Do species evolve in diverse communities, similar to the simple community of our research? We are looking at the evolution that we've seen in a species of sulfate reducers found in our simple laboratory environment and comparing this evolution to that of a large, more complex anaerobic digester of the wastewater treatment plant. In our 'simple community', a methanogen, Methanoccocus maripaludis, and a sulfate reducer, Desulfovibrio vulgaris, were paired. When two species are forced to live together over time we expect to see evolution take place. It was found that after 1000 generations D. vulgaris loses its ability to reduce sulfate in this paired relationship. Using this laboratory pairing as a model, we have looked at the evolution of sulfate reducers in  these two different anaerobic digesters from the University of Washington and Brightwater Municipal Waste Water Treatment Center. Both digesters have similar conditions; they are both anaerobic with low levels of sulfate present. To test our hypothesis we have currently been extracting DNA from weekly samples of both digesters. The dsrAB gene is first amplified, as it is a conserved region throughout sulfate reducers and their relatives. We are now doing Illumina sequencing in order to better determine the diversity of sulfate reducers present. Using this information, we will be able to design primers for the sat apsA and apsB genes. This will show us if any mutations are present among the population of sulfate reducers in both digesters. This will be means for the comparison between real world and experimental conditions. Future work will look for mutations in the sat, apsA and apsB genes. By identifying these loss-of-function mutations in both the digesters, we will be able to see the impact of evolution on sulfate reducers in complex communities.

When isolated subpopulations adapt to the particular features of the predators, prey, or cooperative partners that are specific to their local environment, populations may become specialized to their local partner and less able to interact with other populations. To what extent might this hypothesis explain the diversification of microbial species, which cooperate or compete with each other in complex communities? We tested whether specialization was a common outcome of evolution between the bacterium Desulfovibrio vulgaris and the archaeon Methanococcus maripaludis after they evolved together in a beneficial interaction for 1000 generations. To test if D. vulgaris populations tended to become specialized for the M. maripaludis populations they evolved with (and vice versa), we revived 6 independently-evolved communities, separating the D. vulgaris and M. maripaludis populations from one another, and then paired each population back up with its sympatric partner (evolved in the same community) or with 5 allopatrically-evolved partners (evolved in different communities). We used microscopy to quantify the density of D. vulgaris and M. maripaludis to test whether the absolute fitness of either species was higher in sympatric versus allopatric pairings. We also competed allopatrically-evolved and sympatrically-evolved D. vulgaris against one another. We conclude that variation across cocultures in population yield of D. vulgaris or M. maripaludis is not necessarily correlated with whether they evolved in sympatry or allopatry, suggesting that the first 1000 generations of adaptation to a new mutualism may not be characterized by specialization. Our results show that some sympatric pairings were more efficient than allopatric pairings;however no distinct trend was identified. Broader implications of our research include roles in  byproduct inhibition which is an important factor in global carbon cycling within diverse anaerobic environments, including bio-digestion for fuels, wastewater treatment, and experimental evolution. 

There are many mutualistic interactions that occur naturally in the world. Some of these mutualistic relationships evolve to be obligate, where the presence of one species is dependent on the other for its survival. We study the evolution of an obligate relationship with two model organisms, a sulfate reducer, Desulfovibrio vulgaris, and a methanogen, Methanococcus maripaludis. 22 populations of these organisms have evolved in an environment that has forced their cooperation. When we sequenced these populations, we discovered that at least 13 of the 22 populations of D. vulgaris have lost the ability to reduce sulfate To confirm if these mutations are beneficial or not, we measured the fitness of mutants with knockouts in the sulfate-reducing gene pathway relative to the wild-type strain with either an ancestral or evolved M. maripaludis partner. The final population size of each D. vulgaris mutant will be measured using FREQ-Seq; a technique that quantifies frequencies of various mutations in an amplicon. In previous iterations of this experiment, results presented a discrepancy between replicates (I and II) due to a failed cell lysis technique. We found that only a small fraction of the cells were lysed. With a better cell lysis protocol, we expect to see an increase in D. vulgaris mutant frequencies when paired with either an ancestor or evolved M. maripaludis compared to the wild-type. The data collected from these experiments will help us have a better understanding whether losing the ability to reduce sulfate is a beneficial mutation for D. vulgaris or not.