Material and Methods
Study organism and strainsParamecium caudatum is a freshwater ciliate with a world-wide distribution, feeding on bacteria and detritus. Asexual reproduction occurs by mitotic division and represents the main mode of population growth. Swimming is accomplished through the coordinated movement of ciliary bands on the cell surface (48). Previous work on P. caudatum indicated a genetic basis of dispersal propensity (49). Here, we used 20 Paramecium strains (i.e., clonal cultures derived from a single individual) from various geographic origins (49, 50) and representing different groups of COI genotypes (“mitochondrial haplotypes”; Table S1). All cultures were reared under standard laboratory conditions in lettuce medium with the food bacteriumSerratia marcescens at 23 °C, allowing up to 3 asexual doublings per day (51).
Founder strains measurementsPrior to the start of the long-term experiment, we assayed the 20 founder strains for dispersal and population growth characteristics (Table S1). For the dispersal assay, we placed aliquots of 8 mL of culture (at equilibrium density) in a 2-patch system (additional details below) and let the Paramecium disperse for 3h. Once connections were blocked, we estimated the number of residents and dispersers by taking 150-600 µL samples from the two tubes and counting the number of individuals under a dissecting microscope. Dispersal was taken as the proportion of dispersers of the total number of individuals in the system. Dispersal rates (gaussian posteriors) were then estimated using a generalized linear mixed model (binomial error distribution; glmer in “lme4” R package with bobyqa optimizer (52)) for each strain with time and observation (to account for overdispersion) as random effects. We tested 4 replicates per strain. For the growth assay, we placed ca. 200 individuals (from cultures at equilibrium density) in 20 mL of fresh medium. Over the course of 6 days, we tracked population density by counting the number of individuals in daily samples of 100-200 µL. We tested 3 replicates per strain. Using a Bayesian approach (53), we estimated the intrinsic population growth rate (r0) and equilibrium density (\(\overline{N}\)) for each replicate by fitting a Beverton-Holt population growth model to the time series data. Details of the Bayesian fitting are given in Supplementary Information (SI Appendix).
Evolutionary experiment The evolutionary experiment comprised a sequence of cycles, where dispersal events alternated with periods of population growth. The founder population was created by mixing the 20 strains at equal proportions in a single culture, which was then divided up into 15 replicate lines, assigned to the following three treatments. First, in the range front treatment (6 lines), we placed the Paramecium in one of the two tubes in 2-patch dispersal systems (interconnected 15-mL tubes, Fig. 1A). Connections were opened for 3 h, during which time individuals were allowed to swim to the other tube. We then collected the dispersers and cultured them for 1 week under permissive conditions in 20 mL of fresh medium (in 50-mL plastic tubes), until we initiated a new round of dispersal, again only retaining the dispersers and culturing them for 1 week, and so on. Second, the range core treatment (6 lines) followed the same cycles of dispersal and growth, but only the non-dispersing residents were retained after each dispersal episode. Third, in the control treatment (3 lines), residents and dispersers were mixed after each dispersal event and then cultured for 1 week, as in the other treatments. In corollary, the range front treatment mimics the advancing cohort of a spatially expanding population, whereas populations from the core treatment remain in place and constantly lose emigrants. The control treatment is similar to the core treatment, except for the loss of emigrants. This simple 2-patch scenario can capture essential processes occurring at range core and fronts (25), and produce results comparable to those observed in more complex continuous landscapes as shown in previous microcosm studies using freshwater ciliates (54, 55).
A total of 161 cycles were accomplished. Prior to each dispersal event, ca. 1800 individuals (median; 25% / 75% quantile range: 1400 / 2700) were placed in the dispersal systems. After dispersal, the number of individuals starting the 1-week growth period were matched between treatments. Because dispersal rates were low at the beginning, these starting numbers were initially set to 200 individuals (placed in a total volume of 20 mL of fresh medium). During the following 1-week growth period, stable population sizes were typically reached within 3-4 days, with densities of ca. 240 individuals per ml (median; 25% / 75% quantiles: 180 / 360). After cycle 32, when dispersal had already reached higher levels (see Results), we adjusted the starting numbers to ca. 1500 (median; 25% / 75% quantiles: 1100 / 2000).
Data collectionFor each line, dispersal was measured at each dispersal event and equilibrium densities (\(\overline{N}\)) taken at the end of the 1-week growth period at each cycle. Furthermore, growth rate (r0) was determined in assays conducted at cycle 21 (year 1), 78 (year 2) and 160 (year 3), as described above, with 2-3 replicates per line and year. Bayesian model fitting was used to estimate r0 (SI Appendix, Fig. S1). Measurement of swimming behaviour were also taken in the first two years of the experiment (SI Appendix, Fig. S2).