AQUATIC MICROBIAL ECOLOGY Vol. 42: 293–310, 2006 Published March 29 Aquat Microb Ecol

Responses of intermittent pond populations and communities to in situ bottom-up and top-down manipulations

Oksana P. Andrushchyshyn, A. Katarina Magnusson, D. Dudley Williams*

Surface and Groundwater Ecology Research Group, Department of Life Sciences, University of Toronto at Scarborough, 1265 Military Trail, Ontario M1C 1A4, Canada

ABSTRACT: Pond physicochemical characteristics and bottom-up effects were more important than top-down effects in governing ciliate community structure in 2 adjacent intermittent ponds in South- ern Ontario, Canada. The showed a bimodal seasonal pattern with abundances peaking early and late in the hydroperiods, and the communities showed a strong seasonal succession of — only 15% of the 162 ciliate species were present throughout the hydroperiods. Less than half of the species occurred in both ponds. Adding riparian leaf litter to large pond enclosures affected several physicochemical variables, increased bacterial abundance, and promoted the appearance of particu- lar species — many of which are known to be associated with nutrient- or organic matter-enriched conditions. This treatment resulted in higher ciliate abundance (mainly small-sized bacterivores) and lower ciliate diversity in mid-hydroperiod in one of the ponds. The removal of litter generally produced effects in the physicochemical variables that were opposite to those seen in the leaf litter addition, and resulted in a 15% decrease in the proportion of ciliate bacterivores in one pond. The effects of top-down manipulations (i.e. prevention of aerial colonization of insects) were minor. Many treatment effects were season-, and pond-specific. The measured environmental variables (including pond and treatments) explained half of the variation in ciliate abundance, one-third of the species diversity, and one-fifth of the species composition. Pond characteristics and the leaf litter additions were the most important factors for determining ciliate abundance (together with chlorophyll a), diversity (together with dissolved oxygen), and community composition (together with season).

KEY WORDS: Ciliates · Seasonal succession · Temporary ponds · Resource addition · Resource removal · Predator exclusion · Seasonal succession · Top-down · Bottom-up

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INTRODUCTION consume more food, have a higher respiration rate per mass unit, have shorter generation times, and repro- Free-living ciliates are unicellular, eukaryotic, mainly duce much faster, all of which contribute to their domi- phagotrophic that play important metabolic nance in the turnover of pond organic matter. roles in freshwater ecosystems (Fenchel 1987, review Although there is extensive work on ciliate com- by Beaver & Crisman 1989, Finlay & Fenchel 2004). Cil- munities and populations in different freshwater iates and other protists are the most important grazers habitats including ponds (Wang 1928, Goulder 1971, of microbes in aquatic environments, the only grazers Hatano & Watanabe 1981, Kusano et al. 1987, of any importance in anoxic habitats, and usually pre- Madoni 1991, Müller et al. 1991, Guhl et al. 1994, dominate in sediments. Although ciliate biomass typi- Finlay & Esteban 1998b, Madoni & Sartore 2003), cally accounts for slightly less than 10% of total benthic lakes (Goulder 1974, review by Beaver & Crisman invertebrate biomass, ciliate production may equal or 1989, Müller et al. 1991, James et al. 1995), reser- even exceed invertebrate production (Finlay & Esteban voirs (Salvado & Gracia 1991, 2imek et al. 1995), and 1998a). Compared with meio- and macrofauna, ciliates other water bodies (Bick 1973), almost no data exist

*Corresponding author. Email: [email protected] © Inter-Research 2006 · www.int-res.com 294 Aquat Microb Ecol 42: 293–310, 2006

on ciliate communities of intermittent ponds (An- held water from 15 March until 4 July (112 d). Pond II drushchyshyn et al. 2003, but see Picken 1937). was smaller (~0.1 ha) but deeper (maximum 48 cm), Intermittent ponds are bodies of water that experi- and held water from 18 March until 17 June (92 d). ence a recurrent dry phase of varying length that is Mixed deciduous trees surrounded both ponds, predictable in its onset and duration (Williams 1997). although Pond II was more open in exposure. The veg- The most important influences on their biota are length etation in Pond I was heterogeneous whereas in Pond of the aquatic phase (hydroperiod), pattern of disap- II it consisted mainly of the grass Phalaris sp. pearance of the water, degree of intraspecific competi- The same experimental set-up was established in tion and , and the seasonal influx of aerial both ponds for 2 reasons: (1) to achieve proper replica- colonizers (Williams 2006). Thus, the particular envi- tion, and (2) as a means to assess whether the same ronment in intermittent ponds makes these habitats manipulations would yield similar results in ponds ideal arenas for testing hypotheses about community- with different vegetation structure. The ciliate data regulating mechanisms. The relative roles of bottom- presented here are a part of an ongoing, major study of up (i.e. resource availability) and top-down (i.e. preda- top-down and bottom-up responses on intermittent tion) mechanisms versus physicochemical constraints pond community structure. for governing intermittent pond communities are not Experimental design. The experimental design in- known. Ciliates are ideal organisms for such studies cluded 2 replicates per pond of each of 3 manipulations because they have short generation times and respond (1 top-down and 2 different bottom-up manipulations), rapidly to environmental change. Previous research on and 2 controls in which no manipulations were per- protists in permanent waters suggests that top-down formed (namely, an enclosure control, and a natural control from zooplankton is stronger than bottom-up ‘pondwater’ control). All manipulations were performed effects from nutrient addition (review in Sanders & in enclosures made of circular galvanized-steel sheets Wickham 1993), and that bottom-up effects may be (each enclosing an area of pond bed measuring 4.2 m2) important primarily in oligotrophic waters, whereas that were placed in homogeneous areas of each pond. top-down effects are more important in eutrophic The enclosures were assembled so as to prevent any waters (Berninger et al. 1993). exchange of water and organisms between the inside The aim of this study was to explore key factors influ- and outside (pond) water, and were set 5 cm into the encing ciliate communities in 2 intermittent ponds. To pond beds. The enclosure controls (‘c’) consisted of do this, we determined, in situ: (1) the natural, tempo- enclosed areas, in which no manipulations were per- ral variation in ciliate abundance, diversity, and com- formed, which served as reference areas to the top-down munity structure in 2 adjacent ponds, and related these and bottom-up manipulations. The pondwater controls to environmental factors; and tested (2) the role of bot- (‘o’) comprised 2 non-enclosed areas of the pond that tom-up effects by indirect manipulation of resource were used to assess any potential enclosure effects. availability (leaf-litter addition vs leaf-litter removal; To study top-down effects on the ciliate community, and phytoplankton are the main prey for cili- invertebrate aerial colonization was prevented by a ates) in large enclosures, and (3) the role of top-down fine mesh-net (1 mm) that covered the openings of 2 effects by excluding predatory aerial colonizers from enclosures in each pond. This treatment (referred to as other enclosures. We hypothesize that an increase of top-down manipulation, ‘p–’) chiefly excluded adult leaf litter material will lead to increasing resources for beetles and odonates, but also reduced the number of the ciliates, which will be reflected in the ciliate spe- chironomid and mosquito larvae. This treatment was cies abundance, diversity, and community composi- designed primarily to examine the effects of predators tion. We expect the removal of leaf litter to have the on pond macroinvertebrates, but the manipulation was opposite effect to the resource addition. We further also useful in examining indirect top-down effects on expect that the removal of aerial colonizers will propa- the ciliate communities. gate down through the food web, via zooplankton, and The resource manipulation consisted of 2 treatments: influence the ciliate communities. addition and removal of riparian leaf- and plant litter (‘r+’ and ‘r–’, respectively). In the latter, riparian leaf litter and decomposing plant material (1.5 ± 0.2 kg MATERIALS AND METHODS [±1 SD] in Pond I and 1.5 ± 0.04 kg in Pond II) were removed the previous autumn (late-October). Double Sampling sites. The ciliate populations were sam- the amount of material that was removed from pled weekly throughout the hydroperiod in 2002 in 2 the resource removal enclosures was added (mid- fishless, intermittent ponds located in Vandorf, Ontario November) to the resource addition enclosures in the (44° 00’ N, 79° 23’ W). Pond I was approximately form of dried (100°C for 24 h) riparian leaf litter 0.25 ha in area with a maximum depth of 39 cm, and collected from the pond banks. Andrushchyshyn et al.: Intermittent pond ciliate populations 295

The 2 ponds were subjected to identical experimen- analysis to better meet the underlying assumptions. tal set-up; thus, each pond contained 8 enclosures (i.e. Since the assumption of non-sphericity was violated (i.e. 2 replicates of each of the 3 manipulations and the samples in the time series were not independent), we enclosure control) and 2 non-enclosed areas (i.e. 2 used the Greenhouse-Geiser (G-G) adjustment on all replicates of the pondwater control). time interaction probabilities (Scheiner & Gurevitch Routine sampling. A weekly sampling programme 2001). To minimize the risk of type I errors due to multi- comprised analyses for ciliates (Protista: Ciliophora), ple independent tests, all statistically significant main water chemistry (using a Hydrolab H2O multiprobe, and interaction effects were Bonferroni corrected. Hydrolab; and portable Hach Kit spectrophotometer Planned post-hoc tests for interaction effects were con- DR2000: Hach), bacteria (0.25 to 1 ml pondwater ducted on days collapsed into early-, mid-, and late sea- stained with acridine orange, filtered, and counted son, to increase the power of the analysis. under a epifluorescence microscope; Sorokin 1999), The influences of the various measured environmen- and chlorophyll a (chl a) (0.25 l pondwater filtered and tal variables on total ciliate abundance, diversity, and extracted in acetone: American Public Health Associa- species richness were analyzed using stepwise (for- tion 1995). Ciliate samples were taken by submerging ward) regressions. The variables were log-transformed an empty, closed 150 ml container adjacent to the sub- when necessary. The longitudinal scale did not cause strate; removing the lid and sealing it once full. Two any serious violation of the assumption of indepen- such replicates were taken at each sampling site and dence (>1.2 Durbin Watson, and <0.7 autocorrelation). these were pooled in the field. For enumeration, sam- The effects of the environmental variables, pond, sea- ples were preserved with mercury chloride (2.5% final son (i.e. days after flooding), and treatments on the cil- concentration, Sime-Ngando & Groliere 1991) immedi- iate species composition were evaluated with Canoni- ately after filling the containers. In the laboratory, after cal Correspondence Analysis (CCA). Prior to the CCA, thorough mixing, 10 ml were withdrawn from each the data were run in an unconstrained ordination, sample, and placed in an Utermöhl chamber (Phyco Detrended Correspondence Analysis (DCA), to find Tech) for counting ciliates under an inverted micro- the overall variability in the ciliate data, and to test the scope. Identification of ciliate species was done mostly gradient length (which was >1.5, i.e. long enough to on live samples (from a subsample extracted prior to use a unimodal distribution analysis). The first CCA preservation) on the same day they were collected. was run without any covariables on 300 samples × 162 The internal organelles were visualized using Klein- species. A second CCA was run with days after flood- Foissner silver nitrate method for silverline system ing and ponds as covariables in order to evaluate the (Foissner et al. 1999). The following keys were used in specific treatment effects. In biplots, the length of an the identifications: Kahl (1930, 1931, 1932, 1935), Bick arrow indicates the importance of an environmental (1972), Corliss (1979), Curds et al. (1982, 1983), Foiss- variable, and the direction indicates what the axes rep- ner et al. (1991, 1992, 1994, 1995, 1999), Foissner & resent. All ciliate abundances and environmental vari- Berger (1996). Biomass (wet mass) for individual spe- ables were log-transformed before the analyses cies was calculated using data presented in Foissner (except days after flooding and water depth) and rare and Berger (1996) or using ciliate shape and size ciliate species were down-weighted. The scaling was according to Chislenko (1968). At least 20 individuals focused on unit variance, and the scores optimized for for each species were measured to get a mean, which inter-sample differences. The significance of each axis together with appropriate ciliate shape were used to was estimated using the Monte Carlo test (199 permu- compute volume (1 µm3 = 1 pg, i.e. specific gravity of tations under the full model). The data were analyzed the protoplasm is 1.0, Foissner & Berger 1996). using STATISTICA (StatSoft) and CANOCO for Win- Organisms, other than ciliates, present in the sam- dows v.4.0 (Centre for Biometry). ples (e.g. algae, rotifers, flagellates, amoebae, gas- trotrichs, and crustaceans) were recorded as: absent; present; or abundant, and were given the numerical RESULTS values of 0, 1, and 4 respectively. Statistical methods. Pond-, time-, and treatment ef- Physicochemical environment fects on water chemistry, ciliate abundance, species rich- ness (i.e. number of species), species diversity (Shan- The water depth in Pond I was 20 to 35 cm for most non’s diversity index), and proportion of size-, and of the hydroperiod before the pond rapidly dried out feeding groups, were analyzed using repeated measures 112 d after flooding (Fig. 1). For the first 10 wk in Pond ANOVA (ANOVAR) with 2 grouping factors: Pond (Pond II, the water depths were 25 to 50 cm, before the pond I and Pond II), and Treatment (‘r+’, ‘r–’, ‘c’, ‘o’, ‘p–’). slowly dried out 3 wk later (92 d hydroperiod length). Abundance and biomass were log-transformed prior to Water temperatures steadily increased as the season 296 Aquat Microb Ecol 42: 293–310, 2006

40 2 )

30 –1

20 Resource addition 1

Resource removal l -N (mg 3

Depth (cm) 10 Top-down manipulation

Enclosure control NH Pondwater control 0 0

30 2 ) –1 20 1 -N (mg l -N (mg

10 3 NO Temperature (°C) 0 0

200 2 ) ) –1

–1 150 1

(µS cm 100 Conductivity Total P (mg l 50 0

150

7 100 pH 50 Turbidity (FTU) 6 0

20 ) 150 o 286

–1 238,186 )

–1 100 (µg l

10 a 50 DO (mg l 0 0 1 8 15 23 29 36 43 50 56 63 70 77 84 91 98 105112 1 8 15 23 29 36 43 50 56 63 70 77 84 91 98 105 112

March April May June Chlorophyll March April May June

Month and days after flooding

Fig. 1. Seasonal variation in physicochemical parameters in Pond I (mean, n = 2) progressed in both ponds, reaching maxima of 15 to Dissolved oxygen (DO) decreased with time in both 23°C (higher in Pond I, Figs. 1 & 2). Turbidity, nitrate, ponds (but had a higher initial concentration in Pond I, and ammonia were generally higher in Pond I, Figs. 1 & 2, p < 0.0001, time × pond effect, ANOVAR). whereas pH and conductivity were higher in Pond II Chl a ranged between 3 and 66 µg l–1 in Pond I, and (p < 0.001, main pond effect, ANOVAR). The nutrients between 4 and 163 µg l–1 in Pond II (pondwater con- slowly increased with time in both ponds (pondwater trols), and peaked during the days prior to drying. controls) until they reached maxima prior to drying Compared with the pondwater controls, the enclo- (Figs. 1 & 2). In Pond II, a drop in these variables on sure controls had lower total phosphorus (–31% and Day 64 was related to unusually high water depth. –19% in Ponds I and II, respectively), higher chl a Andrushchyshyn et al.: Intermittent pond ciliate populations 297

50 1.0 ) –1

25 Resource addition 0.5

Resource removal -N (mg l Top-down manipulation 3

Depth (cm)

Enclosure control NH Pondwater control 0 0.0

1.0 )

20 –1

e (°C)

0.5

10 -N (mg l 3 NO

Temperatur 0 0.0

) 2 500 –1 ) –1 1 300 (µS cm Conductivity Total P (mg l 100 0

8 75

50

pH 7 25 Turbidity (FTU) 6 0

15 ) 100 163 –1 ) –1

10 (µg l a 50 5 DO (mg l 0 0 1 8 14 21 30 36 43 50 57 64 71 78 85 92 99 1 8 14 21 30 36 43 50 57 64 71 78 85 92 99 March April May June Chlorophyll March April May June

Month and days after flooding Fig. 2. Seasonal variation in physicochemical parameters in Pond II (mean, n = 2)

(+300% and +120%), lower ammonia in Pond I early-mid season in both ponds). The resource removal (–26%), and lower conductivity in Pond II (–36%, p < resulted in higher DO (+20% and +70% in early sea- 0.001, planned comparison of pond × treatment effects, son in Ponds I and II) and lower nitrate (–20% and ANOVAR, Figs. 1 & 2). The resource addition resulted –39%). In Pond II, the same treatment resulted in in higher nitrate (+50% in early season in both ponds), higher pH (+0.37 units in early season), but lower phosphorus (+70% and +40% in early-mid season in ammonia (–55% in early-mid-season), total phospho- Ponds I and II), turbidity (+43% in early season), and rus (–46% early-mid-season), and chl a (–55%). The conductivity in Pond I (+46%), but lower DO (–59% top-down manipulation did not significantly affect any and –67% in Ponds I and II) and pH (–0.2 units in physicochemical variables (p > 0.01). 298 Aquat Microb Ecol 42: 293–310, 2006

The biota based on the macrosystem of Corliss 1979), 65 species (40%) occurred in both ponds. 58% of the species in The bacterial abundance in the water column of the Pond I and 72% of the species in Pond II were present pondwater controls ranged from 1.2 to 4.8 × 106 ml–1 in in both replicates. Persistent species (i.e. those present Pond I, and 1.1 to 5.5 × 106 ml–1 in Pond II (Fig. 3), and during the entire hydroperiod) comprised ~15% of the was 70 and 100% higher within the enclosures (Ponds ciliates, whereas 25% of the species occurred sporadi- I and II, respectively, p < 0.01, planned contrast of main cally (i.e. appeared in only 1 or 2 of the weeks). treatment effect). The resource addition resulted in a Total ciliate abundance (Fig. 5b) was significantly 100 vs. 40% higher bacterial abundance compared higher and Shannon’s diversity index lower (Fig. 5c) in with the control enclosures (in Ponds I and II), whereas Pond I compared with Pond II (p < 0.05, Main pond the resource removal resulted in a 30 vs. 70% reduc- effect ANOVAR, Table 1). Ciliate abundance in the tion (early-mid season in Ponds I and II, p < 0.005). pondwater controls (i.e. outside the enclosures) was Crustaceans were more common in Pond II than in usually below 5 to 10 cells ml–1, and peaked early and –1 Pond I (p < 0.005, F1,10 = 18.1, pond effect ANOVAR), late in the hydroperiods (maxima of 49 cells ml in whereas flagellates and amoebae were more common Pond I, and 89 cells ml–1 in Pond II). Total ciliate abun- in Pond I (p < 0.05, F1,10 = 7.1, Fig. 4a). The enclosures dance in Pond I enclosures followed the pattern prevented the occurrence of cladocerans in Pond I and observed in the pondwater controls for the first 29 d. reduced ostracods in Pond II (Fig. 4b). Crustaceans Thereafter, ciliate abundance inside the enclosures peaked in April in both ponds (p < 0.0001, F12,120 = 8.1, exceeded that of the pond water considerably, primar- time effect ANOVAR, Fig. 4c), and in early season the ily due to small ciliates (peaking at ~102 cells ml–1, p < abundance of crustaceans was lower within the enclo- 0.001). Thus, the resource addition resulted in higher sures compared with the outside pondwater in Pond II ciliate abundance (~325 cells ml–1) but lower diversity (p < 0.0001, planned contrast of treatment × time effect (Shannon’s diversity index) in Pond I (mid-season, p < ANOVAR). The resource addition resulted in an 0.0005, Fig. 5c). Abundances in the resource removal increase of crustaceans in Pond II compared with the and top-down (partial predator removal) treatments enclosure controls (mid-, and late-season, p < 0.005). did not differ significantly from the enclosure controls The resource removal resulted in more flagellates and in any of the ponds. amoebae in Pond I (late-season, p < 0.0001) and more The pondwater controls in Pond I were dominated by algal cells (late-season, p < 0.0005) and crustaceans in small (<50 µm, indicated as size group ‘1’ in Appendix Pond II (mid-season, p < 0.0005). The top-down mani- 1) Urotricha and Cyclidium species, which made up 9 pulation enclosures produced a lower occurrence of to 95% of the total community (numerically) through- crustaceans in Pond I (early-, and mid-season, p < 0.05) out the hydroperiod (average 50%). Most medium- and fewer flagellates and amoebae in Pond II (late- sized species (50–200 µm, group ‘2’) belonged to season, p < 0.005). Hypotricha. Although large (>200 µm) species, such as those belonging to Pleurotricha, , and , were generally less common, they contributed Species composition and abundance significantly to overall ciliate biomass by virtue of their size (especially S. ambiguum). In Pond II, the small Full data on the occurrence of cliates in the ponds species of Urotricha and Cyclidium were less dominant are given in Appendix 1. The number of ciliate species than in Pond I, but still made up 5 to 54% (average each week ranged from 5 to 36 in Pond I and 10 to 33 24%) of the total ciliate abundance. Other small-sized in Pond II (average, n = 2, Fig. 5a), and was higher ciliates that occasionally contributed to more than 10% within the enclosures than outside (mid-season, p < of the total abundance in Pond II included , 0.001, planned contrast, ANOVAR, Table 1). The Chilodonella, and Halteria. Strobilidium and cumulative number of species in the pondwater con- species were the most abundant medium-sized ciliates trols was 74 in Pond I (58 ± 2 per site, mean ± 1 SD, n = in Pond II, whereas large-sized ciliates were poorly 2) and 72 in Pond II (62 ± 1). The enclosures added 47 represented. A distinctive feature of Pond II was the more species to Pond I and 34 species to Pond II: result- appearance and predominance of representatives of ing in 61 ± 2 vs. 50 ± 2 species in the enclosure controls the order Hypotricha before drying: especially the (Pond I and II respectively); 75 ± 5 vs. 57 ± 10 in the genera Keronopsis, Oxytricha, Stichotricha, Stylony- resource additions; 59 ± 11 vs. 45 ± 2 in the resource chia, Tachysoma, and Uroleptus. removals; and 54 ± 1 vs. 58 ± 2 in the top-down manip- Small ciliates also dominated inside the enclosures in ulations. Of the total 162 species in the 2 ponds com- Pond I throughout the hydroperiod (with peaks in May bined (43% Kinetofragminophora species, 36% Poly- to June, Fig. 6a). In the resource additions of Pond I, hymenophora, and 21% Oligohymenophora species, small-sized species initially made up only 30% of the Andrushchyshyn et al.: Intermittent pond ciliate populations 299 )

-1 Pond I Pond II Resource addition 100 100 Resource removal ml

6 Enclosure control Top-down manipulation Pondwater control 10 10

1 1 12336506391112 1 213650647892 March April May June March April May June

Bacterial abundance (10 Month and days after flooding Fig. 3. Bacterial development in the different treatments in the ponds (means + 1 SD, n = 2)

Rotifera Pond I Pond II Flagellates + Amoebae 120 120 a e Gastrotricha Crustaceans Algae 80 80

40 40

Relative Abundanc 0 0 r+ r- c p- o r+ r- c p- o

Copepoda Cladocera

e 60 60 b Harpacticoida Ostracoda Nauplii 40 40

20 20

Relative Abundanc 0 0 r+ r- c p- o r+ r- c p- o Treatments

c r+ Resource addition

e 10 10 r- Resource removal c Enclosure control p-Top -down manipulation o Pondwater control 5 5

Relative Abundanc 0 0 March AprilMay June J March April May June

Month Fig. 4. (a) Relative abundance of major taxonomical groups from all dates combined (means + 1 SD, n = 2) within each treatment, (b) major crustacean groups, and (c) seasonal development of the crustaceans (means, n = 2) within each treatment: Figures are based on semi-quantitative abundance (abundant = 4, present = 1, absent = 0) 300 Aquat Microb Ecol 42: 293–310, 2006

Pond I Pond II a 40 40

20 20

No. of species

0 0

Resource addition b 1000 1000 Resource removal Top-down manipulation Enclosure control ) 100 100 –1 Pondwater control

l

10 10

(cells m

Abundance

1 1

c 1.5 1.5

’)

H 1.0 1.0

index (

0.5 0.5 Shannon’s diversity Shannon’s 1 8 15 23 29 36 43 50 56 63 70 77 84 91 98 105112 1 8 14 21 30 36 43 50 57 64 71 78 85 92 March April May June March April May June

Month and days after flooding

Fig. 5. Seasonal development of (a) number of ciliate species, (b) ciliate abundance, and (c) Shannon’s diversity index (means, n = 2) community (compared with 80% in the enclosure con- arrow in Fig. 7b). The leaf-litter removal resulted in trol, p < 0.0021, planned contrast of time × pond × treat- increasing abundance in some species (see ‘Discus- ment interaction, ANOVAR, Table 1, and Fig. 6b), but sion’ and Fig. 7b). The top-down manipulation did not dominated the community from mid April to late May influence the ciliate community composition signifi- (80 to 97%), which was higher than in the en- cantly (the arrow of p- is close to c in Fig. 7b), except closure controls (50 to 86%, p < 0.0021). The peak of for a +20% increase in the dominance of Urotricha and small-sized ciliates comprised Cyclidium glaucoma Cyclidium species in Pond I. (~260 cells ml–1), Urotricha farcta, U. furcata, and Hal- teria grandinella. The medium-sized species were less abundant than the smaller size group, but showed a Ciliate feeding groups distinct seasonal pattern in the resource addition: Col- pidium kleini (37 cells ml–1) and C. peaked at Most ciliate species in the ponds feed on bacteria the beginning of the hydroperiod, and Pelagolacry- and algae (except diatoms, but inclusive of auto- maria rostrata (40 cells ml–1) in the middle. Large-sized trophic flagellates; prey preferences of each ciliate caudatum and medium-sized Vorticella are noted in the ‘prey’ column in Appendix 1; based species dominated prior to drying. The leaf-litter addi- on Foissner & Berger 1996). On average, one-third of tion was the treatment that most affected the ciliate the ciliate species were bacterio-algivores, one-tenth community composition (i.e. the r+ has the longest facultative bacterivores, 15% predators (feeding on Andrushchyshyn et al.: Intermittent pond ciliate populations 301

Table 1. Results (F-values and * indicating statistical significance) of repeated measures ANOVA, of treatment and pond effects on ciliate community properties. Statistical significant p-values are indicated by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001; G- G adjusted for all interaction effects). Below significant F-values, the outcome of Bonferroni corrected planned contrasts are sum- marized in italics (Pond I [I], Pond II [II]; early-, mid-, late season; control enclosure [c]. pondwater control [o], resource addition [r+], and resource removal [r–]; non-significant [ns]). Mean square (MS) values of residuals are given for main effects and interaction effects

df Critical Abundance Number H’ Size group 1 Algivores Facultative Omnivores ppost-hoc of species <50 µm bacterivores

Main effects Pond 1 0.05 ***25.11*** 0.018 ***65.29*** ***82.81*** **13.9** ***51.25*** ***64.7*** I > II II > II> II I > II I > II II > I

Treatment 4 0.0125 **8.67** 1.93 1.45 1.89 **6.87** ***13.05*** 2.48 o < cr+ c

Pond 4 0.0063 **7.22** *4.01* 0.15 1.27 *4.02* 1.62 3.12 × Treatment oI < cI oI < cI ns

MS error 10 0.322 68.39 0.037 346 271 136 159.9

Interaction effects Time 12 ***6.00*** *4.74* **3.00** **3.93** 1.83 **3.65** 1.02

Time 12 0.0083 *2.96* 2.75 **2.8** ***9.15*** 1.85 1.33 2.27 × Pond Imid-late > II

Time 48 0.0042 ***3.29*** *2.51* *2.19*** **3.09** **2.50** *1.75* 1.82 × Treatment r+mid > c > omid omid < cr+mid-late < cr+early < c ns omid < c

Time 48 0.0021 **3.14** 1.74 1.40 *1.99* *1.94* *2.73** 1.02 × Pond r+ I,mid > o,r+I,early < cnsr+I,II,mid > c × Treatment c > oI,med r+ I,mid > cr– I,mid < c

MS error 120 0.07 24.53 0.015 143 118 91 66.8

protozoans, mostly ciliates, and small metazoans), and Hypotricha. In addition, the omnivore Oxytricha hy- 17% omnivorous (i.e. feeding on autotrophic organ- menostoma was very abundant in Pond II prior to dry- isms, small protozoans and metazoans). In Pond I the ing (Fig. 6a). Predatory ciliates peaked at the begin- algivores comprised 58% of the total ciliate abun- ning of the hydroperiod especially in Pond I, then dance (50% in Pond II), whereas 92% were bacteri- declined in the middle and increased again slightly vores (78% in Pond II), 28% facultative bacterivores prior to dry-up. (17% in Pond II), 4% were omnivores (16% in Pond II), and less than 2% were predators (in both ponds; Fig. 6a,b, p < 0.01, pond effect ANOVAR). In mid- Environmental influence on community hydroperiod, the proportion of facultative bacterivores characteristics increased with ~15% in the resource addition in both ponds, and decreased with the same percentage in Ciliate abundance (log) and the species richness the resource removal (p < 0.01). The proportion of from all enclosures and outside were combined into a algivores followed the seasonal trend in phytoplank- larger dataset for analysis of the possible influence of ton (as measured by chl a, Figs. 1 & 2). There were environmental factors (i.e. the ‘global solution’). In few facultative algivores in either pond (usually less both ponds combined, the environmental variables than 5%) although their abundances increased as the together with pond identity and treatment explained ponds dried up (e.g. Uroleptus gallina in Pond II and 49% of ciliate abundance (stepwise linear regression, Pelagohalteria cirrifera in Pond I). The most abundant log[abundance] = 0.94 + 0.55 chl a + 0.35 ‘resource representatives of omnivorous ciliates belonged to the addition’ – 0.33 Pond; F11,119 = 11.12, p < 0.001), and 302 Aquat Microb Ecol 42: 293–310, 2006 U. furcata and U. farcta osures in Ponds I and II. Main food/ spp. includes Urotricha feeding strategy in brackets (Ba = bacteria, Al algae, Fl flagellata, O omnivores, P = predators). Fig. 6. Seasonal development of dominant species (relative abundance) in (a) enclosure controls, and (b) resource-addition encl Andrushchyshyn et al.: Intermittent pond ciliate populations 303

a DO r+ p-Depth Pond II Pond I NH o Conductivity 3 r- c LogP pH Turbidity Chla b NO3 Temperature Day

Col col Did nas pH Depth Sty vor p- Lit cry c Lac olo DO Oxy set Sty pus Col kle Col kle Eup mus Temperature Lem bul Col col Conductivity r- Vor cam Uro pel Hol dis Ste ign Oxy chl Psi vir Eup moe Str vir Hol dis Uro ova r+ Met es Vor mar NO3 Chla Hol gra Chi unc Uro pel Ker spe Str cau Turbidity Vor con Mon bal

CCA2 12.8% Phosphorus CCA2 21.3% Spa vir Met es Oxy hym Oxy sim Parvir o NH Gla sciHal gra Ble lat Tac pel 3 Sty myt Sti sec Fro acu Rha nud Vor mar Dre rev Par cau Sty vor Lit cry Ple gra Vor cam Ste coe Par aur Ste pol Pel lac Uro gal Kah ver

CCA1 43.7% CCA1 21.4%

Fig. 7. Species-environment biplot diagram from the CCA with (a) the whole dataset in the lower diagram (species with lower than 10% fit are not shown; the abbreviated species names correspond to the species in bold in Appendix 1), and the projection of environmental variables in the upper diagram (most important variables are underlined), and (b) the same data set but with Pond and Days after flooding as covariables (species with lower than 5% fit are not shown; the abbreviated species names correspond to the underlined species in Appendix 1)

34% of the species diversity index (H’ = 0.41 + 0.39 was 0.96, 0.85, 0.80, and 0.73 respectively. The treat- LogDO + 0.29Pond – 0.25 ‘resource addition‘ – 0.21 ment effects were further explored in a second CCA

‘top-down manipulation’; F11,119 = 5.59, p < 0.0001). with pond and days after flooding as covariables When the same analysis was done on each pond sepa- (Fig. 7b). In this analysis, 21.4% of this variation could rately, the environmental variables and treatments be explained by a bottom-up gradient (resource addi- explained 62 vs. 32% of the ciliate abundance (in tion versus resource removal, pondwater control, and Ponds I and II, respectively); and 31 vs. 24% of the spe- DO) and 12.8% by a top-down/productivity gradient cies diversity (in Pond I and II; DO was the most impor- (top-down manipulation and enclosure control versus tant factor in Pond I and pH in Pond II). nutrients and pondwater control). The 2 following axes One-fifth of the total variation in the ciliate species were a temperature/DO/resource removal gradient data (22.2%) could be summarized by 4 axes in the (10%) and a pH gradient (9.6%). The species environ- unconstrained ordination. The measured environmen- mental correlations were 0.80, 0.75, 0.71, and 0.65. tal variables accounted for most of this variation (CCA, Similar gradients were found when the CCA was run p = 0.005). The CCA axes represented a pond gradient on each pond separately (data not shown but differ- (43.7%; i.e. pond, pH, and conductivity, Fig. 7a), a sea- ences in species assemblages are addressed in the son gradient (13.5%; i.e. days after flooding and chl a, ‘Discussion’). Furthermore, rotifers and zooplankton Fig. 7a), a bottom-up gradient (9.3%; resource addi- predators had weak correlations with ciliate commu- tion, resource removal), and a top-down gradient nity composition, when added as 2 independent vari- (5.6%; top-down manipulation, and water depth). The ables in the CCA (for simplicity these variables are not species-environment correlation in each CCA axes shown in the figures). 304 Aquat Microb Ecol 42: 293–310, 2006

DISCUSSION Influential factors

Ciliates provide an important link between The spatial and temporal distributions of ciliates are resources and higher trophic levels in many fresh usually governed by seasonal factors, by temporal and waters. They are key organisms in many freshwater spatial availability of food and habitat resources, by food webs and could potentially absorb and reflect any the distribution of oxygen, and by competition and perturbations; hence, ciliates are highly suitable for predator-prey relations (e.g. Bick 1973, Kusano et al. studying community regulating processes such as top- 1987, review by Beaver & Crisman 1989, Guhl et al. down and bottom-up control. The relative importance 1994). Several of these factors were influential for the of top-down and bottom-up mechanisms is thought to ciliate community structure in Vandorf ponds: the change along the habitat duration gradient, but also measured environmental variables (including pond depends on the species composition of both the prey and treatments) accounted for half of the variation in and the predator communities. Intermittent ponds, ciliate abundance, one-third of the species diversity, which are near the extreme short end of the habitat and one-fifth of the community composition. Ciliate duration gradient, are characterized by rapid changes abundance and diversity were best explained by pond in abiotic and biotic factors and the importance of characteristics and the leaf litter addition treatment. bottom-up and top-down control is considered to be The most influential factors, however, were chl a for relatively low. However, this has not been well studied ciliate abundance, and dissolved oxygen for species in the field. diversity. Ciliate community composition was best explained by pond characteristics, followed by season and treatment. Natural development of ciliates The differences in ciliate species composition between the 2 ponds could be attributed to differences Total ciliate abundance, number of species, and in food availability and vegetation structure (mixed diversity showed a bimodal seasonal distribution in the emergent, submergent, and floating in Pond I, pondwater controls, with maxima in spring and late but mainly Phalaris sp. in Pond II); water chemistry summer. The peak in early spring coincided with (e.g. higher levels of nitrogen and turbidity in Pond I, raised temperature and release of nutrients from de- and higher pH, conductivity, and DO in Pond II); and composition of dead aquatic vegetation and riparian predation pressure (zooplankton density was higher in leaf litter, whereas the peak prior to drying was a con- Pond II). For example, an overall higher bacterial centration effect caused by the shrinking water vol- abundance in Pond I was accompanied by higher ume. The lower ciliate abundance and diversity in occurrence of ciliate bacterivores, and the facultative mid-hydroperiod co-occurred with low phytoplankton bacterivores in Pond I comprised 6 species with a total concentrations, and with higher abundance of crus- abundance 20 times higher than in Pond II, which had taceans (peaked in mid-late April). The ciliate commu- 9 species of facultative bacterivores. The lower abun- nities underwent rapid species succession and con- dance of ciliates in Pond II compared with Pond I was tained many rare species (temporally and spatially, likely coupled to predation pressure because zoo- Andrushchyshyn et al. 2003, this study). The low over- plankton (e.g. cladocera and harpacticoids) and omni- lap in ciliate species composition between the 2 inter- vorous hypotricha species (which potentially feed on mittent ponds suggests that, although ciliates may small-sized ciliates) were more abundant in Pond II have cosmopolitan distributions (Finlay & Fenchel than in Pond I. Several previous studies have reported 2004), species composition in adjacent habitats can tight correlation between the abundances of zooplank- vary considerably. Moreover, despite the visual and ton predators and ciliates (Gilbert 1989, review by measured homogeneous appearance of each pond (in Stoecker & Capuzzo 1990, Pace & Funke 1991, Gilbert terms of within-pond depth, substrate, water chem- & Jack 1993, Sanders & Wickham 1993, Sipura et al. istry, light regime, and macrophyte composition), the 2003). spatial heterogeneity of ciliate populations within each The seasonal development of ciliate community pond was pronounced (one-third of the species present composition in the ponds was strong and correlated occurred in only 1 of 2 replicates). Such variation could with the increasing temperatures. The seasonal be related to continuous reciprocal interactions with change in the relative feeding groups reflected the environment, alongside the ability of ciliates to changes in food composition and abundance (leaf lit- quickly and repeatedly exploit suitable, and novel, ter, detritus, bacteria), whereas the change in size dis- niches (e.g. Finlay & Esteban 1998b) — as where, for tribution indicated selective predation by zooplankton, example, the species counts rose in the Vandorf enclo- and other ciliates. In addition, light availability mostly sures compared with the pondwater itself. restricted the distribution of species containing symbi- Andrushchyshyn et al.: Intermittent pond ciliate populations 305

otic algae (e.g. some species in the genera Strombid- 3 wk after snowmelt in this treatment. Thus, the effects ium, Halteria, , Stichotricha, Oxytricha, and of the resource addition differed between the 2 ponds, Paraurostyla) to early in the hydroperiod before turbid- suggesting that the outcome of food web manipula- ity increased in the ponds, and the high density of tions was more related to differences in abiotic and Hypotricha (most of them contain symbiotic zoochlor- biotic factors than to habitat proximity. ellae) in Pond II (which was less turbid than Pond I). Since leaf-litter is not a direct food resource for ciliates, treatment effects were likely mediated by alterations in the habitat and in food composition. The addition of leaf Treatment effects litter resulted in higher bacterial abundance in the water column, higher nutrient concentrations, and lower dis- The use of enclosures led to higher species richness solved oxygen (due to increasing decomposition), which and ciliate abundance, possibly due to more favour- was accompanied by an increasing dominance of ciliate able physicochemical environments, and/or by broad- bacterivores (which sometimes comprised 60 to 85% of ening the range of niches available, by restricting the ciliates). These results agree with the finding of movement of zooplankton predators, and by the cumu- Hatano & Watanabe (1981), who showed a strong lative effect of the ciliates themselves. relationship between bacterivore protists and leaf-litter Although the importance of leaf litter decomposition decomposition in permanent ponds. for bacteria and abundance can be strong in The resource removal resulted in decreasing levels ponds (Hatano & Watanabe 1981, Kusano et al. 1987), of nutrients and bacteria, and a lower proportion of cil- many studies suggest that the addition of resources or iate bacterivores in Pond I. Ciliate abundance and spe- nutrients can have weak effects on protists due to indi- cies richness also tended to be lower in this treatment rect effects, interactions with other zooplankton, pre- than in the enclosure controls, however, these effects dation (especially by Daphnia), and internal feedback were not statistically significant. Other effects included mechanisms (Pace & Funke 1991, Havens & Beaver higher biomass of the large species of Holophrya and 1997, Marchessault & Masumder 1997, Hillebrand et Stentor in Pond I, and some species of Vorticella in al. 2002). However, the addition of leaf litter was the Pond II. Also, the abundance of Halteria, Strobilidium, treatment that had most influence on the ciliate com- and Strombidium increased when leaf and plant litter munity in the Vandorf ponds. This treatment increased were removed. These taxa are either algivorous or total ciliate abundance significantly (especially small- contain symbiotic algae which keeps them directed sized ciliates in mid-season), and affected ciliate com- towards light, thus an increase in these species may munity composition. In Pond I, this treatment resulted be attributed to the lower turbidity in this treatment in the appearance of 11 rare species and 2 abundant compared with the enclosure controls. species (Urotricha ovata and Colpidium colpoda). The top-down manipulation excluded the entry of Some of these species possibly originated from cysts aerially colonizing insects (e.g. culicids, chironomids, introduced with riparian leaf litter. Moreover, some and Chaoborus) whose larvae could feed directly upon species (e.g. Cyclidium glaucoma, Urotricha farcta, U. ciliates and potentially have a substantial effect on furcata, and Halteria grandinella) attained very high their prey communities (review in Sanders & Wickham densities in this treatment, especially early in the 1993). The nets also excluded Coleoptera and Odo- hydroperiod and coincided with increasing tempera- nata, whose larvae feed on zooplankton, and thereby, tures and decomposition of terrestrial material. Several indirectly, may affect ciliate dynamics. However, this species within the genera Colpidium, , Lox- manipulation did not affect the ciliate community sig- odes, Metopus, Spirostomum, and Stentor are known nificantly, indicating a decoupling of the top-down to survive well in low oxygen environments (Foissner & effect at a higher trophic level. The weak top-down Berger 1996), and the contribution of these taxa was control on the ciliate community was consistent for 2 considerably higher when oxygen levels were low in consecutive years (see Andrushchyshyn et al. 2003), the resource addition in Pond I (especially early in the suggesting that abiotic parameters were more impor- hydroperiod). In addition, the increase of small ciliates tant in governing the ciliate communities than the top- was accompanied by an increase of medium-sized down manipulation tested here, and that the treatment Pelagolacrymaria rostrata (peaked in abundance dur- failed to reduce predation pressure on the ciliates. ing Days 75 to 85) — a predator that feeds on Urotricha Contrary to what was expected, however, this treat- and oligotrichs (Foissner et al. 1999). In Pond II, the ment resulted in a decrease in crustaceans in Pond I resource additions affected ciliate abundance at times, and less flagellates and amoebae in Pond II. Although but had less influence on the species composition. macrofauna are known to predate upon meiofauna However, medium-sized bacterivores (especially Col- (Schmid-Araya & Schmid 2000), and meiofauna pre- pidium kleini and C. colpoda) peaked during the first date upon ciliates (McCormick & Cairns 1991), unex- 306 Aquat Microb Ecol 42: 293–310, 2006

pected or absent cascading effects from invertebrate Bick H (1973) Population dynamics of associate with predator removal have been reported from several the decay of organic materials in fresh water. Am Zool 13: studies. For example, Pace & Funke (1991) showed that 149–160 Chislenko LL (1968) Nomographs for determination of weight top-down mechanisms were ‘truncated’ at the level of of water organisms by sizes and body shape. Academy of protists, and Sipura et al. (2003) attributed the absence Science of USSR, Moscow (in Russian) of community-level cascades to consumer recycling of Corliss JO (1979) The ciliated protozoa — characterization, resources and trophic level heterogeneity. However, classification and guide to the literature. Pergamon Press, Oxford Wickham et al. (2004) found that ciliate abundance Curds CR, Gates MA, Roberts DM (1982) British and other decreased and the ciliate composition changed when freshwater ciliated protozoa. Part I. Ciliophora: Kineto- macrozooplankton predators were removed from lake fragminophora. Cambridge University Press, Linnean enclosures. Thus, the outcome of food web manipula- Society of London, London tions is likely habitat specific, partly due to differences Curds CR, Gates MA, Roberts DM (1983) British and other freshwater ciliated protozoa. Part II. Ciliophora: Oligohy- in relative abundance of predators and prey, selective menophora and Polyhymenophora. Cambridge University feeding, and presence and abundance of alternative Press, Linnean Society of London, London prey (review by Sanders & Wickham 1993), and possi- Fenchel T (1987) Ecology of Protozoa — the biology of free- bly due to the different mechanisms that prevail in living phagotrophic protists. Springer Verlag, New York Finlay BJ, Esteban GF (1998a) Freshwater protozoa: biodiversity permanent versus intermittent waters. and ecological function. Biodivers Conserv 7:1163–1186 Since ciliates are an intermediate link between bac- Finlay BJ, Esteban GF (1998b) Planktonic ciliate species teria and zooplankton, high ciliate abundance and diversity as an integral component of ecosystem function strong seasonal community development, as observed in a freshwater pond. Protist 149:155–165 in the Vandorf ponds, likely significantly contribute to Finlay BJ, Fenchel T (2004) Cosmopolitan metapopulations of free-living . Protist 155:237–244 the seasonal variation in other components of the food Foissner W, Berger H (1996) A user friendly guide to the cili- web. We conclude that although the Vandorf ciliate ates (Protozoa, Ciliophora) commonly used by hydrobiolo- populations showed pronounced natural temporal and gists as bioindicators in rivers, lakes, and waste waters, spatial heterogeneity, together with rapid species suc- with notes on their ecology. Freshw Biol 35:375–482 Foissner W, Blatterer H, Berger H, Kohmann F (1991) Taxono- cession, responses to some of the manipulations were mische und ökologische Revision der Ciliaten des Sapro- nonetheless detectable, suggesting that pond physico- biensystems — Band I: Cyrtophorida, Oligotrichida, Hypo- chemical characteristics and bottom-up trophic effects trichia, . Informationsberichte Des Bayerischen were important in governing the ciliate community Landesamtes für Wasserwirtschaft 1:1–478 structure in these 2 intermittent ponds. Foissner W, Berger H, Kohmann F (1992) Taxonomische und ökologische Revision der Ciliaten des Saprobiensy- stems — Band II: Peritricha, Heterotricha, Odontostomat- ida. Informationsberichte Des Bayerischen Landesamtes Acknowledgements. We gratefully acknowledge funding for für Wasserwirtschaft 1:1–502 this project from the Natural Sciences and Engineering Foissner W, Berger H, Kohmann F (1994) Taxonomische und Research Council of Canada to D.D.W. We thank K. Wilson ökologische Revision der Ciliaten des Saprobiensy- and M. Roy for help in the field, A. Tavares for editorial assis- stems — Band III: Hymenostomata, Prostomatida, Nassu- tance, and 2 anonymous referees for useful comments on the lida. Informationsberichte Des Bayerischen Landesamtes manuscript. We are also very grateful to Dr. and Mrs. P. van für Wasserwirtschaft 1:1–548 Nostrand for permission to work on their property. Foissner W, Berger H, Blatterer H, Kohmann F (1995) Taxono- mische und ökologische Revision der Ciliaten des Sapro- biensystems —Band IV: Gymnostomatea, Loxodes, Sucto- LITERATURE CITED ria. Informationsberichte Des Bayerischen Landesamtes für Wasserwirtschaft 1:1–540 American Public Health Association, American Water Works Foissner W, Berger H, Schaumburg J (1999) Identification and Association, and Water Pollution Control Federation ecology of limnetic plankton ciliates. Informationsberichte (1995) Standard methods for the examination of water and Heft, Report, Issue 3 wastewater, 19th edn. Washington, DC Gilbert JJ (1989) The effect of Daphnia interference on a nat- Andrushchyshyn O, Magnusson AK, Williams DD (2003) Cili- ural rotifer and ciliate community: short-term bottle ex- ate populations in temporary freshwater ponds: seasonal periments. Limnol Oceanogr 34:606–617 dynamics and influential factors. Freshw Biol 48:548–564 Gilbert JJ, Jack JD (1993) Rotifers as predators on small cili- Beaver JR, Crisman TL (1989) Review — the role of ciliated ates. Hydrobiologia 255:247–253 protozoa in pelagic freshwater ecosystems. Microb Ecol Goulder R (1971) The effects of saprobic conditions on some 17:111–136 ciliated Protozoa in the benthos and hypolimnion of a Berninger UG, Wickham SA, Finlay BJ (1993) Trophic cou- eutrophic pond. Freshw Biol 1:307–318 pling within the microbial food web: a study with fine tem- Goulder R (1974) The seasonal and spatial distribution of poral resolution in a eutrophic freshwater ecosystem. some benthic ciliated protozoa in Esthwaite Water. Freshw Biol 30:419–432 Freshw Biol 4:127–147 Bick H (1972) Ciliated protozoa — an illustrated guide to the Guhl BE, Finlay BJ, Schink B (1994) Seasonal development of species used as biological indicators in freshwater biology. hypolimnetic ciliate communities in a eutrophic pond. World Health Organization, Geneva FEMS Microbiol Ecol 14:293–306 Andrushchyshyn et al.: Intermittent pond ciliate populations 307

Hatano H, Watanabe Y (1981) Seasonal change of protozoa Müller H, Schöne A, Pinto-Coelho RM, Schweizer A, Weisse and micrometazoa in a small pond with leaf litter supply. T (1991) Seasonal succession of ciliates in Lake Con- Hydrobiologia 85:161–174 stance. Microb Ecol 21:119–138 Havens KE, Beaver JR (1997) Consumer vs. resource control Pace ML, Funke E (1991) Regulation of planktonic microbial of ciliate protozoa in a copepod-dominated subtropical communities by nutrients and herbivores. Ecology 72: lake. Arch Hydrobiol 140:491–511 904–914 Hillebrand H, Kahlert M, Haglund AL, Berninger UG, Nagel Picken LE (1937) The structure of some protozoan communi- S, Wickham S (2002) Control of microbenthic communities ties. J Ecol 25:368–384 by grazing and nutrient supply. Ecology 83:2205–2219 Salvado H, Gracia MD (1991) Response of ciliate populations James MR, Burns CW, Forsyth DJ (1995) Pelagic ciliated pro- to changing environmental conditions along a freshwater tozoa in 2 monomictic, southern temperate lakes of con- reservoir. Arch Hydrobiol 123:239–255 trasting trophic state: seasonal distribution and abun- Sanders R, Wickham S (1993) Planktonic protozoa and meta- dance. J Plankton Res 17:1479–1500 zoa: predation, food quality and population control. Mar Kahl A (1930) Urtiere oder Protozoa I: Wimpertiere oder Microb Food Webs 7:197–223 Ciliata (Infusoria) 1. Allgemeiner Teil und Prostomata. Scheiner SH, Gurevitch J (2001) Design and analysis of eco- Tierwelt Deutschlands 18:1–180 logical experiments. Oxford University Press, Oxford Kahl A (1931) Urtiere oder Protozoa I: Wimpertiere oder Schmid-Araya JM, Schmid PE (2000) Trophic relationships: Ciliata (Infusoria) 2. ausser den im 1. Teil integrating meiofauna into a realistic benthic food web. behandelten Prostomata. Tierwelt Deutschlands 21: Freshw Biol 44:149–163 181–398 Sime-Ngando T, Groliere C (1991) Effets quantitatifs des fixa- Kahl A (1932) Urtiere oder Protozoa I: Wimpertiere oder teurs sur la conservation des ciliés planctoniques d’eau Ciliata (Infusoria) 3. Spirotricha. Tierwelt Deutschlands douce. Arch Protistenkd 140:109–120 25:399–650 2imek K, Bobkova J, Macek M, Nedoma J, Psenner R (1995) Kahl A (1935) Urtiere oder Protozoa I: Wimpertiere oder Ciliate grazing on picoplankton in a eutrophic reservoir Ciliata (Infusoria) 4. Peritricha und Chonotricha. Tierwelt during the summer phytoplankton maximum — a study at Deutschlands 30:651–886 the species and community-level. Limnol Oceanogr 40: Kusano H, Kusano T, Watanabe Y (1987) Seasonal succession 1077–1090 of a microphagotroph community in a small pond during Sipura J, Lores E, Snyder RA (2003) Effect of copepods on litter decomposition. Microb Ecol 14:55–66 estuarine microbial plankton in short-term microcosms. Madoni P (1991) Seasonal changes of ciliate protozoa in a Aquat Microb Ecol 33:181–190 small pond covered by floating macrophytes. Arch Hydro- Sorokin YI (1999) Aquatic microbial ecology — a textbook for biol 121:449–461 students in environmental sciences. Backhuys Publishers, Madoni P, Sartore F (2003) Long-term changes in the struc- Leiden ture of ciliate communities in a small isolated pond. Ital J Stoecker DK, Capuzzo JM (1990) Review — predation on pro- Zool 70:313–320 tozoa: its importance to zooplankton. J Plankton Res 12: Marchessault P, Mazumder A (1997) Grazer and nutrient 891–908 impacts on epilimnetic ciliate communities. Limnol Wang CC (1928) Ecological studies of the seasonal distribu- Oceanogr 42:893–900 tion of protozoa in a fresh-water pond. J Morph Phys 46: McCormick PV, Cairns J (1991) Effects of micrometazoa on 431–478 the protistan assemblage of a littoral food web. Freshw Wickham SA, Nagel S, Hillebrand H (2004) Control of epiben- Biol 26:111–119 thic ciliate communities by grazers and nutrients. Aquat Muylaert K, Van der Gucht K, Vloemans N, De Meester L, Microb Ecol 35:153–162 Gillis M, Vyverman W (2002) Relationship between bacte- Williams DD (1997) Temporary ponds and their invertebrate rial community composition and bottom-up versus top- communities. Aquat Conserv 7:105–117 down variables in 4 eutrophic shallow lakes. Appl Environ Williams DD (2006) The biology of temporary waters. Oxford Microbiol 68:4740–4750 University Press, Oxford 308 Aquat Microb Ecol 42: 293–310, 2006

Appendix 1. Summary of ciliate occurrence in Ponds I and II (average ciliates per ml in all enclosures and pondwater control per sampling date, n = 10 replicates, 17 sampling dates for Pond I and 14 dates for Pond II), with notes on ciliate size (1 = 10–50 µm; 2 = 50–200 µm; 3 > 200 µm), habitat preference (p indicates planktonic, all other ciliates are benthic), and sporadic species (s), individual biomass (see ’Materials nad methods: Routine sampling’), feeding preference (a = algae, b = bacteria, c = , s = sulphur bacteria, d = diatoms, h = hetero- trophic flagellates, o = omnivorous, p = predator [mostly ciliates and some small metazoans], Hi = histophagous), and presence in treatments (+ = resource addition, – = resource removal, p = top-down manipulation, c = enclosure control, o = pond-water control). Species identified as important in the first CCA analysis (>10% fit) are denoted in bold, and the second CCA analysis (>5% fit) are underlined

Abundance Indiv. Treatment Ciliate species (×10–3 cells ml–1) Size biomass Prey Pond I Pond II Pond I Pond II 10–6 mg ++––ppccoo ++––ppccoo

I. Kinetofragminophora Actinobolina radians (Stein 1867) Strand 1928 20.6 2 125 p – – p p c o o Actinobolina vorax (Wenrich 1929) Kahl 1930 0.6 12.9 2 s 250 p + p p Amphileptus pleurosigma (Stokes 1884) Foissner 1984 5.3 2.1 3 150 p – c + p Askenasia volvox (Eichwald 1852) Kahl 1930 55.3 49.3 1 p 35 ad + – – p c o o + – – p p c c o Balanion planctonicum Foissner, Oleksiv & Müller 1990 2.1 1 ps 2 a + (Ehrenberg 1838) Strand 1928 45.9 306.4 1 11 b + + – – p p c c o o + + – – p p c c o o Chilodontopsis depressa (Perty 1852) Blochmann 1895 77.6 20.7 2 10 bad + + – – p c c o o p p c o o nasutum (Müeller 1773) Stein 1859 10.6 6.4 2 500 p + + p c c o o p p amphileptoides Kahl 1931 1.4 3 s 390 p + Dileptus anguillula Kahl 1931 2.1 2 s 80 p c Dileptus anser Müeller 1786 1.2 3 s 700 p c Dileptus bivacuolatus Da Cunha 1915 0.6 0.7 2 s 130 p + c Dileptus conspicuus Kahl 1931 1.2 2 s 180 p o Dileptus monilatus Stokes 1886 3.6 3 340 o o o Dileptus mucronatus Penard 1922 4.3 3 260 o o o Drepanomonas revoluta Penard 1922 62.9 131.4 1 1 b + – p p c c o o + + p p c c o o Enchelyodon vorax Penard 1922 2.9 3 180 + c Enchelys gasterosteus Kahl 1926 20.0 2 62 o + – p p c o Enchelys pupa Müeller–Ehrenberg–Schewiakoff 1893 110.0 24.3 2 82 o + + – – p p c c o o + + – – p p c c o Holophrya discolor Ehrenberg 1833 125.3 124.3 2 290 o + + – – p p c o o + + – – p p c c o o Holophrya gracilis Penard 1922 168.2 9.3 2 110 + + – – p p c c o o p o Holophrya kessleri Mereschowsky 1878 1.8 2 900 + + Holophrya ovum Ehrenberg–Kahl 1930 12.4 2 320 bca + o Holophrya teres (Ehrenberg 1833) Foissner et al. 1994a 1.2 10.7 2 1300 o c o + p c o o (Müeller 1786) Bory de Saint–Vincent 1824 85.3 44.3 3 33 p + + – – p c + + – – p c c o o Lagynus elegans (Engelmann 1862) Quennerstedt 1867 2.9 2 200 o c o Litonotus crystallinus Vuxanovici 1960 2.4 40.0 2 45 p – p – – p p c c o o Litonotus lamella Müeller 1773 23.6 2 15 p + p c o o Loxodes magnus Stokes 1887 2.1 3 960 o p p Loxodes rostrum (Müeller 1773) Ehrenberg 1830 6.5 10.0 2 250 o + + c – p c Loxodes striatus (Engelmann 1862) Penard 1917 1.8 3 s 200 adc + c Loxophyllum loxophylliforme Kahl 1927 6.4 2 100 p p p Loxophyllum utriculariae Penard 1922 0.6 2 s 90 p – Monodinium balbiani Fabre–Domergue 1888 28.2 11.4 2 55 p + + – p c c o o + + – o ornata Ehrenberg 1833 1.2 3 s 1600 c c Nassula picta Greeff 1888 4.1 2 224 c (o) + – p c Paradileptus elephantinus Svec 1897 2.1 3 s 1000 o o Pelagolacrymaria rostrata Kahl 1935 1148.2 29.3 2 30 + + – – p p c c o o + + – – p c c o o Pelatractus lacrymarieformis Kahl 1930 34.1 2 80 + + – c c Phialina coronata Claparede & Lachmann 1858 0.6 3 s 230 p + Phialina jankowski Foissner 1984 4.1 2 s 30 p o Phialina lagenula Claparede & Lachmann 1858 4.7 2 150 p – c o Phialina pupula Müeller 1786 19.4 6.4 2 170 p + + – – p o + p o Phialina sapropelica Kahl 1927 5.3 2.9 2 70 p – p o p o Placus luciae Kahl 1930 14.1 8.6 2 25 o + + o c c Podophrya fixa (Müeller 1786) Ehrenberg 1833 17.1 1 50 p + – c Prorodon cinereus Penard 1922 12.9 3 1500 + – p p o o Prorodon elipticus Kahl 1930 15.3 2 190 p – p c Prorodon margaritifer Claparede & Lachmann 1858 10.0 3 700 – p c c o Prorodon niveus Ehrenberg 1833 2.9 3 2500 p + p c Pseudochilodonopsis fluviatilis Foissner 1988 2.9 2 s 15 d o Pseudochilodonopsis piscatoris (Blochmann 1895) Foissner 1979 2.1 2 s 19 ad p Andrushchyshyn et al.: Intermittent pond ciliate populations 309

Appendix 1 (continued)

Abundance Indiv. Treatment Ciliate species (×10–3 cells ml–1) Size biomass Prey Pond I Pond II Pond I Pond II 10–6 mg ++––ppccoo ++––ppccoo

Rhagadostoma nudicaudatum Kahl 1926 42.9 2 45 + + – p p c o o Rhopalophrya cirrifera Penard 1922 2.1 2 s 30 p Spathidium breve Kahl 1930 44.7 25.7 1 60 p + + – p p o o – p p c o o Spathidium chlorelligerum Kahl 1930 3.6 3 310 p + p Spathidium cithara Penard 1912 2.9 2 110 p p c o Spathidium faurei Kahl 1930 22.4 2 80 p + p o Spathidium spathula (Müeller 1786) Woodruff & Spenser 1922 14.1 5.0 2 150 p + + – – c + o Spathidium viride (Penard 1922) Kahl 1926 29.3 2 90 p + + – p p c c o o Supraspathidium vermiforme Penard 1922 1.2 3 s 330 p + Trachelophyllum apiculatum (Perty 1852) Claparede & Lachmann 1859 39.4 48.6 2 39 o + + – – p p c o o + + – – p p c c o o Trachelophyllum vestitum Stokes 1884 7.6 12.9 3 100 o + – p c o + – – o o Urotricha agilis (Stokes 1886) Kahl 1930 1190.0 431.4 1 0,5 bh + + – – p p c c o o + + – – p p c c o o Urotricha farcta Claparede & Lachmann 1859 9075.3 1737.1 1 5 bah + + – – p p c c o o + + – – p p c c o o Urotricha furcata Schewiakoff 1892 3034.1 956.4 1 p* 3,5 ba + + – – p p c c o o + + – – p p c c o o Urotricha ovata Kahl 1926 176.5 40.7 1 15 a + + + p p c c o o Urotricha pelagica Kahl 1935 1319.4 305.0 1 p 26 ba + + – – p p c c o o + + – – p p c c o o Urotricha venatrix Kahl 1935 4.3 2 s 30 +

II. Oligohymenophora Astylozoon fallax Engelmann 1862 0.6 2 ps 30 b + Carchesium pectinatum (Zacharias 1897) Kahl 1935 7.9 1 p 60 b p o o Colpidium colpoda (Losana 1829) Stein 1860 401.2 2 130 bha + + – Colpidium kleini Foissner 1969 831.8 2 65 b + + – – p p c c o o Cyclidium glaucoma Müeller 1773 9594.7 939.3 1 3 b + + – – p p c c o o + + – – p p c c o o Disematostoma buetschlii Lauterborn 1894 7.1 0.7 2 p 400 ab + p Epystylis anastatica (Linnaeus 1767) Kent 1881 1.8 2 s 25 o Epistylis chrysemydis Bishop & Jahn 1941 7.9 2 p 300 ba o o Epistylis digitalis (Linnaeus 1758) Ehrenberg 1830 11.2 2 30 b + c c Frontonia acuminata (Ehrenberg 1833) Büetschli 1889 242.9 36.4 2 100 o + + – – p p c c o o + + – – p c o o Frontonia leucas Ehrenberg 1838 15.3 11.4 3 270 o + – – p p c c o o + + p c o Glaucoma scintillans Ehrenberg 1830 84.7 3.6 2 25 b + + – – p p c c o o c Kahlilembus verminus Mueller 1786 17.9 2 15 b + o o Lembadion bullinum (Müeller 1786) Perty 1849 40.6 46.4 2 200 o + – – p p c c o o + + – – p p c o Lembadion lucens (Maskell 1887) Kahl 1931 2.4 2 40 o + + Ophryoglena atra Lieberkuhni 1856 7.1 3 550 Hi p p o Ophryoglena flava Ehrenberg 1833 4.3 3 400 Hi + + Ophryoglena macrostoma Kahl 1928 16.4 3 350 Hi + p o Opisthonecta henneguyi Faure–Fremiet 1906 1.2 2 s 1000 bh o Paramecium aurelia – Komplex Foissner et al.1994a 111.2 2 150 b + + – p p c c Paramecium caudatum Ehrenberg 1833 859.4 3 500 ba + + – – p p c c o o Paramecium trichium (Stokes 1885) Wenrich 1926 2.1 2 s 95 b p Philasterides armatus Kahl 1931 2.4 2 s 25 Hi + Pleuronema coronatum Kent 1881 4.7 1.4 2 60 o + – o + Pleuronema crassum Dujardin 1841 1.8 9.3 2 60 bad + + + + p p Stokesia vernalis Wenrich 1929 4.1 2 p 400 bad c pyriformis – Komplex Foissner et al. 1994a 95.9 11.4 1 15 b Hi + + – – p p c o o + + – Urocentrum turbo (Müeller 1786) Nitzsch 1827 1.2 2 s 70 bd o Uronema nigricans (Müeller 1786) Florentin 1901 196.5 121.4 1 5 bh + + – – p p c c o o + + – – p p c c o o Vorticella aquadulcis – Komplex Foissner et al. 1992a 8.8 40.0 1 15 ba p o o + + – p c o o Vorticella campanula Ehrenberg 1831 515.9 487.9 2 135 ba + + – – p p c c o o + + – – p p c c o o – Komplex Foissner et al. 1992a 117.1 2 63 b + – – p p c c o o Vorticella marginata Stiller 1931 494.7 2 100 b + + – – p p c c o o Vorticella microstoma – Komplex Foissner et al. 1992a 24.7 2 30 ba + + – c

III. Polyhymenophora Aspidiska lynceus (Müeller 1773) Ehrenberg 1830 21.2 7.1 1 17 b + + – p c o + + lateritium (Ehrenberg 1831) Stein 1859 17.1 182.9 2 250 ba + + – p c o o + + – – p p c c o o Blepharisma steini Kahl 1932 38.8 22.9 2 300 o + + – p p c c o + + – – p p c

(Appendix continued on next page) 310 Aquat Microb Ecol 42: 293–310, 2006

Appendix 1 (continued)

Abundance Indiv. Treatment Ciliate species (×10–3 cells ml–1) Size biomass Prey Pond I Pond II Pond I Pond II 10–6 mg ++––ppccoo ++––ppccoo

Brachonella spiralis (Smith 1897) Jankowski 1964 1.8 2 s 110 o Bursaridium pseudobursaria (Faure-Fremiet 1924) Kahl 1927 3.5 12.1 2 p 342 a + c + – p c o o minimum Foissner 1980 2.1 2 ps 350 p o moebiusi Kahl 1932 31.4 2 23 bdh + + – p p c o o Euplotes muscicola Kahl 1932 41.8 2 18 – – c o o Euplotes patella (Müeller 1773) Ehrenberg 1831 98.2 5.7 2 93 o + + – – p c o o + – c Halteria grandinella (Müeller 1773) Dujardin 1841 4101.2 750.7 1 27 ba + + – – p p c c o o + + – – p p c c o o Holosticha kessleri Wrzesniowski 1877 16.5 2 66 bd – p o Holosticha monilata Kahl 1928 336.5 2 52 bda c Holosticha pullaster (Müeller 1773) Foissner et al. 1991a 91.8 2 12 bda – – p p c c o Hypotrichidium conicum Ilowaisky 1921 0.6 2 s 150 o c Kerona pediculus (Müeller 1773) Blochmann 1886 1.4 2 s 230 ad p Keronopsis spectabilis Kahl 1932 253.6 3 140 o + + – – p p c c o o Linostoma vorticella (Ehrenberg 1833) Jankowski 1978 5.9 0.7 2 p 1000 o + + – – – Metopus barbatus Kahl 1927 2.4 2 85 bha + c Metopus campanula Kahl 1932 29.4 55.0 2 75 bha + + – p c c + + – p p c c o o Metopus es (Müeller 1776) Lauterborn 1916 14.1 129.3 2 170 bha + + + + – p p c c o o Metopus fuscus Kahl 1927 1.8 2 200 bha + Metopus hyalinus Kahl 1932 2.9 2 150 bha + c Oxytricha chlorelligera Kahl 1932 142.4 57.9 2 35 bhd + + – p p c o + + – p p c o o Oxytricha ferruginea Stein 1859 1.2 3 s 125 bcad + Oxytricha hymenostoma Stokes 1887 668.6 2 30 o + + – – p p c c o o Oxytricha setigera Stokes 1891 190.0 184.3 2 8 bh + + – – p p c c o o + – – p p c c o o Oxytricha similis Engelmann 1862 477.6 2 14 b + + – – p p c c o o Paraurostyla viridis (Stein 1859) Borror 1972 14.1 137.9 2 87 b + p o + + – – p p c c o o Paraurostyla weissei (Stein 1859) Borror 1972 17.6 3 240 o – c Pelagohalteria cirrifera Kahl (1932) Foissner et al. 1988a 582.4 117.1 1 p 35 a + + – – p p c c o o + + – – p p c c o o Pleurotricha grandis Stein 1859 91.8 2.1 3 1300 da + + – – p p c c o o c c Pseudoblepharisma tenue Kahl 1926 15.9 30.7 2 30 b + + – – p c + + – p p c c o Psilotricha viridis Penard 1922 354.7 362.9 2 12 + + – – p p c c o o + + – – p p c c o o Rimostrombidium lacustris Petz & Foissner 1992 10.0 2 p 50 ab o Spirostomum ambiguum (Müeller 1786) Ehrenberg 1835 32.4 3 14600 bha + + – – p c c o Spirostomum minus Roux 1901 54.7 10.0 3 425 b + + – p c o + p c o o Spirostomum teres Claparede & Lachmann 1858 78.8 7.1 3 p 380 sbad + + – – p p c c o o + p o Stentor coeruleus (Pallas 1776) Ehrenberg 1831 6.5 3 12000 o o Stentor igneus Ehrenberg 1838 63.5 10.0 3 450 bad + + – – p p c c o o + – – p c o Stentor multiformis (Müeller 1786) Ehrenberg 1838 59.4 7.9 3 600 ab + + – – p c c o o + – p o o Stentor polymorphus (Müeller 1773) Ehrenberg 1830 20.0 3 4500 o o Stichotricha marina Stein 1867 0.7 2 s 50 – Stichotricha opisthotonoides Smith 1897 2.9 2 s 15 o Stichotricha secunda Perty 1852 167.1 2 30 bad + + – – p p c c o o Strobilidium caudatum (Fromentel 1876) Foissner 1987 87.6 344.3 2 45 dab + + – – p o o + + – – p c c o o Strobilidium humile Penard 1922 1.8 12.1 1 p 4 d – c c Strobilidium hyalinum Mirabdulajev 1968 5.0 1 ps 16 da + Strombidium viride Stein 1867 150.6 6.4 2 p 50 dab + – p p c c o o + o mytilus - Komplex Foissner et al. 1991a 272.4 2 400 o + + – – p p c o o Stylonychia pustulata (Müeller 1786) Ehrenberg 1835 295.9 312.1 2 80 o + + p p c c o o + + – – p p c c o o Stylonychia vorax Stokes 1885 687.1 2 57 o + + – – p p c c o o Tachysoma pellionellum (Müeller 1773) Borror 1972 247.9 2 15 bcad + + – – p p c c o o Tintinnidium fluviatile (Stein 1863) Kent 1881 2.1 2 ps 50 ad – Uroleptus caudatus Claparede & Lachmann 1858 18.2 2 20 + + p c c Uroleptus gallina (Müeller 1786) Foissner et al. 1991a 211.4 2 72 a – – p p c o o Uroleptus muscorum Kahl 1932 40.7 2 65 a + + – p c o Urostyla grandis Ehrenberg 1830 35.3 3 500 o c Urostyla urostyla Claparede & Lachmann 1858 4.7 2 90 + – Urostyla viridis Stein 1859 9.4 2 70 + c aFoissner, Blatterer, Berger & Kohmann (1991); Foissner, Berger & Kohmann (1992, 1994); Foissner, Skogstad & Pratt (1988)

Editorial responsibility: Fereidoun Rassoulzadegan, Submitted: August 24, 2005; Accepted: January 19, 2006 Villefranche-sur-Mer, France Proofs received from author(s): February 25, 2006