Mutualistic Relationships are critical for the stability of ecosystems but their mechanisms are poorly understood. Local adaptation is an indication of how specialized the organisms are to their mutualistic partner. Most experiments that study locally adapted species focus on host-parasite and trophic interactions, however, we focus on two species, Desulfovibrio vulgaris and Methanococcus maripaludis, existing in a mutualistic relationship. We used 6 independently evolved populations. The individual species in the co-evolved populations were separated and paired with a partner from a different evolved population to see if they grew faster with the partner they evolved with. We tested for local adaptation by frequently measuring the optical density of the cocultures. The OD measurements were used to calculate growth curves. The growth curves were compared to determine if the co-cultures were locally adapted. Our results indicated that neither species was locally adapted.

Sulfate-reducing bacteria are widespread and live in many environments. Some sulfate reducers are capable of growing in a mutualistic relationship with methanogens. In nature, diverse sulfate-reducing bacteria have repeatedly evolved into obligate mutualists. We want to study how this evolution occurred by using a model system for evolution in the lab between a sulfate reducer, Desulfovibrio vulgaris, and a methanogen, Methanococcus maripaludis. After allowing these two organisms to evolve for 1000 generations in an environment that forces their cooperation, 12 out of 22 D. vulgaris populations lost the ability to reduce sulfate (sulfate-minus genotypes). We want to test whether the ratio of sulfate-minus relative to sulfate-plus (a genotype still able to grow on sulfate) genotypes in coculture affects the fitness benefit of the sulfate-minus genotypes. To do this, a sulfate-minus and plus D. vulgaris were placed together and their relative abundance in coculture was tracked over time. The experiment will tell us if some genotypes have the ability to take over when they are rare. To track these changes, we are using a method called FREQ-Seq. FREQ-Seq is an inexpensive way of analyzing the frequency of certain genes or other DNA sequences in a mixed population over time. We can use this method to understand how these populations of D. vulgaris evolved. I originally predicted there would be a higher sulfate-minus to plus ratio at the end of the competition compared to the initial ratio. D. vulgaris is an interesting model organism for studying the evolution of obligate sulfate-reducer/methanogen mutualisms because it can live either alone as a sulfate-reducer or together with other organisms in a syntrophic relationship. The data we collected can help us better understand the mechanisms of natural selection that cause sulfate reducing populations to become obligate mutualists.

Climate change is a crucial factor that affects plant physiology, population dynamics and geographic ranges. Little is known about the effects of changing climate on conifer morphology, whether their key morphological structures have the ability to change in response to environmental differences and what the impact of these changes mean for future ecological population dynamics. Are morphological and physiological traits locally adapted within a species or do they acclimatize to specific environmental conditions? Two studies were conducted to test 1) whether morphological structures of conifers differ between source populations when transplanted across an elevation gradient and 2) whether photosynthetic levels under a light treatment differ between source populations. Tsuga mertensiana seedlings from three study sites, varying from high to low elevation on Mt. Rainier, were each transplanted across and beyond the species' elevation range. For the first study, measurements of shoot to root ratio, specific leaf area (SLA) (m²/g) and leaf mass vs. stem mass were calculated. We expect our results to show that as elevation increases, SLA and shoot to root ratio decrease, however overall values are smaller for trees at higher elevation. This indicates that within a single species, trees from distinctive ecotypes are inherently different morphologically, even though all seedlings have the ability to acclimate to changes in elevation. The second study manipulates light as a single variable and addresses whether the driving force of morphological and physiological characteristics are due to light levels at different elevations or whether T. mertensiana seedlings at different ecotypes are genetically different physiologically.

The focus of this research is on p120 catenin, an essential protein in the development of vertebrate embryos. p120 catenin is associated with the regulation of cell migration during development, which includes cells migrating towards the dorsal axis. The mesodermal cells that migrate towards the dorsal axis will become the notochord and somites. We hypothesize that p120 catenin regulates this movement through its association with cadherins, and its activation of the actin cytoskeleton. p120 catenin binds the cytoplasmic tail of E-cadherin to stabilizes and strengthen adhesion and in the cytosol regulates small Rho GTPases to mediate actin dynamics. The regulatory domain of p120 catenin contains tyrosine and serine/threonine residues that are phosphorylated. We are modifying these sites to amino acid residues that lack the ability to be phosphorylated, phenylalanine or alanine, or are constitutively charged, glutamic acid. A splice site antisense morpholino injected into single celled zebrafish embryos knocks down the expression p120 catenin proteins, which can then be rescued by co-injection of wild-type p120 catenin mRNA. The modified p120 catenin mRNAs are co-injected with splice site RNA morpholino and the embryos’ development is then compared to those embryos that were rescued with wild-type p120 catenin. Thus we are studying the phosphorylation sites essential in the cell migration process. Data from the tyrosine 228 to non-charged phenylalanine mutation rescues the normal phenotype. This suggests phosphorylation of the tyrosine at the 228 site is not required for cell motility. Whether serines 268 and 288 are required for cellular motility is not yet clear. Other phosphorylation sites are currently being studied for their roles in cell adhesion and migration. This research is significant for developmental biology as well as cancer biology and wound healing.

During zebrafish gastrulation, presumptive mesodermal cells internalize beneath the ectodermal cells and migrate toward the dorsal axis to form the notochord and the presomitic mesoderm. Our research focuses on how p120 catenin is involved in the pathways of cell adhesion and migration during zebrafish morphogenesis. p120 catenin is a regulatory molecule which has dual functions. The first is to stabilize cell-cell adhesion by binding to the intracellular and juxtamembrane region of cadherins at the cell membrane which form adherens junctions with other cells within a tissue, holding them together. The second role of p120 catenin is to function upstream of CDC42 and Rac1 GTPases in the cytosol to initiate cell motility by stimulating the formation of lamellipodia and filopodia. Both functions are greatly regulated by p120 catenin phosphorylation. The hypothesis is that tyrosine phosphorylation in p120 catenin mediates its binding to cadherins while serine/threonine phosphorylation leads to activation of CDC42 and Rac1 and thus, cell migration. Our general approach is to apply immunoprecipitation of embryo lysates to determine which proteins interact with p120 catenin when it is phosphorylated on tyrosine or serine/threonine residues. Next we will inject mutated forms of p120 catenin into the embryos and determine how that changes protein interactions. So far, we have been testing different mammalian commercial antibodies on normal embryo lysates to determine which ones work with zebrafish. This research could have important implications on treatments of some cancers and wound healing.