DOI: 10.5061/dryad.wh70rxwww
title: Data from: Competition between mixo- and heterotrophic ciliates under dynamic resource supply

description: 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.
description: <strong>Organisms used and culture
 conditions</strong> We used four freshwater
 ciliates belonging to the genera <em>Coleps</em> and
 <em>Euplotes</em> that differed in trophic mode, feeding
 preference and average cell size (see Table 1 for characteristics and
 origin of the organisms used). <em>Coleps hirtus</em> and
 <em>Euplotes octocarinatus</em> are heterotrophic, whereas
 <em>Coleps</em> <em>viridis</em> and
 <em>Euplotes daidaleos</em> are mixotrophic. Both ciliates
 carry phototrophic symbionts. We did not perform sequence analysis on the
 symbionts of our <em>E. daidaleos</em> and <em>Coleps
 viridis </em>strains. However, <em>Chlorella
 vulgaris</em> has been identified for other <em>E.
 daidaleos</em> strains, whereas <em>Micractinium
 conductrix</em> is the endosymbiont of several <em>Coleps
 viridis </em>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
 <em>Cryptomonas</em> sp. (referred from hereon as
 <em>Cry</em>), a flagellate whose motility is strong enough to
 avoid sedimentation. Both of the <em>Euplotes</em> and the
 <em>Coleps</em> species used in this experiment are also able
 feed on bacteria; however, only <em>Euplotes</em> is able to
 grow on a diet consisting solely of bacteria. Abiotic resources were light
 and mineral nutrients. Cultures of <em>Cry</em> were grown in
 WEES medium (Kies 1967), mineral water (Volvic) served as culture medium
 for ciliates. <strong>Experimental set-up and
 design</strong> 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<sup>-1</sup>. Experimental communities grew in a modified WC
 medium (Guillard and Lorenzen 1972), which was nitrogen limited (120 µmol
 N L<sup>-1</sup>). According to previous experiments,
 <em>Cry</em> grows better if organic compounds are available,
 which can be supplied by adding soil extract. Half (60 µmol N
 L<sup>-1</sup>) 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<sup>-1</sup> were added using the WC Nitrogen stock solution
 (NaNO<sub>3</sub>). 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 <em>Euplotes octocarinatus</em> (<em>E.
 het</em>) – <em>Coleps</em>
 <em>viridis</em>. (<em>C. mixo</em>) and
 <em>Coleps hirtus</em> (<em>C. het</em>) –
 <em>Euplotes daidaleos</em> (<em>E. mixo</em>)
 were investigated in experiment 1 and the combinations <em>E.
 het</em> – <em>E. mixo</em> and <em>C.
 het</em> – <em>C. mixo </em>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 10<sup>5</sup>
 µm<sup>3</sup> ml<sup>-1</sup> in experiment 1 and
 6.30 x 10<sup>5</sup> µm<sup>3</sup>
 ml<sup>-1</sup> 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<sup>-2</sup>
 s<sup>-1</sup> photosynthetic photon flux density (PPFD)).
 Under fluctuating light, phases of high (59 µmol
 m<sup>-2</sup> s<sup>-1</sup> PPFD) and low light
 (7 µmol m<sup>-2</sup> s<sup>-1</sup> 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
 <em>Cry</em> 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 <em>Cry</em> 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.
 <strong>Sample processing and analysis
 </strong> Samples for microscopic analysis were
 fixed with Lugol’s solution (1% final concentration) and stored in brown
 glass bottles. <em>Cry</em> 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<sup>-1</sup>) was assumed
 (Clarke 1998). Since <em>C. het</em> and <em>C.
 mixo</em> were indistinguishable by light microscopy, total
 <em>Coleps</em> concentrations were quantified in the
 <em>C. mixo</em> – <em>C. het</em> treatment. We
 determined the <em>C. mixo</em> to <em>C. het</em>
 ratio via epifluorescence microscopy (Axiophot, Zeiss) using
 Glutaraldehyde preserved samples (final concentration 1 %). Here,
 <em>C. mixo </em>could be distinguished from <em>C.
 hetero</em> under blue light excitation by the presence of algal
 symbionts, which could be clearly distinguished from potentially ingested
 algae of <em>C. hetero </em>at 1000x magnification. The
 <em>C. mixo</em> and <em>C. hetero</em>
 proportions determined were then used to calculate the concentrations of
 the different <em>Coleps</em> 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.   
 <strong>Table 1:</strong> 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. 
 
 <strong>Abbreviation</strong> 
 <strong>Algal and
 ciliate</strong>
 <strong>species</strong> 
 <strong>Class</strong>
 
 <strong>Average</strong>
 <strong>cell size (µm³)</strong>
 
 <strong>Origin</strong> 
 
 <em>Cry</em> 
 <em>Cryptomonas
 </em>sp<em>.</em> 
 Cryptophyceae 
 664 SAG
 <em>C. mixo
 </em> <em>Coleps
 viridis </em> 
 Prostomatea 
 14,050 New
 Brunswick 
 <em>C. het </em> 
 <em>C. hirtus</em> 
 Prostomatea 
 9,850 
 Stuttgart 
 <em>E. mixo </em> 
 <em>Euplotes daidaleos</em> 
 Stichotricha 
 33,920 New
 Brunswick 
 <em>E. het </em> 
 <em>E. octocarinatus</em> 
 Stichotricha 
 26,890 Pisa
  
description: # Competition between mixo- and heterotrophic ciliates under dynamic
 resource supply
 [https://doi.org/10.5061/dryad.wh70rxwww](https://doi.org/10.5061/dryad.wh70rxwww) Data were collected during a 48-day chemostat experiment investigating competitive interactions of different heterotrophic and mixotrophic ciliates of the genera *Euplotes* and *Coleps* under different resource regimes. Prey was provided either continuously or in pulses, and light was either constant or fluctuating, 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. ## Description of the data and file structure The set contains biovolume data (total, mixotrophic and heterotrophic ciliate biovolume, prey (*Cryptomonas*) biovolume), the logratio mixotrophic/heterotrophic biovolume and abundance data. The table below explains the abbreviations used in the data set. | **Factors** | | | :--------------------- | :-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | Organisms | species combinations: Coleps mixo. + E. Octo (*Coleps viridis* + *Euplotes octocarinatus*), E. daid. + Coleps Stu. (*Euplotes daidaleos* + *Coleps hirtus*), Coleps mixo. + Coleps Stu. (*Coleps viridis* + *Coleps hirtus*), E. daid. +  E. octo (*Euplotes daidaleo*s + *Euplotes octocarinatus*) | | Light | light supply modes: continuous (Cont), fluctuating (Fluct) | | Prey | prey supply modes: constant (Const), pulsed (Puls) | | Day | time: sampling day | | | | | **Response Variables** | | | TotBV | total biovolume ml^-1 | | MixBV | mixotrophic biovolume ml^-1 | | HetBV | heterotrophic biovolume ml^-1 | | LogratioMH | Logratio mixotrophic/heterotrophic biovolume | | CryBV | *Cryptomonas* sp. biovolume ml^-1 | | EhetAB | *Euplotes octocarinatus* (heterotrophic) abundance ml^-1 | | EmixoAB | *Euplotes daidaleos* (mixotrophic) abundance ml^-1 | | ChetAB | *Coleps hirtus* (heterotrophic) abundance ml^-1 | | CmixoAB | *Coleps viridis* (mixotrophic) abundance ml^-1 | | CryAB | *Cryptomona*s sp. abundance ml^-1 | ## Code/Software We used a linear mixed model ANOVA to analyse community dynamics of both experiments, where the log-biomass ratio of mixotrophic and heterotrophic ciliates served as response variable. Species combination, and modes of light and prey supply were entered as factors, time (days) was set as trend and as factor for random fluctuations over time. Model assumptions were validated graphically. The analysis (Type III ANOVA) was performed with R version 3.6.3 (R Development Core Team 2020) using RStudio version 1.2.5042 (RStudio, Boston, USA), using R-package lme4 (Bates et al. 2015) with Satterthwaite's method of degrees of freedom in combination with lmerTest (Kuznetsova et al. 2017). Post-hoc tests (contrasts) used Kenward-Roger method for degrees-of-freedom method and Tukey P-value adjustment method for comparing a family of 4 estimates. Code: &gt; mod.LogratioMH = lmer(LogratioMH ~ Organisms*Light*Prey*Day+(1|ID)+(1|day.fac), data=DataHM_Dryad)