DOI: 10.5061/dryad.5dv41nsd3
title: Data from: In the social amoeba Dictyostelium discoideum, shortened stalks may limit obligate cheater success even when exploitable partners are available

description: Cooperation is widespread across life, but its existence can be threatened
 by exploitation. The rise of obligate social cheaters that are incapable
 of contributing to a necessary cooperative function can lead to the loss
 of that function.  In the social amoeba Dictyostelium discoideum,
 obligate social cheaters cannot form dead stalk cells and in chimeras
 instead form living spore cells. This gives them a competitive advantage
 within chimeras. However, obligate cheaters of this kind have thusfar not
 been found in nature, probably because they are often enough in clonal
 populations that they need to retain the ability to produce stalks.
  In this study we discovered an additional cost to obligate
 cheaters. Even when there are wild-type cells to parasitize, the chimeric
 fruiting bodies that result have shorter stalks and these are
 disadvantaged in spore dispersal.  The inability of obligate
 cheaters to form fruiting bodies when they are on their own combined with
 the lower functionality of fruiting bodies when they are not represent
 limits on obligate social cheating as a strategy.
description: <em>Strains and culture conditions </em>
 To prepare food bacteria for <em>D. discoideum
 </em>clones to prey upon, we first spread non-pathogenic
 <em>K. pneumoniae</em> KpGe (Dicty Stock Center,
 dictybase.org) from stocks frozen in 80% KK2 [2.25 g
 KH<sub>2</sub>PO<sub>4</sub> (Sigma-Aldrich) and
 0.67 g K<sub>2</sub>HPO<sub>4</sub> (Fisher
 Scientific) per liter] and 20% glycerol on an SM/5 agar media [2 g glucose
 (Fisher Scientific), 2 g yeast extract (Oxoid), 0.2 g
 MgCl<sub>2</sub> (Fisher Scientific), 1.9 g
 KHPO<sub>4</sub> (Sigma-Aldrich), 1 g
 K<sub>2</sub>HPO<sub>5</sub> (Fisher Scientific),
 and 15 g agar (Fisher Scientific) per liter] and allowed the bacteria to
 grow at room temperature until colonies appeared. We picked a single
 colony with a sterile loop, spread it on a new SM/5 plate, and allowed the
 bacteria to proliferate. We collected these bacteria into KK2 with a
 sterile loop and diluted them to 1.5 OD<sub>600</sub> in KK2
 (~5 x 10<sup>8</sup> cells, measured with an Eppendorf
 BioPhotometer). We used these bacteria as food for amoebas in our
 experiment and repeated this process anew for each of the three replicate
 experiments. To grow NC28.1, the wild-type ancestor
 <em>D. discoideum</em> clone, from freezer stocks for use in
 our experiments we added spores frozen in 80% KK2 and 20% glycerol to
 200ml of 1.5 OD<sub>600</sub> <em>K.
 pneumoniae</em> suspension. We spread the mix of spores and bacteria
 on SM/5 plates with a sterile glass spreader, then incubated the plates at
 room temperature for 7 days under constant overhead light until the social
 cycle was complete and fruiting bodies had formed. We repeated this
 process for each of the three replicate experiments.
 EC2 (also called EC28.2 in (Inglis, Ryu et al. 2017)) was
 selected as an obligate cheater.  It is the result of an experimental
 evolution experiment that used unstructured growth and dispersal to evolve
 a cooperative wild strain called NC28.1 into non-fruiting cheaters.  Past
 work measuring the degree of cheating (the overrepresentation of spores
 within chimeric fruiting bodies) suggests that EC2 is a particularly
 effective cheater, and its recent divergence from a wild isolate should
 render its behavior more natural than cheaters derived from extensively
 lab-adapted strains.  To grow EC2, the RFP-labelled obligate social
 cheater, from freezer stocks for use in our experiments, we added amoebas
 frozen in HL5 (5 g proteose peptone, 5 g thiotone E peptone, 10 g glucose,
 5 g yeast extract, 0.35 g Na2HPO4 * 7H2O, 0.35 g KH2PO4 per liter) to 10%
 DMSO to 200ml of 1.5 OD<sub>600</sub> <em>K.
 pneumoniae</em> suspension. We spread the mix of amoebas and
 bacteria on an SM/5 plate with a sterile glass spreader, then incubated
 the plate at room temperature for 24-48 hours until starving EC2 amoebas
 began aggregating. We then used a sterile loop to transfer a sample to a
 new plate containing fresh <em>K. pneumoniae</em> for them to
 prey upon. These were allowed to grow for 24-48 hours until a vegetative
 front of amoebas had formed. We collected these amoebas with a sterile
 loop into ice-cold KK2 (see “Experimental procedures”) and ensured that
 the amoebas we used were clonal by plating 10 SM/5 plates with about 10
 amoebas each, then picking a single clonal plaque originating from a
 single amoeba. We repeated this process for each of the three replicate
 experiments. <em>Experimental
 procedures</em> In order to obtain cells of
 both <em>D. discoideum </em>clones for experimental mixing, we
 plated amoebas (EC2) or spores (NC28.1) previously grown from freezer
 stocks as described above on separate SM/5 agar plates with 200ml of 1.5
 OD<sub>600</sub> <em>K. pneumoniae
 </em>suspension. We collected amoebas to make the
 mixtures by pouring ice-cold KK2 onto the plates, mixing them into
 suspension with a gloved fingertip, then collecting and centrifuging the
 mixture at 10°C for 3 minutes at 1300 rpm in order to pellet the amoebas
 and leave <em>K. pneumoniae</em> in solution. We decanted the
 pellets, resuspended them in KK2, and measured their density with a
 hemacytometer before making the mixtures. For each treatment, we mixed
 200ml of fresh <em>K. pneumoniae</em> suspension with a total
 of 2x10<sup>5</sup> amoebas then spread the solution evenly
 with an ethanol-sterilized glass spreader on an SM/5 agar plate. We made
 mixtures of EC2 and NC28.1 with various initial frequencies of EC2 (0.0,
 0.1, 0.3, 0.5, 0.7, 0.9, and 1.0). We repeated this experiment three
 times, each on a separate day. We collected fruiting
 bodies after one week at room temperature under constant overhead light to
 allow fruiting bodies to fully develop. On each plate, we selected three
 fruiting bodies at random to represent three independent data points. To
 do this, we placed a plate of fruiting bodies over a grid of 1cm by 1cm
 squares. We selected three squares at random using a random number
 generator and marked each plate at the centers of each of the squares. We
 then individually collected the closest intact (not collapsed) fruiting
 body to each mark with fine tweezers. For each, we pressed the sorus,
 which contains the spores, against the side of a tube containing 100ml of
 KK2 to dislodge the spores, then laid the stalk on a glass microscope
 slide. After three fruiting bodies were collected from a single plate, the
 stalks were covered with a cover slip and sealed with nail polish for
 later imaging. Stalk length was individually recorded by imaging picked
 stalks under a Leica S8AP0 dissecting microscope with Leica application
 suite software v4.1 using the “draw line” tool.
 <strong> </strong><em>Analysis</em> We excluded several data points from the analysis for which we could not accurately measure stalk height due to damage incurred during collection. In order to test whether increasing cheater frequency yields shorter fruiting bodies, we used a linear mixed-effects model with the function lme<em> </em>in the nlme package in R version 4.2.1 (Team 2013) with stalk height as the response variable, the initial cheater frequency as a fixed effect, the total number of spores per sorus as a fixed effect, and the day of the experiment as a random effect (stalk height ~ initial cheater frequency + total spores + 1|day).  In this model, we included total number of spores as a fixed effect in case fruiting body height could vary due to random variation in the size of aggregates that form across the plate.  We then compared this model with one lacking the random effect of day (stalk height ~ initial cheater frequency + total spores) using the <em>anova</em> function in base R. The two models were not significantly different (p = 0.27, with day: AIC = 105.67, without day: AIC = 104.89), so we proceeded with the simpler model without the effect of day. We then further simplified the model by removing the effect of total number of spores because it did not significantly affect stalk height (p = 0.20).
description: # Data from: In the social amoeba Dictyostelium discoideum, shortened
 stalks may limit obligate cheater success even when exploitable partners
 are available
 [https://doi.org/10.5061/dryad.5dv41nsd3](https://doi.org/10.5061/dryad.5dv41nsd3) These data represent an attempt to look for effects of the presence of obligate cheaters in Dictyostelium discoideum chimeras on resulting fruiting body stalk lengths. A cooperative strain (NC28.1) and an obligate cheater descendant (EC2) were combined in various proportions, allowed to fruit, and the resulting fruiting bodies were measured using microsophy. ## Description of the data and file structure Filename: Medina2024datafinal.csv Short description: Heights of fruiting bodies **Column names:** sampleDescription - Factor - a unique name for each sample dateCollected - Factor - date of data collection stalk_length - Number - length (in mm) of fruiting body stalks, measured via light microscopy day - Factor - Each experiment was performed on 3 different days, representing 3 replicates. This column defines whether they are replicate 'A', 'B', or 'C'. initial_cheater_proportion - Factor - Fruiting bodies developed from chimeras created at different known proportions of the cooperative stran NC28.1 and the cheater strain EC2. This column represents the proportion of EC2 in the mix. replicate - Factor - Each experiment involved measuring 3 different randomly selected fruiting bodies