Figure 1.
Experimental design for the evolution experiments.
(i) Stocks of wild isolates were grown up, each comprising a single starting genotype of each species. (ii) Experiments were started with each species in monoculture or in polyculture (all five species mixed together). (iii) To stimulate active growth and promote adaptation to the laboratory conditions, each culture was diluted 20-fold in fresh medium twice weekly for 8 wk. Tubes were shaken to prevent the formation of biofilms and maintain spatial homogeneity. Numbers of generations ranged from 60.9 to 82.2 across cultures and effective population sizes ranged from 5.3×105 to 9.9×106 (Table S3). (iv) Final cultures were plated on agar. (v) Single colonies of each species were isolated for growth assays described in the main text.
Figure 2.
Maximum growth rates of isolates after evolution under each diversity treatment.
Maximum rate of growth from low densities, VMAX, of each species grown on unused beech tea under assay conditions. Dark bars, growth rates of ancestral isolates. Mid grey bars, growth rates of monoculture isolates. Pale bars, growth rates of polyculture isolates. Standard error bars are shown. Tukey Honest Significant Difference test contrasts between treatments: *** p<0.001, ** p<0.01, * p<0.05; n.s., not significant (see also Table S4). Species A evolved slower maximum growth rates in polycultures compared to its ancestral and monoculture isolates. Species B and C evolved faster maximum growth rates on unused beech tea in monocultures, but far slower maximum growth rates in polycultures compared to ancestral isolates. Species D evolved faster maximum growth rates in monocultures compared to its ancestral isolate and even faster maximum growth rates in polycultures.
Figure 3.
The interspecific impacts of resource use on relative growth.
Interspecific effect on relative growth among species inferred from their ability to grow on sterile beech tea previously used by each other species, shown separately for each treatment. Blue arrows indicate negative effects on growth, and red arrows indicate positive effects on growth. The width of the arrow represents the maximum growth rate (VMAX) on used tea minus the maximum growth rate on unused tea (underlying data in Figure S1 and linear model in Table S4). Dashed lines indicate that growth on used tea was not significantly different from growth on unused tea.
Figure 4.
Trajectories of evolution in monoculture (solid black arrows) and polyculture (grey dashed arrows) of each species with respect to the first two principal components summarizing variation in their ability to use and produce compounds identified by NMR. The start of each arrow indicates the position of the ancestral isolates along these axes. Increasing PC1 is correlated with using more glucose, choline, formate and succinate, and producing more pyruvate (Figure S4). Increasing PC2 is correlated with using more acetate, formate, and producing more lactate. Species resource use evolved more in polyculture than in monoculture (dashed grey arrows are longer than solid black ones), and polyculture isolates display greater divergence in resource use and production than either ancestral or monoculture isolates (dashed grey arrows point towards the four corners of the plot).
Figure 5.
Correspondence between compounds being generated and compounds being used up by other species in polycultures.
The data summarize results from assays growing one species on beech tea medium, filtering that medium, and then growing a second species on the used medium. We calculated two quantities: δ0,1 = the amount of compound in the filtrate from species 1 minus the amount of compound in beech tea (relative to the amount of the DSS standard); δ1,2 = the amount of compound in the filtrate from species 2 minus the amount of compound in filtrate from species 1. Positive δ indicates production of compounds during the assay and negative δ indicates consumption. We then compared δ between evolved and ancestral isolates for different species pairs: each point shows the comparison for a given species pair and either monoculture (black circles) or polyculture (grey crosses) treatments. The x-axis is δ0,1 of the evolved isolate minus δ0,1 of the corresponding ancestral isolate. More positive values indicate that the evolved isolate of the first species produced more of that compound than did its ancestral isolate. To focus on waste products as potential targets of cross-feeding, only compounds that were produced by the evolved isolate were included. The y-axis is δ1,2 for evolved isolate minus δ1,2 for the corresponding ancestral isolate. More negative values indicate that the evolved isolate of the second species used more of the compound than did its ancestral isolate. For example, the point indicated by the arrow represents increased production of acetate by species A in polyculture relative to ancestral isolates (x-axis) and its increased use by species D in polyculture relative to ancestral isolates (y-axis, all changes shown separately by species and compound in Figure S5). There is a general negative trend: if the first species produces more of a compound, the second species is likely to use more of it. However, the effect is significantly stronger in polyculture isolates (grey dashed line) than in monocultures (black line): polyculture isolates have evolved increased consumption of compounds that have increased in production in polyculture isolates of other species. Linear model of y = x * treatment (monoculture or polyculture), interaction term coefficient = −1.13, t = −5.4, p<0.0001.
Figure 6.
The effect of diversity during evolution on ecosystem function of reassembled communities.
The mean rate of CO2 release over a 6-h period by communities reassembled from isolates that evolved under the two diversity treatments. Standard errors are shown. Tukey Honest Significant Difference test contrasts between interaction types: ** p<0.01.