Meta-population structure and the evolutionary transition to multicellularity

The evolutionary transition to multicellularity has occurred on numerous occasions, but transitions to complex life forms are rare. While the reasons are unclear, relevant factors include the intensity of within versus between group selection that are likely to shape the course of life cycle evolution. A highly structured environment eliminates the possibility of mixing between evolving lineages thus ensuring strong competition between groups. Less structure intensifies competition within groups decreasing opportunity for group-level evolution. Here we present experiments that contrast two ecological frameworks that differ in the way in which nascent multicellular groups, and their constituent cells, compete. Groups of the bacterium Pseudomonas fluorescens were propagated under a regime requiring reproduction via a life cycle with developmental and dispersal phases. By controlling the extent of mixing during the dispersal phase it was possible to alter the relative emphasis on the two phases. While all groups possessed ‘paradigmatic’ features of multicellular individuals (e.g., bottleneck and germ line), the mode of group interaction substantially affected the strength and direction of selection operating at both group and cell levels. Constraints on meta-population structure may therefore explain the observation that multicellular aggregates rarely complete the transition to individuality.


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Life is hierarchically structured. Multicellular organisms are comprised of cells, cells 50 contain organelles; the nucleus contains chromosomes comprised of genes (1, 2). The 51 origin of this structure reflects successive evolutionary transitions in individuality (ETIs) 52 with lower level particles being subsumed within higher-level self-replicating entities 53 (2). Central to each transition was the emergence of Darwinian properties at the new 54 Theoretical studies on the origins of cells provide clues (14, 15). Theoretical models 73 show that selection at the higher level can be achieved through specific population 74 structures: when replicators are individually isolated, selection remains focussed on the 75 lower level -that of the individual replicators (14). However, when replicators are 76 localized in reproducing compartments within a meta-population of compartments, the 77 focus of selection is the higher level (15, 16). We therefore hypothesize that a specific 78 population structure that embraces innovation in primordial life cycles while escaping the 79 from our previously published results (9) (Non-Mixed Ecology treatment; Figure 1a) -111 with an identical two-phase life cycle that incorporates competition (mixing) during the 112 propagule phase of emerging multicellular groups. This environmental manipulation, 113 which we term the Mixed Ecology treatment (Figure 1b), was performed simultaneously 114 with the earlier study. We show that competition effected during the propagule phase of a 115 two-stage life cycle leads selection to favour traits that promote cell growth at the 116 expense of traits underlying group fitness. This conflict between the two levels of 117 selection is due to a trade-off between traits underlying the fitness of groups and their 118 cells. While the existence of a germ line can bring about the decoupling of fitness 119 required to achieve a higher level of individuality, intense competition between cells 120 nevertheless skews selection towards traits that enhance the competitive ability of cells, 121 rather than towards traits that enhance the functioning of the life cycle as a whole. 122 123

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The evolution of simple groups has been observed in experiments using populations of P. 125 fluorescens propagated in spatially structured microcosms (17-20). Such groups 126 constitute a plausible starting position from which to explore transitions in Darwinian 127 individuality from cells to multicellular groups (7, 8, 18). In these experiments, 128 cooperative groups arise from single mutant "wrinkly spreader" (WS) cells that 129 overproduce a cell-cell glue; the failure of cells to separate at cell division leads to the 130 formation of mats that colonize the air-broth interface (19, 21-23). Glue production is 131 costly to individual cells, but the trait spreads because cells within the mat reap a reward 132 (access to oxygen) that is denied to free-living cells (24). Cooperating groups are 133 therefore inevitably short-lived (18, 25-29). Selection continues to act at the level of 134 individual cell by favouring mutant "smooth" (SM) types that cheat by no longer 135 producing glue, but nonetheless gain the advantages of being part of the group (18). 136

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The negative frequency-dependent selection described above creates a cycling between 138 cooperative WS cells and non-cooperative SM cells (10). When viewed from the long-139 term perspective of the group, this oscillation drives a rudimentary life cycle that confers 140 on the group the potential to reproduce by means of dispersing 'germ line' cells (8). The 141 group may thus evolve by natural selection (9). Each generation begins with WS cells 142 forming a mat at the air-liquid interface (Figure 1). For a mat to reproduce it must be both 143 viable and fecund, i.e., produce SM germ line cells (Phase I in Figure 1

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We have shown previously that this life cycle results in the 'decoupling' of fitness 1 153 between groups and their cells (9), however this alone does not guarantee the emergence 154 of individuality. In the present study we examine this life cycle in two ecological 155 scenarios in order to understand how the partitioning of variation (and therefore 156 competition and selection) can help or hinder the emergence of individuality. In the Non-157 Mixed Ecology treatment, competition between groups resulted from a death-birth 158 process: following an extinction event (usually due to the lack of SM production (9)), a 159 group was randomly replaced by a surviving competitor group. SM cells were harvested 160 separately from each surviving group at the end of the Maturation Phase (Phase I). 161 Extinction/replacement of groups occurred with high frequency (9) and therefore imposed 162 potent between-group selection. By contrast, competition between surviving groups in the 163 Mixed Ecology resulted from a mixing step: following Phase I extinction events all

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Trade-off between group and cell fitness 191

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In both ecologies, the life cycle began in Phase I with a single-celled bottleneck, and 217 therefore variation existed only between groups (regardless of population ecology) until 218 within-group variation emerged during maturation ( Figure 1). Phase II Non-Mixed 219 groups are also seeded with dispersal cells originating from single parent groups, 220 resulting again in high levels of between-group variation and relatively low levels of 221 within-group variation. This between-group variation led to competition and therefore 222 selection between groups, resulting in increased group fitness. A consequence of strong 223 group-level selection was a reduction in cell fitness due to the negative relationship 224 between cell and group fitness ( Figure 3). 225

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The direction of selection along this negative fitness spectrum was more complicated in 227 the Mixed Ecology. Phase II began with eight identical groups so variation, competition 228 and therefore selection occurred only within groups (Figure 1), leading to increased cell 229 fitness. The mixing step has consequences not only for the partitioning of variation but 230 also has downstream effects on the overall amount of variation. Competition within 231 groups (between cells) during Phase II likely resulted in the same single WS cell type 232

Ancestor
Non-Mixed Mixed ultimately seeding all eight groups in Phase I of the next generation. Consequently, the 233 limited between-group competition in Phase I did not overcome the considerable within-234 group selection towards the 'high cell fitness/low group fitness' corner of the fitness 235 spectrum during Phase II, because between-group variation is essentially removed during 236 Phase II of the previous generation. 237

Changes in life cycle parameters 239
To explore specific adaptations contributing to differences in fitness between the two 240 ecologies, we assayed parameters expected to be key to thriving in the multicellular life

Trade-off between WS-SM cell transition rate and WS density 302
A negative relationship (trade-off) exists between WS Density (which is linked to cell 303 fitness) and WS-SM transition rate (which is linked to group fitness) in the ancestral 304 population (r=-0.705, P=0.003, N=15; Figure 6, left panel). The nature of the association 305 between these two traits explains both the negative relationship between the two levels of 306 fitness observed above (Figure 3), and the opposing direction of selection in the two 307    Life cycles underpin evolutionary transitions in individuality (1, 7-9). The particular 349 mode by which the earliest multicellular groups acquired the capacity to reproduce has 350 implications for their ability to transition to groups that come to participate in the process 351 of evolution by natural selection in their own right ( Figure 8) (1, 5, 8, 10, 31). The

Experimental regime 517
We have previously published the Non-Mixed Ecology treatment in a study that 518 compared its effect relative to a life cycle without reproductive specialisation (9). Here 519 we compare the effect of meta-population structure on the potential for an ETI. were no WS colonies on the plate, the microcosm was deemed extinct. Figure 1 contrasts 541 the death-birth process of group competition in the Non-Mixed Ecology, with the 542 physical mixing mode of competition in the Mixed Ecology. 543 544

Fitness assay and life cycle parameters 545
Cell-level and group-level fitness were assayed after ten life cycle generations: 15 546 representative clones (one per replicate population) were generated from each of the 547 evolved treatments, in addition to 15 ancestral WS lines (each independently isolated 548 from the earliest mats to emerge from the ancestral SM strain SBW25) (as described in 549 detail in (9). Three replicate competition assays were performed for one group generation 550 against a neutrally marked ancestral competitor (46). 551 Our proxy for group-level fitness is the proportion of evolved 'offspring' mats relative to 552 the marked reference strain, and cell-level fitness the total number of cells in the mat after 553 Phase I. Density of WS and SM cells, and Proportion of SM cells were also assayed after 554 Phase I. The growth rate of SM cells was determined from three biological replicate SM 555 colonies per line (for details on how the SM were obtained, see (9)) in 96-well microtitre 556 plates shaken at 28°C, and absorbance (OD600) measured in a microplate reader 557 (BioTek) for 24h. The experiment was repeated three times and the maximum growth 558 rate (Vmax) was calculated from the maximum slope of absorbance over time. The 559 transition rate between WS and SM cells, i.e., the level of SM occurrence in Phase I, and 560 WS occurrence in Phase II, was determined in a separate experiment, where static 561 microcosms were individually inoculated with single colonies of the representative WS 562 types (Phase I). Phase I was extended from 6 to 12 days, and Phase II from 3 to 6 days. 563 At day six, SM cells were collected for Phase II, and microcosms inoculated. Each day, 564 three replicate microcosms per line were destructively harvested and the occurrence, i.e. 565 the microcosms with SM, and number of SM and WS colony forming units recorded. 566 567

Statistical analysis 568
For detecting differences in group-level fitness and transition rate between cells of the 569 evolved and ancestral lines, generalized linear models (error structure: binomial; link 570 function: logit) with the explanatory variables Ecology, and representative clone (nested 571 within Ecology) were calculated. Analyses of variance (ANOVA) were used to test for 572 differences in cell-level fitness, density of WS cells, and density, proportion, and growth 573 rate of SM cells between the evolved and ancestral lines. Explanatory variables were 574 Ecology, and representative clone (nested within Ecology). Posthoc tests revealed 575 differences between the evolved and ancestral lines. Relationships between the traits and 576 cell and group-level fitness were tested using the mean per representative type accounting 577 for regime. Pearson correlations and regressions were performed. The sample size was 578 chosen to maximise statistical power and ensure sufficient replication. Assumptions of 579 the tests, i.e., normality and equal distribution of variances, were visually evaluated. All 580 tests were two-tailed. Effects were considered significant at the level of P = 0.05. All