The outcome of species competition strongly depends on the traits of the competitors and associated trade-offs, as well as on environmental variability. Here we investigate the relevance of consumer trait variation for species coexistence in a ciliate consumer – microalgal prey system under fluctuating regimes of resource supply. We focus on consumer competition and feeding traits, and specifically on the consumer’s ability to overcome periods of resource limitation by mixotrophy, i. e. the ability of photosynthetic carbon fixation via algal symbionts in addition to phagotrophy. In a 48-day chemostat experiment, we investigated competitive interactions of different heterotrophic and mixotrophic ciliates of the genera Euplotes and Coleps under different resource regimes, providing prey either continuously or in pulses under constant or fluctuating light, entailing periods of resource depletion in fluctuating environments, but overall providing the same amount of prey and light. Although ultimate competition results remained unaffected, population dynamics of mixotrophic and heterotrophic ciliates were significantly altered by resource supply mode. However, the effects differed among species combinations and changed over time. Whether mixotrophs or heterotrophs dominated in competition strongly depended on the genera of the competing species and thus species-specific differences in the minimum resource requirements that are associated with feeding on shared prey, nutrient uptake, light harvesting and access to additional resources such as bacteria. Potential differences in the curvature of the species’ resource-dependent growth functions may have further mediated the species-specific responses to the different resource supply modes. Overall, our study demonstrates that genus- or species-specific traits other than related to nutritional mode may override the relevance of acquired phototrophy by heterotrophs in competitive interactions, and that the potential advantage of photosynthetic carbon fixation of symbiont-bearing mixotrophs in competition with pure heterotrophs may differ greatly among different mixotrophs, playing out under different environmental conditions and depending on the specific requirements of the species. Complex trophic interactions determine the outcome of competition, which can only be understood by taking on a multidimensional trait perspective.

Organisms used and culture conditions

We used four freshwater ciliates belonging to the genera Coleps and Euplotes that differed in trophic mode, feeding preference and average cell size (see Table 1 for characteristics and origin of the organisms used). Coleps hirtus and Euplotes octocarinatus are heterotrophic, whereas Coleps viridis and Euplotes daidaleos are mixotrophic. Both ciliates carry phototrophic symbionts. We did not perform sequence analysis on the symbionts of our E. daidaleos and Coleps viridis strains. However, Chlorella vulgaris has been identified for other E. daidaleos strains, whereas Micractinium conductrix is the endosymbiont of several Coleps viridis strains (Pröschold et al. 2011, Pröschold et al. 2021). None of the cultures used are axenic and are accompanied by a microbial community consisting of bacteria and heterotrophic flagellates. All ciliates were fed with a non-axenic culture of the microalga Cryptomonas sp. (referred from hereon as Cry), a flagellate whose motility is strong enough to avoid sedimentation. Both of the Euplotes and the Coleps species used in this experiment are also able feed on bacteria; however, only Euplotes is able to grow on a diet consisting solely of bacteria. Abiotic resources were light and mineral nutrients. Cultures of Cry were grown in WEES medium (Kies 1967), mineral water (Volvic) served as culture medium for ciliates.

Experimental set-up and design

The chemostat system used consisted of 24 culture vessels (culture volume 900 ml) and corresponding medium and waste containers, tubing and peristaltic pumps (Ismatec, Wertheim, Germany). The medium inflow and the culture suspension outflow were established via a port in the cap of the culture vessel. A compressor provided the air pressure necessary to push the culture suspension through the outflow (Del Arco et al. 2020). Magnetic stirrers were used to keep the organisms in suspension. The dilution (flow through) rate was 0.1 d^-1. Experimental communities grew in a modified WC medium (Guillard and Lorenzen 1972), which was nitrogen limited (120 µmol N L^-1). According to previous experiments, Cry grows better if organic compounds are available, which can be supplied by adding soil extract. Half (60 µmol N L^-1) of the N concentration in the modified WC medium, therefore, originated from a soil extract prepared following the instructions of Kies (1967). An additional 60 µmol N L^-1 were added using the WC Nitrogen stock solution (NaNO₃). Temperature was kept constant (18 °C), and illumination of the cultures vessels was from the side, with a light to dark cycle of 12:12 hours.

Due to material, space and time constraints, we divided the experimental investigation of the competition between mixotrophic and heterotrophic ciliates into two parts. The combinations Euplotes octocarinatus (E. het) – Coleps viridis. (C. mixo) and Coleps hirtus (C. het) – Euplotes daidaleos (E. mixo) were investigated in experiment 1 and the combinations E. het – E. mixo and C. het – C. mixo in experiment 2. We chose a 2 x 2 x 2 x 3 design for the experiments, exposing two different species combinations to two light regimes (constant and fluctuating) and two regimes of prey supply (continuous and pulsed), each of those replicated three times, resulting in 24 experimental units per experiment. The initial total ciliate biovolume was 10.76 x 10⁵ µm³ ml^-1 in experiment 1 and 6.30 x 10⁵ µm³ ml^-1 in experiment 2, with each of the ciliate species being initially incubated at equal biovolume. Constant light was supplied at an intermediate intensity (33 µmol m^-2 s^-1 photosynthetic photon flux density (PPFD)). Under fluctuating light, phases of high (59 µmol m^-2 s^-1 PPFD) and low light (7 µmol m^-2 s^-1 PPFD) alternated at the interval length of 8 days, resulting in an equal amount of light provided over the course of the experiments under both constant and fluctuating light conditions. Experimental light intensities were chosen based on previous experiments on the effect of light limitation and fluctuation on phytoplankton population and community dynamics (Huisman 1999, Flöder et al. 2002, Flöder and Burns 2005). To establish the light intensities, the PPFD was determined inside of the empty culture vessels. Intermediate and low light intensities were realized by covering the culture vessels of the chemostat systems by black netting of different mesh size. The microalgal prey was added manually. To ensure the cells were in a comparable condition each time, we used dense batch cultures of Cry that were in or close to steady state condition. Abundance was determined microscopically (see below), before adding the appropriate volume of culture suspension to the chemostats. Continuous prey supply consisted of 1000 cells/ml of Cry every day, while under pulsed prey supply 8000 cells/ml were fed every eighth day, with the first pulse supplied the day following inoculation (day 1). This way the time average of light intensity and prey supply was the same in all treatments. The interval length of 8 days corresponds to 4-5 generations of the ciliates used in our experiment (see Kusch and Kuhlmann 1994, Foissner et al. 1999). Resource pulses and light fluctuations interfering with generation times have been shown to promote coexistence, since they cause repeated interruptions and reversions of the exclusion process. Interval lengths shorter than one generation time result in physiological responses, and interval lengths exceeding 20 generations are likely to lead to competitive exclusion (Reynolds 1988, Flöder and Burns 2005). Duration of the experiments was 48 days.

Experimental units were sampled every second day (starting on day 2) using a hypodermic syringe and cannula (1.0 x 200 mm, BD Plastipak, B. Braun, Melsungen, Germany; neoLab Migge, Heidelberg, Germany). Constant prey treatments were sampled before the daily prey addition, and treatments with pulsed prey supply were sampled the day after a prey pulse was given. While this ensured that the time interval between prey addition and sampling was the same in both treatments, neither daily additions nor pulses of prey were captured. The total sample volume (60 ml) was subsampled as follows: Subsamples for microscopic analyses (30 ml) of ciliate and microalgal abundance were taken every second day, and subsamples for Glutaraldehyde fixation (9 ml) every fourth day.

Sample processing and analysis

Samples for microscopic analysis were fixed with Lugol’s solution (1% final concentration) and stored in brown glass bottles. Cry concentration was analysed microscopically (Leica DMIL) using a subsample of 1 ml. If possible, at least 400 cells per sample were counted in grids at 100x magnification (Lund et al. 1958). In cases where algal concentrations were too low following this method, two 0.5 mm transects at 100x magnification (equalling a sixth of the counting chamber or a subsample of 0.335 ml) were counted. Ciliate concentrations were counted in a subsample sized 2 or 3 ml. If no ciliates were detected, a concentration of 0.5 x the detection limit (0.25 cell ml^-1) was assumed (Clarke 1998). Since C. het and C. mixo were indistinguishable by light microscopy, total Coleps concentrations were quantified in the C. mixo – C. het treatment. We determined the C. mixo to C. het ratio via epifluorescence microscopy (Axiophot, Zeiss) using Glutaraldehyde preserved samples (final concentration 1 %). Here, C. mixo could be distinguished from C. hetero under blue light excitation by the presence of algal symbionts, which could be clearly distinguished from potentially ingested algae of C. hetero at 1000x magnification. The C. mixo and C. hetero proportions determined were then used to calculate the concentrations of the different Coleps species. The dimensions of 20 individuals of each ciliate and algal species were initially determined using a digital image system program (Cell-P) to calculate the average specific biovolume (Hillebrand et al. 1999). These data were used to calculate population biovolume throughout the experiments.   

Table 1: Abbreviations, taxonomy, average cell size, and origin of algal and ciliate cultures used in the experiment. SAG: Culture Collection of Algae at Göttingen University, Germany; New Brunswick: Prof. P. Morin, Rutgers University, New Brunswick, NJ, USA; Pisa: Dr G. Di Giuseppe, University of Pisa, Italy; Stuttgart: Dr M Schweikert, University of Stuttgart, Germany.