| 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.From flowering plants and their pollinators to the microbial endosymbionts of insects, there are many examples in nature of obligate mutualists (1, 2), or species dependent upon a mutually beneficial interaction for their survival or reproduction. How these interactions evolve is a mystery because much theory predicts that cooperative interactions should be unstable (3) and because of the difficulty of inferring evolutionary events that occurred in the distant past (4). Although there are few, if any, empirical observations of evolution toward dependence on mutualism, there are now several examples of mutualisms evolving de novo in the laboratory (5–8). This advancement has provided researchers an experimental framework to study populations and ecological conditions in the early stages of evolution (5–8).Here, we describe our observations of rapid and repeated evolution of increased dependency on a mutualism through natural selection. This interaction is similar to a widespread relationship between prokaryotes that plays a pivotal role in the decomposition of carbon in many oxygen-free environments. In these syntrophic mutualisms, bacteria ferment organic acids, producing hydrogen or formate as by-products, which are then used by hydrogen-consuming species, often methanogenic archaea (9). Removal of hydrogen and formate benefits the bacteria because the free energy (ΔG) available decreases with increasing concentrations of these products (9). A variety of bacterial species have been described that seem to be specialized for fermenting organic acids in syntrophic association with hydrogen-consuming species (10–13). Notably, most clades of characterized syntrophs share a recent common ancestry with sulfate reducers (10, 14). Some retain vestiges of the sulfate-reducing pathway, and several lines of evidence hint at the possibility that specialized syntrophs were once sulfate reducers (11, 14).Sulfate-reducing bacteria gain energy from organic acids, such as lactate, in the absence of oxygen by coupling their oxidation to the reduction of sulfate to sulfide. These bacteria play a critical role in sulfur and carbon cycling, contribute to corrosion in the petroleum industry and wastewater treatment plants, and have been used for bioremediation of toxic heavy metals (15). The ability of sulfate reducers to grow in syntrophic association with methanogens was first demonstrated in laboratory studies (16) and is now generally recognized to be of environmental relevance (17–19). Many sulfate reducers would therefore be better described as facultative syntrophs, well adapted to environments of fluctuating electron acceptor availability (17, 20). Past evolutionary transitions of sulfate-reducing bacteria between obligate and facultative syntrophs is also indicated by comparative analyses indicating horizontal transfer of genes in the pathway of sulfate respiration (21, 22). Thus, the evolutionary adaptive flexibility of sulfate-reducing bacteria suggests that they offer an attractive experimental system to study the evolution of mutualism.To understand how mutualisms, and syntrophic interactions in particular, might evolve from their origin, we paired the sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, with the archaeon, Methanococcus maripaludis S2, and propagated 22 initially isogenic planktonic cocultures for 1,000 generations in medium with lactate but no sulfate or added hydrogen. In this environment, neither species can gain energy from the oxidation of lactate without syntrophic cooperation.Within the first 300 generations of evolution, the cocultures evolved increased stability, higher yields, and higher growth rates, with both species contributing to these changes (6), a trend that continued through 1,000 generations. We describe a common evolutionary outcome of these experiments. Many of the independently evolved D. vulgaris accumulated loss-of-function mutations in genes required for the reduction of sulfate, suggesting strong selection for mutations resulting in loss of the ability to respire sulfate during evolution in syntrophy. |
