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Intraspecific trait variation alters the outcome of competition in freshwater ciliates


Floeder, Sabine et al. (2022), Intraspecific trait variation alters the outcome of competition in freshwater ciliates, Dryad, Dataset,


Trait variation among heterospecific and conspecific organisms may substantially affect community and food web dynamics. While the relevance of competition and feeding traits have been widely studied for different consumer species, studies on intraspecific differences are more scarce, partly owing to difficulties in distinguishing different clones of the same species. Here, we investigate how intraspecific trait variation affects the competition between the freshwater ciliates Euplotes octocarinatus and Coleps hirtus in a nitrogen-limited chemostat system. The ciliates competed for the microalgae Cryptomonas sp. (Cry) and Navicula pelliculosa (Nav), and the bacteria present in the cultures over a period of 33 days. We used monoclonal Euplotes and three different Coleps clones (Col 1, Col 2, Col 3) in the experiment that could be distinguished by a newly developed rDNA-based molecular assay based on the internal transcribed spacer (ITS) regions. While Euplotes feeds on Cry and on bacteria, the Coleps clones cannot survive on bacteria alone but feed on both Cry and Nav with clone-specific rates. Experimental treatments comprised two-species mixtures of Euplotes and one or all of the three different Coleps clones, respectively. We found intraspecific variation in the traits “selectivity” and “maximum ingestion rate” for the different algae to significantly affect the competitive outcome between the two ciliate species. As Nav quickly escaped top-down control and likely reached a state of low food quality, ciliate competition was strongly determined by the preference of different Coleps clones for Cry as opposed to feeding on Nav. In addition, the ability of Euplotes to use bacteria as an alternative food source strengthened its persistence once Cry was depleted. Hence, trait variation at both trophic levels co-determined the population dynamics and the outcome of species competition. --


Organisms used and culture conditions

We used the freshwater ciliate predator species Euplotes octocarinatus (monoclonal) and three different clones of Coleps hirtus (Col 1, Col 2, Col 3). The cryptophyte Cryptomonas sp. and the diatom Navicula pelliculosa served as prey (see Table 1 for characteristics and origin of the organisms used). Prior to the experiment all species and clonal ciliate cultures were fed Cry.

Mineral water (Volvic) was used as culture medium for our ciliate clones, while microalgae were grown in WEES culture medium (Kies, 1967). None of the cultures were axenic and free of heterotrophic flagellates. Bacterial biomass was well below 5% of the total biomass of the stock cultures. Biomass of heterotrophic flagellates was comparably low. Their abundance was deemed negligible, since it was close to or below the detection limit of our microscopical analysis. The ciliates differed in average cell size and in their feeding preferences, while microalgae differed in average cell size and edibility (Table 1). The feeding preferences of the ciliates were characterised by the trait value maximum ingestion rate (Imax) (Table 1). Imax was calculated based on the data published in Flöder et al. (2018) following Frost (1972), Heinbokel (1978) and Michaelis-Menten (see Appendix A for details). Euplotes feeds and grows only on Cry, whereas our Coleps clones feed and grow on Cryptomonas (Cry) and on Navicula (Nav). Imax for Cry and Nav, however, differ among the Coleps clones. Col 2 has a higher Imax for Cry and a lower Imax for Nav than the other clones. Col 1 and Col 3 show no difference in Imax, neither for Nav nor for Cry.

Experimental set-up and design

The chemostat system used consisted of 16 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, Woltermann, & Becks, 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 & Lorenzen, 1972), which was nitrogen limited (120 µmol N L-1). According to previous experiments, both microalgae grow 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 (100 µmol m-2 s-1 photosynthetic photon flux density), with a light to dark cycle of 12:12 hours. The experiment lasted 33 days.

We chose a 4 x 4 (4 treatments, 4 replicates) design to study the competition between Euplotes and mono- and polyclonal Coleps (Col poly) populations, resulting in the following combinations: Treatment 1: EuplotesCol 1, Treatment 2: EuplotesCol 2, Treatment 3: EuplotesCol 3, Treatment 4: EuplotesCol poly (Col 1, Col 2, Col 3). In each treatment, the experimental communities were supplied with the same mixture of the microalgae Cry and Nav, added once at the beginning of the experiment. The initial total ciliate biovolume in the experimental units was 1.3 x 106 µm3 ml-1, and the total microalgal biovolume was 14.4 x 106 µm3 ml-1. Different ciliate and microalgal species were inoculated with equal biovolume, respectively. Culture vessels were sampled every second day using a hypodermic syringe and cannula (1.0 x 200 mm, BD Plastipak, B. Braun, Melsungen, Germany; neoLab Migge, Heidelberg, Germany). The total sample volume (60 ml) was subdivided as follows: Subsamples for microscopic analyses (30 ml) of ciliate and microalgal abundance were taken every second day. Subsamples for nutrient analyses (20 ml) were taken every fourth day. On dates without nutrient sampling, bacteria (10 ml) or subsamples for molecular biological analysis (30 ml) were taken alternating every eighth day starting with bacteria samples on day 5 and molecular samples on day 9.

Sample processing and analysis

Plankton samples were fixed with Lugol’s solution (1% final concentration) and stored in brown glass bottles. Algal abundance was analysed microscopically (Leica DMIL) counting at least 400 cells per sample in randomly placed squares (Lund, Kipling, & Le Cren, 1958) if possible. Subsample size was 0.1 ml for the highly abundant Navicula pelliculosa and 1-2 ml for Cryptomonas sp. In cases where algal abundance was too low following this method, either two 0.5 mm transects at 100x magnification in a subsample of 2 ml (equalling a sixth of the counting chamber or a subsample of 0.335 ml) or the entire subsample was counted. Ciliate abundance was counted in a subsample sized 2 ml. If no ciliates were detected, an abundance of 0.5 x the detection limit (0.25 cells ml-1) was assumed (Clarke, 1998). The different cell size dimensions of 20 individuals of each ciliate and algal species were once determined using a digital image system program (Cell-P) to calculate the average specific biovolume (Hillebrand, Dürselen, Kirschtel, Pollingher, & Zohary, 1999). These data were used to calculate population biovolume.

Bacteria samples were preserved with Glutaraldehyde (final concentration 1 %). Diluted (1:15) subsamples (3 ml) were stained with DAPI (Porter & Feig, 1980), collected on black polycarbonate membrane filters (diameter: 25 mm) of 0.2 µm pore size (Whatman Cyclopore) and analysed by epifluorescence microscopy (Axiophot, Zeiss) counting the bacteria in 10 grids at 1000 x magnification (0.1 mm2).

Cell volume to carbon convertions (see Table 1) were done using empirically established biovolume to carbon relationships (Menden-Deuer and Lessard, 2000; Putt and Stoecker, 1989; Loferer-Krößbacher, Klima, and Psenner, 1998)

Samples for analysis of soluble reactive fractions of nitrogen, phosphorus and silicate concentrations were filtered using syringe filters (0.2 µm, cellulose acetate, Macherey-Nagel) and stored frozen (-20°C). They were analysed using a Scalar analytical auto-analyser (San++ System, Scalar Analytical, Breda, The Netherlands), following the methods published by Grasshoff et al. (1999).

Data Analyses

In one of the replicates of the treatment EuplotesCol 3, population dynamics especially of Euplotes differed greatly from the other three replicates (population size was up to 2.5 times higher than the average of the other replicates). Since this was due to problems concerning the flow through system of this particular chemostat (medium was pumped in, but culture suspension did not flow off), we removed the results for this replicate from the analysis.

We used a linear mixed model ANOVA to analyse experimental community dynamics, where the log-ratio of Euplotes and Coleps biomass (LogratioCEBM) served as response variable. Treatment (Combo) was a factor, time (Days) was set as trend and as factor for random fluctuations over time. The analysis was performed with R version 3.6.3 (R Development Core Team, 2020) using RStudio version 1.2.5042 (RStudio, Boston, USA).   

mod.LogratioCEBM = lmer(LogratioCEBM ~ Combo*Days  + (1|ID) + (1|Days.fac), data=eup_col)


Clarke, J. U. (1998). Evaluation of Censored Data Methods To Allow Statistical Comparisons among Very Small Samples with Below Detection Limit Observations. Environmental Science & Technology, 32(1), 177-183. doi:10.1021/es970521v

Flöder, S., Bromann, L., & Moorthi, S. (2018). Inter- and intraspecific consumer trait variations determine consumer diversity effects in multispecies predator-prey systems. Aquatic Microbial Ecology, 81(3), 243-256.

Frost, B. W. (1972). Effects of size and concentration of food particles on the feeding behavior of the marine planktonic copepos Calanus pacificus. Limnology and Oceanography, 17, 805-815.

Grasshoff, K., Kremling, K., & Ehrhardt, M. (1999). Methods of seawater analysis. Weinheim: Wiley-VCH.

Guillard, R. R. L., & Lorenzen, C. J. (1972). Yellow-green algae with chlorophyllide c. Journal of Phycology, 8, 10-14.

Heinbokel, J. F. (1978). Studies of the functional role of tintinnids in the Southern Californian Bight. I. Grazing and growth rates in laboratory cultures. Marine Biology, 47, 177-189.

Hillebrand, H., Dürselen, C. D., Kirschtel, D., Pollingher, U., & Zohary, T. (1999). Biovolume calculation for pelagic and benthic microalgae. Journal of Phycology, 35, 403-424.

Loferer-Krößbacher, M., Klima, J., & Psenner, R. (1998). Determination of bacterial cell dry mass by transmission electron microscopy and densitometric image analysis. Applied and Environmental Microbiology, 64(2), 688-694.

Menden-Deuer, S., & Lessard, E. J. (2000). Carbon to volume relationships for dinoflagellates, diatoms, and other protist plankton. Limnology and Oceanography, 45(3), 569-579.

Putt, M., & Stoecker, D. K. (1989). An experimentally determined carbon: volume ratio for marine “oligotrichous” ciliates from estuarine and coastal waters. Limnology and Oceanography, 34(6), 1097-1103.


Usage Notes

Table 1: Abbreviations, origin, food preference, maximum ingestion rate (I max), average cell size, volume to carbon relationship and carbon to volume conversion factor of algal and ciliate cultures used in the experiment. SAG: Culture Collection of Algae at Göttingen University, CCAP: Culture Collection of Algae and Protozoa; Salzburg: Dr UG Berninger, University of Salzburg, Austria. Stuttgart: Dr M Schweikert, University of Stuttgart, Germany; Pisa: Dr G. Di Giuseppe, University of Pisa, Italy; M-D&L: Menden-Deuer and Lessard (2000); P&S: Putt and Stoecker (1989); L-K: Loferer-Krößbacher, Klima, and Psenner (1998) assuming C = 0.5·DW. Note that when adjusted for the difference in ciliate biovolume, Imax of E. octocarinatus for Cry is 1.72 fold the average Imax of the Coleps clones.



Short form

Algal and ciliate



Food preference

Max. Ingestion rate (I max )


cell size (µm³)

Volume-to-carbon relationship


Conversion factor

pg C/µm3

Cells ciliate-1 d-1

pg C ciliate-1 d-1






Cryptomonas sp.








C [pg] 0.216∙V[μm3]0.939




Navicula pelliculosa








C [pg] 0.288∙V[μm3]0.811




Coleps hirtus


Cry, Nav






C [pg]0.14∙V[μm3 ]



Col 1

Col 2

Col 3

C. hirtus clone 1

C. hirtus clone 2

C. hirtus clone 3





17.6 ± 0.44

38.8 ± 0.51

1.7 103

0.47 103






18.6 ± 0.31

12.1 ± 1.89

1.79 103

0.15 103


17.0 ± 0.64

35.1 ± 4.98

1.64 103

0.43 103


Euplotes octocarinatus



116 ± 5.4


11.2 103



C [pg]0.14∙V[μm3 ]












C [pg] 0.5·0.435∙V[μm3]0.86




Deutsche Forschungsgemeinschaft, Award: 394736697