, squares are Woodfrog and circles are Rough- are the <62 lm fraction. The lines represent a LOWESS wood. smoother with tension of 0.5. In both panels, triangles are for West Gull Pond, squares for Woodfrog and circles for Roughwood. high in April, average C : Chlorophyll was 144, but in May this ratio averaged 681. light levels was associated with decreases in chloro- In all three ponds, the concentrations of TN and TP phyll concentrations (RM ANOVA – time, F1,9 = 56.7; greatly increased from April to May (Fig. 2a, RM P < 0.0001, Fig. 1b); furthermore, the large differences ANOVA – time, TN, F1,9 = 58.8, P < 0.0001; TP among ponds in April chlorophyll essentially disap- F1,9 = 28.0, P = 0.0005). The ponds differed in mean peared in May as total chlorophyll dropped to nutrient concentrations, (RM ANOVA – pond, TN, )1 <1 lgL (RM ANOVA – pond*time interaction, F2,9 = 23.1, P = 0.0003; TP, F2,9 = 48.1, P < 0.001) and

F2,9 = 9.5, P = 0.006). Despite seasonal declines in they retained their rank differences through time (TN chlorophyll, seston carbon stayed relatively constant pond*time interaction F2,9 = 2.38, P = 0.15; TP time*- throughout the sampling period (RM ANOVA – time, pond interaction F2,9 = 2.36, P = 0.15). TP displayed

F1,9 = 0.02; P = 0.88), although the ponds differed in greater proportionate increases than did TN. average seston carbon as reflected by their productiv- The three ponds were clearly distinguished in C : P ity (RM ANOVA – pond, F1,9 = 8.1, P = 0.01). Conse- of their seston (Fig. 2b, RM ANOVA – pond; quently, the C : Chlorophyll ratio underwent a drastic F2,9 = 20.1, P = 0.0005). Furthermore, sestonic C : P increase in all ponds (Table 1). When light levels were decreased seasonally in all three ponds (RM ANOVA –

2008 The Authors, Journal compilation 2008 Blackwell Publishing Ltd, Freshwater Biology, 53, 1291–1302 1296 C. E. Ca´ceres et al. time; F1,9 = 9.2, P = 0.014), with no significant pond* (a) 300 time interaction (F2,9 = 1.8, P = 0.22). Hence, in both 250 comparisons among ponds, or temporally within each 200 pond, seston C : P reflected changes in supply rates of Roughwood P (measured by TP), as predicted by stoichiometric 150 theory. The C : N of the seston also differed among ponds (Fig. 2b; RM ANOVA – pond F = 16.4, 2,9 100 P = 0.001) and seasonally (RM ANOVA – time West Gull F1,9 = 12.9, P = 0.006), but in the opposite direction. Woodfrog, which had the lowest seston C : P values, Seston C : P had the highest C : N ratios. Through time, pond 50 Woodfrog C : N values increased instead of decreasing as with C:P. We tested for a relationship between C : P ratio of seston and the light : TP ratio in the ponds (Fig. 3a). (b) 12 As a joint consequence of both decreased light from Woodfrog 11 tree leaf shading, and increased TP in the water, the light : TP ratio decreased by more than an order of 10 magnitude in each pond during April and May. The 9 ponds showed the predicted decrease in seston C : P 8 with decreased light : TP ratio (ANCOVA F1,20 = 6.2, P = 0.02, Fig. 3a). Surprisingly, seston C : N also 7 exhibited a marked change in response to decreased Seston C : N West Gull light : TP ratio (F1,20 = 4.6, P = 0.04, Fig. 3b), but in 6 Roughwood the opposite direction of seston C : P. The qualitative pattern of decreasing seston C : P and increasing 5 seston C : N is similar for both light : TP or light : TN as the surrogate for light : nutrient ratio. However, since there was much less variance over time in the 0.0001 0.0010 0.0100 light : TN as compared to the light : TP ratio, signif- LIGHT : TP icance values were 0.068 C : P and 0.098 for C : N for the models considering the effect of light : TN. Fig. 3 Seston (<62 lm) carbon : phosphorus ratio (a) and nitro- We also tested for a relationship between the N : P gen : phosphorus ratio (b) as a function of the light : TP ratio. For each pond, the arrow represents the change in values over ratio of the seston and the TN : TP ratio (F1,31 = 7.89, time, from early April to mid May. The ellipses indicate bivar- P = 0.001; Fig. 4). West Gull exhibited little change in iate SE. TN : TP, but for the two other ponds (Roughwood and Woodfrog) a strong seasonal decrease in TN : TP produced the expected positive relationship with Birge was found in small numbers in West Gull seston N : P. However, seston N : P is consistently pond. Most taxa were rare; hence we only consider lower than TN : TP (below a 1 : 1 line) in these ponds. the dynamics of the three most abundant taxa. In West Gull, which was the richest in TP (lowest in Daphniopsis ephemeralis peaked first in all three ponds, TN : TP), the seston N : P was initially higher than and this was also the first species to return to expected but quickly decreased to match TN : TP dormancy and disappear from the water column by within a few weeks. early May. This species largely completed its life cycle Zooplankton abundance fluctuated in all three ponds prior to pond shading. In contrast, D. pulex and (Fig. 5). Daphniopsis ephemeralis Schwartz and Her- cyclopoid copepods dominated the water column bert, Daphnia pulex, Ceriodaphnia sp., Scapholeberis sp., after the reduction in light caused by tree leaf-out. Simocephalus sp., cyclopoids, ostracods and mosquito Although both the D. pulex population and the larvae were found in all ponds and Daphnia laevis cyclopoids declined sharply during May, a second

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(a)

1:1 –1 108 Roughwood West Gull 7 pond 10 WestGull Roughwood Woodfrog 106 105 104

D. ephemeralis 0 10 (b) 108 –1

Seston N : P Molar ratio 107

Woodfrog pond 106 105 4 10 100 D. pulex 10 TN:TP Molar ratio 0 (c) Fig. 4 l Seston (<62 m) nitrogen : phosphorus ratio as a function 108 of supply rates (Total N : P). For each pond, the arrow repre- –1 sents the change in values over time, from early April to mid 107 May. The ellipses indicate bivariate SE. 106 105 cohort of each was observed in all three ponds, 104 suggesting the availability of suitable food resources Cyclopoids pond well after the canopy closed. 0 Estimated grazing rates suggest that at peak 21 Mar 10 Apr 30 Apr 20 May 9 Jun

Daphnia densities in late April, grazers should be Fig. 5 Seasonal dynamics of the three most abundant zoo- ) imposing a strong mortality on the seston. Peak plankton taxa. Densities are expressed as number pond 1 to estimates of clearance rates were 0.37 of the pond avoid confounding temporal changes in population dynamics ) ) water d 1 in Woodfrog, 0.90 d 1 in Roughwood and with a concentration of individuals as the pond dries. ) 2.9 d 1 in West Gull. However, the population abun- dance of the Daphnia underwent dramatic changes period of transition from light to shade, we examined over short periods of time. Hence, estimates of its population dynamics in more detail. Such an grazing rates were highly variable throughout the analysis can suggest times at which the grazers may season. The realized impact of this grazing on seston be food-limited. In late March and early April, the abundance is apparently small; only in West Gull, population growth rate of D. pulex was greater than which had extremely high grazing rates, was there expected from birth rates. Consequently, negative any evidence of a reduction in seston carbon during death rates were calculated (Fig. 6), suggesting hatch- April (Fig. 1b). Moreover, the seasonal variability in ing from dormant eggs. During April, the populations grazing did not appear to influence seston stoichio- grew quickly as a result of extremely high birth rates, metry; we found no relationship between our estimate despite a drastic increase in death rates. In May, the of grazer biomass and the N : P or C : P ratio of the switch to diapausing egg production (data not shown) seston (ANCOVA C:N, F1,31 = 1.1, P = 0.28; C : P, reduced birth rates. This reduction, combined with

F1,31 = 1.32, P = 0.26). increasing death rates, resulted in sharp declines in Although seston stoichiometry did not seem to be population abundances. In mid-May, we observed determined by grazer abundance, we were also birth rates of zero because adult D. pulex were not interested in whether changes in seston stoichiometry detected in the water column. The continued presence resulted in food-limitation for the grazers. Because of water in the ponds allowed a second cohort of D. pulex was the dominant grazer throughout the D. pulex to invade the water column from the egg

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(a) 30 cohort, death rates increased dramatically once the population became reestablished.

Discussion

20 Our results illustrate how forested ponds undergo rapid temporal change in the balance of light and nutrient supply and that this results in a shift in the elemental composition of seston. Despite large differ- 10 ences in the initial conditions with respect to trophic Temperature (ºC) status, the three ponds exhibited nearly identical patterns of seasonal change in seston C : N : P and in grazer population dynamics. These parallel changes in ponds of varying size and trophic state suggest that 0 the temporal responses to shading that we observed (b) 1.5 are robust. b It was not unexpected that we would record rapid d temporal changes in physical, chemical and ecological 1.0 conditions in these vernal ponds. However, temporal changes in ephemeral aquatic habitats have received far less study than have similar seasonal dynamics in permanent lakes and reservoirs. Several studies point 0.5 to the importance of leaf-out and decomposition in changing light and nutrients supply to forest ponds (Wiggins, Mackay & Smith, 1980; Colburn, 2004), but Birth or death rate 0.0 quantitative data such as we present are rare. Simi- larly, prior studies of plankton population dynamics and succession in temporary ponds corroborate our observations of synchronized hatching from dormant –0.5 stages driving seasonal phenologies (Hairston & Olds, 21 Mar 10 Apr 30 Apr 20 May 9 Jun 1984; Taylor & Mahoney, 1990). Day To a large extent, changes in seston stoichiometry Fig. 6 (a) Maximum (triangle) and minimum (star) tempera- were as predicted by the LNH (Sterner et al., 1997). tures of all three ponds, as recorded by a max–min thermometer The C : P ratio of the seston declined rapidly in all located in a shady location in each pond. Note the midseason three ponds in response to decreased light and decline in temperature prior to the onset of the second cohort (highlighted by the vertical line). The lines represent a LOWESS increased phosphorus supply (measured as TP) as smoother with tension of 0.5. (b) Birth and death rates for the the season progressed. However, we also observed three populations of Daphnia pulex. The dotted line indicates changes in seston C : N : P that were inconsistent the average birth rate of the three populations, and is a with simple stoichiometric predictions. Seston N : P LOWESS smoother with tension of 0.5. Grey triangles highlight was typically less than expected based on nutrient the two periods of negative death rate whereas the stippled triangles highlight the rapid increase in death rates for both supply in the water column, and despite a large cohorts. temporal increase in nitrogen supply and little or no change in seston carbon, the C : N ratio of the bank in late May. As with the first cohort, negative seston increased. Prior studies have also reported death rates were commonly estimated in late May that the N : P ratio of natural phytoplankton assem- until an adult population was well established in the blages does not always match that expected from water column. Birth rates increased rapidly again supply rates of N and P (Hall et al., 2005). There are until stabilizing in early June as some females began at least three potential explanations for these pat- producing diapausing eggs again. As with the first terns.

2008 The Authors, Journal compilation 2008 Blackwell Publishing Ltd, Freshwater Biology, 53, 1291–1302 Stoichiometry in temporary ponds 1299 The most common explanation for a lack of consis- phytoplankton are expected to have different N and P tency between seston stoichiometry and changing requirements than are heterotrophic or mixotrophic supply rates has been physiological constraints of the organisms (Katechakis et al., 2005). Hence, seasonal algae, especially a greater plasticity to store P com- changes in stoichiometric ratios would be expected pared to N. It is also likely that much of the increase in based on seasonal changes in community structure. TN produced by decomposition was in the form of We also observed dramatic changes in C : Chloro- dissolved organic nitrogen. The ability of phytoplank- phyll ratio when leaf-out shaded the pond, with the ton to take up this form of nitrogen may be limited. initial values in early April being indicative of an Further, A˚ gren (2004) showed that N : C is expected autotrophic assemblage (Sterner & Schulz, 1998). A to increase linearly, while P : C should increase dramatic shift to very high ratios occurred after quadratically as a function of growth rate of algae. canopy closure. If this were a high light environment, This also will give rise to different patterns of N : P increasing C : Chlorophyll ratio would be considered depending on the nutrient supply rate. Hence, phys- evidence of nutrient limitation, but clearly that is not iological constraints may contribute to why total and the case in these ponds. Visual observations of the seston N : P ratios did not match; the N : P of the phytoplankton in our study ponds and in forest ponds seston was almost always less than what was pre- in general (Colburn, 2004), reveal a dominance of dicted based on the supply rate ratio. Still, the seston cryptomonads, chrysophytes, dinoflagellates and N : P did decline seasonally with the decline in euglenoids; many of these taxa are believed to be supply rate of TN : TP, confirming an important mixotrophic and can shift from autotrophic to hetero- prediction of ecological stoichiometry. trophic nutrition with changes in light and nutrient However, it was unexpected that TN : TP would supply. We suggest that changes in nutritional mode decline seasonally. Interestingly, it occurred only in or perhaps species compositional shift to greater the two less productive ponds, and was driven by a heterotrophy in the phytoplankton assemblage, disproportionate increase in TP compared to TN. It is contributed to the unexpected increase in C : N of likely that at the whole pond basin scale, nitrogen may the seston despite increased supply of N from not accumulate as readily as phosphorus due to decomposition (Jansson et al., 1996; Rothhaupt, 1996). differential uptake by forest trees and shrubs. In A third factor considers the grazers and top-down addition, the ponds were observed to become quite influences on plankton composition. Hall et al. (2007) low in oxygen in May suggesting that microbial suggested that these top-down factors could influence denitrification might contribute to loss of nitrogen. producer stoichiometry through a variety of mecha- We observed not just a decline in N : P ratio of nisms including reducing standing stocks, recycling of seston and nutrient supply, but an increase in the nutrients and forcing an increased turnover rate. The C : N ratio of seston. Others have also reported an transition from open to closed canopy was also increase in C : N ratio of seston with a decrease in associated with dramatic increases in daphniid abun- light : nutrient ratio. Frenette, Vincent & Legendre dance; grazing rates in at least two of the ponds (1998) observed that small-celled phytoplankton would be expected to impose very high mortalities on increased C : N after a typhoon decreased water phytoplankton. Because the ponds had similar sized transparency and Schiesari (2006) found that the animals and similar temperatures, among-pond dif- C : N composition of algae sampled from open ferences in our estimates of grazing rates were driven canopy ponds was lower than that of nearby closed by grazer abundance. However, we found no rela- canopy ponds. We know of no obvious physiological tionship between changes in grazer biomass and mechanism for why C : N should increase under seston stoichiometry. The fact that seston carbon lower irradiance, but these results from other diverse remained relatively stable suggests that turnover rate systems suggest the phenomenon is broader than just of phytoplankton was very rapid. The increased our study. C : Chlorophyll may reflect more than a change in A second, general factor to consider in under- nutrition; it is possible that the phytoplankton shifted standing relationships between nutrient supply and (via species replacement or phenotypic plasticity) to seston elemental composition in these ponds is forms that invest more in defensive structures to nutritional mode and species turnover. Autotrophic minimize grazing loss (Agrawal, 1998). This seems

2008 The Authors, Journal compilation 2008 Blackwell Publishing Ltd, Freshwater Biology, 53, 1291–1302 1300 C. E. Ca´ceres et al. unlikely in these ponds given the evidence of high Acknowledgments productivity of the grazers and prior studies docu- We thank P. Woodruff, S. Mulvany and A. Jaurigue menting high food quality (Tessier & Woodruff, 2002). for assistance in the laboratory and field. S.R. Hall and A related question asks how the observed seasonal C.T. Milling provided helpful discussion on earlier changes in seston stoichiometry influence the drafts of the manuscript. Support for this project was dynamics of the grazers. The C : P ratio of the provided by NSF grants DEB-9816047, DEB-9816191, seston, which is often used as an indicator of food DEB- 0235118 and DEB-0237438. Any opinions and quality was well above the thresholds at which conclusions expressed in this paper are those of the P-content is thought to become limiting for Daphnia authors and do not reflect the views of the National (Sterner & Elser, 2002; DeMott & Pape, 2005). The Science Foundation. This is contribution number 1452 apparent availability of P, coupled with the fact that of the Kellogg Biological Station. birth rates of the grazers remain high for most of the season, suggests that neither food quantity nor quality are limiting. Hence, zooplankton dynamics References are largely governed by hatching and production of Agrawal A.A. (1998) Algal defense, grazers, and their diapausing eggs. Daphniopsis ephemeralis had a single interactions in aquatic trophic cascades. Acta Oecolog- cohort that was driven by a hatch of females, which ica-International Journal of Ecology, 19, 331–337. essentially produced males and then diapausing A˚ gren G.I. (2004) The C:N:P stoichiometry of auto- eggs. Daphnia pulex had a more extensive occupation trophs: theory and observations. Ecology Letters, 7, of the water column, but even this was composed of 185–191. two episodes of diapausing egg hatch and produc- Andersen T. (1997) Pelagic Nutrient Cycles: Herbivores as tion. Although birth rate declined just when leaf-out Sources and Sinks. Ecological Studies #129. Springer, and shading took effect in late April, the decline in Berlin. birth rates can be attributed primarily to the popu- Andersen T. & Hessen D.O. (1991) Carbon nitrogen and lations switching to diapausing egg production, phosphorus content of freshwater zooplankton. Lim- nology and Oceanography, 36, 807–814. rather than food limitation (Ca´ceres & Tessier, APHA. (1980) Standard Methods for the Examination of 2004). Our estimates of birth and death rates for this Water and Wastewater. 15th edn American Public species were similar in all three ponds and indicate Health Association, Washington, DC. rich food conditions (high birth rates) and high Bachmann R.W. & Canfield D.E. (1996) Use of an extrinsic mortality. This has been reported previ- alternative method for monitoring total nitrogen ously by Dudycha (2004) and reflects high level of concentrations in Florida lakes. Hydrobiologia, 323, predation on the zooplankton. Hence, vernal ponds 1–8. appear to be highly productive environments with Bottrell H.H., Duncan A., Gliwicz Z.M., Grygierek E., strong top-down (mortality) influences that create Herzig A., Hillbricht-Ilkowska A., Kurasawa H., Lars- rapid turnover in prey assemblages. son P. & Weglenska T. (1976) A review of some The light : nutrient hypothesis (Sterner et al., 1997) problems in zooplankton production studies. Norwe- provides a valuable framework for assessing the gian Journal of Zoology, 24, 419–456. Ca´ceres C.E. & Tessier A.J. (2004) To sink or swim: transfer of nutrients and energy among trophic levels. variable diapause strategies among Daphnia species. To date, most empirical and theoretical attention has Limnology and Oceanography, 49, 1333–1340. focused on permanent lakes (but see Hall et al., 2007). Chrzanowski T.H. & Grover J.P. (2001) The light:nutrient Ephemeral ponds, however, experience much more ratio in lakes: a test of hypothesized trends in bacterial rapid temporal changes in light levels, nutrient nutrient limitation. Ecology Letters, 4, 453–457. supply rates and grazer dynamics; changes that are Colburn E.A. (2004) Vernal Pools: Natural History and central to many of the predictions of ecological Conservation. The McDonald & Woodward Publishing stoichiometry. Hence, the temporal dynamics of these Company, Blacksburg. forested ponds provide an excellent opportunity to DeMott W.R. & Pape B.J. (2005) Stoichiometry in an examine ecological stoichiometry at the scale of whole ecological context: testing for links between Daphnia food webs, and at the interface of terrestrial and P-content, growth rate and habitat preference. Oecolo- aquatic ecosystems. gia, 142, 20–27.

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