Erosion of functional independence early in the evolution of a microbial mutualism

Abstract

Many species have evolved to function as specialized mutualists, often to the detriment of their ability to survive independently. However, there are few, if any, well-controlled observations of the evolutionary processes underlying the genesis of new mutualisms. Here, we show that within the first 1,000 generations of initiating independent syntrophic interactions between a sulfate reducer (Desulfovibrio vulgaris) and a hydrogenotrophic methanogen (Methanococcus maripaludis), D. vulgaris frequently lost the capacity to grow by sulfate respiration, thus losing the primary physiological attribute of the genus. The loss of sulfate respiration was a consequence of mutations in one or more of three key genes in the pathway for sulfate respiration, required for sulfate activation (sat) and sulfate reduction to sulfite (apsA or apsB). Because loss-of-function mutations arose rapidly and independently in replicated experiments, and because these mutations were correlated with enhanced growth rate and productivity, gene loss could be attributed to natural selection, even though these mutations should significantly restrict the independence of the evolved D. vulgaris. Together, these data present an empirical demonstration that specialization for a mutualistic interaction can evolve by natural selection shortly after its origin. They also demonstrate that a sulfate-reducing bacterium can readily evolve to become a specialized syntroph, a situation that may have often occurred in nature.

Keywords: coevolution; experimental evolution; sulfate-reducing prokaryote; syntrophy; trade-offs.

Fig. 1.

Average growth rate (A) and yield (B) of ancestral cocultures (D_AM_A; blue dots), cocultures where only D. vulgaris (D_EM_A; mustard triangles) or M. maripaludis (D_AM_E; orange triangles) was 1,000-generation evolved, and cocultures containing both evolved populations (D_EM_E; green squares). Error bars indicate the SD of four measurements.

Fig. 2.

(A) Enzymes in the sulfate reduction pathway. APS is adenosine 5′-phosphosulfate. (B) Occurrence, frequency, and predicted effects of mutations in these genes after 1,000 generations of evolution in syntrophy. Height of bars indicates the frequency of the allele within the population. The predicted effect of the mutation on the amino acid sequence or functionality of the protein is indicated by color. Predicted loss-of-function mutations (red) include both premature stop codons and frameshift mutations. Missense mutations are blue and synonymous mutations are tan.

Fig. 3.

Growth rate (A) and yield (B) of cocultures of D. vulgaris strains (aps, qmo, sat, JW710, DA, D+, D−) paired with each of three M. maripaludis populations. Results for pairings containing M+ (blue diamonds), M− (red squares), and MA (green triangles) are shown. Asterisks (*) or X (colored according to the M. maripaludis strain) indicate a significant difference (after sequential Bonferroni correction, P < 0.05 per panel for all comparisons against appropriate control) between the mean for that coculture and a control coculture in the full ANOVA model (*) or a submodel (x) containing only data with open symbols. The control coculture for D+ and D− cocultures were DA cocultures (solid symbols). The control coculture for mutants was JW710 (open symbols). M+ and D+ are evolved partners from coculture H2 dilutions that have retained the ability to respire sulfate; M− and D− from H2 dilutions that have lost that capability. MA and DA refer to ancestral strains. Error bars indicate 95% confidence interval.

Fig. 4.

Comparison of cocultures with sulfate reduction (SR)-negative and SR-positive phenotypes. Each point is the average growth rate (A) or yield (B) of three replicates of an evolved coculture (solid squares) or an EPD coculture that was derived from evolved coculture U9 (green), H6 (aqua), or H2 (purple) and had either a SR-positive (+ symbol) or SR-negative (− symbol) phenotype. Error bars indicate 95% confidence intervals calculated from the ANOVA (see SI Methods and Table S5), and asterisks (*) indicate a significant difference between the EPD and the evolved coculture from which it was derived after a sequential Bonferroni correction (P < 0.05 for all 11 tests in the panel).