DOI: 10.5061/dryad.hdr7sqvj8
title: Intraspecific trait variation alters the outcome of competition in freshwater ciliates

description: 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. --
description: <i>Organisms used and culture
 conditions</i> We used the freshwater ciliate
 predator species <i>Euplotes octocarinatus</i> (monoclonal)
 and three different clones of <i>Coleps hirtus
 </i>(<i>Col</i> 1, <i>Col</i> 2,
 <i>Col</i> 3). The cryptophyte <i>Cryptomonas</i>
 sp.<i> </i>and the diatom <i>Navicula
 pelliculosa</i> 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 <i>Cry</i>. 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
 (<i>I</i><sub>max</sub>) (Table 1).
 <i>I</i><sub>max</sub> 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).
 <i>Euplotes</i> feeds and grows only on
 <i>Cry</i>, whereas our <i>Coleps </i>clones feed
 and grow on <i>Cryptomonas</i> (<i>Cry</i>) and on
 <i>Navicula</i> (<i>Nav</i>).
 <i>I</i><sub>max</sub> for <i>Cry</i>
 and <i>Nav</i>, however, differ among the
 <i>Coleps</i> clones. <i>Col</i> 2 has a higher
 <i>I</i><sub>max</sub> for <i>Cry</i>
 and a lower <i>I</i><sub>max</sub> for
 <i>Nav</i> than the other clones. <i>Col</i> 1 and
 <i>Col</i> 3 show no difference in
 <i>I</i><sub>max</sub>, neither for
 <i>Nav</i> nor for <i>Cry</i>.
 <i>Experimental set-up and design</i> 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<sup>-1</sup>.
 Experimental communities grew in a modified WC medium (Guillard &
 Lorenzen, 1972), which was nitrogen limited (120 µmol N
 L<sup>-1</sup>). 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<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 (100 µmol
 m<sup>-2</sup> s<sup>-1</sup> 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
 <i>Euplotes</i> and mono- and polyclonal
 <i>Coleps</i> (<i>Col</i> poly) populations,
 resulting in the following combinations: Treatment 1:
 <i>Euplotes</i> – <i>Col</i> 1, Treatment 2:
 <i>Euplotes</i> – <i>Col</i> 2, Treatment 3:
 <i>Euplotes</i> – <i>Col</i> 3, Treatment 4:
 <i>Euplotes</i> – <i>Col</i> poly
 (<i>Col</i> 1, <i>Col</i> 2,
 <i>Col</i> 3). In each treatment, the experimental communities
 were supplied with the same mixture of the microalgae
 <i>Cry</i> and <i>Nav,</i> added once at the
 beginning of the experiment. The initial total ciliate biovolume in the
 experimental units was 1.3 x 10<sup>6</sup>
 µm<sup>3</sup> ml<sup>-1</sup>, and the total
 microalgal biovolume was 14.4 x 10<sup>6</sup>
 µm<sup>3</sup> ml<sup>-1</sup>. 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.
 <i>Sample processing and analysis
 </i> 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 <i>Navicula pelliculosa</i> and 1-2 ml
 for <i>Cryptomonas</i> 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<sup>-1</sup>) 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
 mm<sup>2</sup>). 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<sup>++</sup> System,
 Scalar Analytical, Breda, The Netherlands), following the methods
 published by Grasshoff et al. (1999). <i>Data
 Analyses</i> In one of the replicates of the
 treatment <i>Euplotes</i> – <i>Col</i> 3,
 population dynamics especially of <i>Euplotes</i> 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 <i>Euplotes</i> and <i>Coleps</i>
 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) References Clarke, J. U.
 (1998). Evaluation of Censored Data Methods To Allow Statistical
 Comparisons among Very Small Samples with Below Detection Limit
 Observations. <i>Environmental Science & Technology,
 32</i>(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. <i>Aquatic Microbial Ecology,
 81</i>(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.
 <i>Limnology and Oceanography, 17</i>, 805-815.
 Grasshoff, K.,
 Kremling, K., & Ehrhardt, M. (1999). <i>Methods of seawater
 analysis</i>. Weinheim: Wiley-VCH. Guillard, R. R.
 L., & Lorenzen, C. J. (1972). Yellow-green algae with
 chlorophyllide c. <i>Journal of Phycology, 8</i>,
 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.
 <i>Marine Biology, 47</i>, 177-189. Hillebrand, H.,
 Dürselen, C. D., Kirschtel, D., Pollingher, U., & Zohary, T.
 (1999). Biovolume calculation for pelagic and benthic microalgae.
 <i>Journal of Phycology, 35</i>, 403-424. Loferer-Krößbacher, M., Klima, J., & Psenner, R. (1998). Determination of bacterial cell dry mass by transmission electron microscopy and densitometric image analysis. <i>Applied and Environmental Microbiology, 64</i>(2), 688-694. Menden-Deuer, S., & Lessard, E. J. (2000). Carbon to volume relationships for dinoflagellates, diatoms, and other protist plankton. <i>Limnology and Oceanography, 45</i>(3), 569-579. Putt, M., & Stoecker, D. K. (1989). An experimentally determined carbon: volume ratio for marine “oligotrichous” ciliates from estuarine and coastal waters. <i>Limnology and Oceanography, 34</i>(6), 1097-1103.  
description: Table 1: Abbreviations, origin, food preference, maximum
 ingestion rate (<i>I</i> <sub>max</sub>), 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,
 <i>I</i><sub>max</sub> of <i>E.
 octocarinatus</i> for <i>Cry </i>is 1.72 fold the
 average <i>I</i><sub>max</sub> of the
 <i>Coleps</i> clones. <pre style="margin-left:-19px;">  </pre> <pre style="margin-left:-19px;">  </pre> <b>Short form</b> <b>Algal and ciliate</b> <b>Species</b> <b>Origin</b> <b>Food preference</b> <b>Max. Ingestion rate (<i>I</i></b><b><sub> max</sub></b><b> )</b> <b>Average</b> <b>cell size (µm³)</b> <b>Volume-to-carbon relationship</b> <b>Reference</b> <b>Conversion factor</b> <b>pg C/µm<sup>3</sup></b> <b>Cells ciliate<sup>-1</sup> d<sup>-1</sup></b> <b>pg C ciliate<sup>-1</sup></b><b> d<sup>-1</sup></b> <b>Cry</b> <b>Nav</b> <b>Cry</b> <b>Nav</b> <i>Cry</i> <i>Cryptomonas </i>sp<i>.</i> SAG /         664 <i>C [</i><i>pg]</i> <i>≅</i> <i>0.216∙</i><i>V[</i><i>μ</i><i>m</i><sup><i>3</i></sup><i>]</i><sup><i>0.939</i></sup> M-D&L 0.145 <i>Nav</i> <i>Navicula pelliculosa</i> SAG /         100 <i>C [</i><i>pg]</i> <i>≅</i><i>0.288</i><i>∙V[</i><i>μ</i><i>m</i><sup><i>3</i></sup><i>]</i><sup><i>0.811</i></sup> M-D&L 0.121   <i>Coleps hirtus</i>   Cry, Nav         9,850 <i>C [</i><i>pg]</i><i>≅</i><i>0.14∙V[</i><i>μm</i><sup><i>3</i></sup><i> ]</i> P&S 0.14 <i>Col</i> 1 <i>Col</i> 2 <i>Col</i> 3 <i>C. hirtus</i> clone 1 <i>C. hirtus</i> clone 2 <i>C. hirtus</i> clone 3 Salzburg Stuttgart CCAP   17.6 ± 0.44 38.8 ± 0.51 1.7 10<sup>3</sup> 0.47 10<sup>3</sup>           18.6 ± 0.31 12.1 ± 1.89 1.79 10<sup>3</sup> 0.15 10<sup>3</sup>   17.0 ± 0.64 35.1 ± 4.98 1.64 10<sup>3</sup> 0.43 10<sup>3</sup> <i>Eup</i> <i>Euplotes octocarinatus</i> Pisa Cry 116 ± 5.4 -- 11.2 10<sup>3</sup> -- 26,890 <i>C [</i><i>pg]</i><i>≅</i><i>0.14∙V[</i><i>μm</i><sup><i>3</i></sup><i> ]</i> P&S 0.14   Bacteria             0.05 <i>C [</i><i>pg]</i> <i>≅</i>0.5·<i>0.435∙</i><i>V[</i><i>μ</i><i>m</i><sup><i>3</i></sup><i>]</i><sup><i>0.86</i></sup> L-K 0.33