Our research is dedicated to understanding the link between thermostability and genetic robustness. Genetic robustness is defined as the ability to accumulate mutations without a change in phenotype. We evolved the bacterial virus φ6 for thermostability by doing 5 minute heat shock treatments over 100 generations. We then sequenced the evolved strains and compared these sequences to the ancestor’s sequence. The majority of my participation in the research was dedicated to identifying single mutations which we thought could confer thermostability in the virus, then reverse engineering these mutations into new phage particles, and finally evaluating whether increased thermostability was achieved. Future work in this project will test whether thermostable mutants are genetically robust. To determine genetic robustness, we will engineer new mutations into both the ancestor and the mutants with similar amounts of random mutations, and test survivability in both. If the genetically engineered thermostable viruses have a higher survivability when hit with random mutations, they will be said to be more genetically robust than the ancestor. The goal of this project is to try and establish a mechanism by which genetic robustness might evolve, as it is not yet clear in the literature what would be selected for for robustness to evolve. The project also has big picture implications in understanding how viruses might evolve in response to treatments.

Gene duplication provides raw material for evolution, allowing duplicate genes to change functions. However, relatively few detailed case studies have shown how duplicate genes contribute to evolutionary change. Among several species of Chilean Mimulus (monkeyflowers), gene duplication may underlie the repeated gain in petal anthocyanin pigmentation. Previous work implicates a set of duplicated Myb genes as top candidates for causing pigmentation gain in at least two Mimulus species. We hypothesized that increased Myb gene expression is responsible for anthocyanin pigmentation. To test this hypothesis, we compared the expression of two candidate genes, Myb 4 and Myb 5, in the highly pigmented Mimulus luteus var. variegatus and in the sparsely pigmented M. l. luteus. We predicted if expression changes in either candidate gene were responsible for controlling anthocyanin pigmentation, then gene expression would be correlated with pigmentation intensity in tissues with more anthocyanin pigments. RNA was extracted from leaf, outer petal, and floral nectar guide tissues from inbred lines of M. l. variegatus and M. l. luteus. The RNA was converted to cDNA and tested using PCR for the presence of unwanted secondary products. Quantitative PCR was used to compare gene expression levels among these tissues. Data were analyzed with ANOVAs in SPSS. Results from PCR indicated no unwanted secondary products. Quantitative PCR showed that higher Myb 5 gene expression was correlated with increased anthocyanin pigmentation, while basal levels of Myb 4 gene expression indicate Myb 4 may be nonfunctional. Within the larger context of evolution, these findings support a growing body of evidence that changes in gene expression can be a major driver in evolution. This provides an alternative to the classical evolutionary paradigm, which holds that changes to amino acid sequences largely drive evolution.

Heterotypic cooperation, an interaction where two types of organisms promote each other’s fitness at a cost, is widely observed in nature. For example, legumes feed metabolites to rhizobia and are reciprocated with energetically-expensive fixed nitrogen. It is unclear why many forms of heterotypic cooperation exist, given that individuals who increase their consumption of benefits from partners and decrease their production of costly benefits will have a competitive advantage. Furthermore, extant heterotypic cooperation could have evolved over millions of years, making it difficult to retrace their evolutionary trajectories. Here, we used an engineered microbial system to examine how incipient heterotypic cooperation could evolve. The system is composed of two reproductively-isolated Saccharomyces cerevisiae strains: a red-fluorescent strain that requires lysine and releases adenine and a green-fluorescent strain that requires adenine and releases lysine. The ancestral coculture is viable, able to grow from low to high density in the absence of adenine and lysine supplements, only if the initial total cell density exceeds a minimal “viability threshold.” All cocultures rapidly evolved to improve viability by reducing the viability threshold. Furthermore, the evolved lysine auxotrophic cooperator was sufficient for this viability improvement. Deep sequencing of these evolved cooperators revealed duplication of the yeast chromosome 14. Sufficiency experiments revealed that this mutation promoted the “self-serving” trait of improved growth under the lysine-limited coculture environment. Moreover, this duplication event generated the “partner-serving” phenotype of increasing the rate of adenine release to the environment. Both these phenotypes also arose in lysine auxotrophic cooperators evolved as monocultures in lysine-limited chemostats. Thus pleiotropy, the control of multiple phenotypes by a single genetic element, can promote incipient heterotypic cooperation by creating “win-win” phenotypes.

The Pleistocene ice-ages profoundly influenced the abundances and distributions of boreal gadid fishes in both the north Atlantic and Pacific Oceans. Although the evolutionary selection pressures underlying these demographic processes are not known, DNA sequence analyses of the nuclear pantophysin (Pan I) gene in two sister taxa, walleye pollock (Gadus chalcogrammus) and Atlantic cod (G. morhua), show evidence of positive Darwinian selection, characterized by selective sweeps of more recently derived Pan I lineages in contemporary populations. The distribution of Pan I lineages is correlated with water temperature in both species, suggesting that adaptive responses to temperature played a role in their historical demographies. Here we analyze variation at the Pan I locus in Pacific cod, G. macrocephalus, using maximum-likelihood methods to examine the evidence for positive selection in this genus and phylogenetic congruency of gene and species trees for these three ecologically and commercially important species.

Organochlorines (OCs) bind to fats and persist in the environment. These persistent organic pollutants (POPs) biomagnify up the food chain and can be transferred from females to their offspring. Maternal transfer of POPs has been studied in pinnipeds (seals, sea lions, and walruses) during the lactation period, but little work has been done on cetaceans (whales and dolphins). Cetaceans differ in life history from pinnipeds, so a study on cetaceans is warranted. The purpose of this study was to examine the dynamics of POP transfer, specifically pesticides, from delphinid mothers to calves over the lactation period. Serum and milk samples were taken from mother-calf bottlenose dolphin (Tursiops truncatus) pairs at the US Navy Marine Mammal Program from birth until 14-15 months post-partum. Lipid content and concentrations of HCB, Mirex, DDT and its metabolites, as well as chlordane and its metabolites (all pesticides) were quantified in all serum and milk samples. The most prevalent OCs were identified, including those which were preferentially transferred from mothers to calves during the lactation period. Biomagnification of contaminants in calf serum relative to both maternal serum and to milk were also calculated for each sampling period. Results show that sum DDTs were the most prevalent OC in milk comprised primarily of ppDDE. Biomagnification factors varied across metabolites. The results from this study will help us understand which pesticides are preferentially transferred during lactation from female bottlenose dolphins to calves. Furthermore, because bottlenose dolphins and killer whales are expected to be physiologically similar, this information will provide some insight into one of the major threats facing endangered Southern Resident killer whales (Orcinus orca).