We know that climate sensitivity can vary within species, but the factors that influence which climatic variables influence population dynamics remain unclear. In this study, I assess whether macroclimate influences the climate sensitivity of four high elevation conifer species: Pacific silver fir (Abies amabilis), mountain hemlock (Tsuga mertensiana), Alaska yellow cedar (Callitropsis nootkatensis), and sub-alpine fir (Abies lasiocarpa). I also investigate if climate sensitivity of tree communities Mt. Rainier is changing over time. To address these questions, I analyzed tree core data from three high elevation sites at Mt. Rainier National Park. Each site is located in a distinctly different climatic zone on the mountain. I compared standardized tree growth to climate variables (mean growing-season temperature, total growing-season precipitation, annual snowpack, climate moisture deficit, etc.) using linear mixed effect models and “treeclim”, a tree ring analysis program in R. With these methods, I determine how influential climate variables differ by location, by species and over time. Gaining more insight into which climate variables have the greatest influence on growth in different areas will allow us to better predict how specific plant populations will react to ongoing climate change. Moreover, studying tree communities at high elevations is particularly important, since behavior at treeline will determine whether a species’ range expands, contracts or shifts, which in turn has implications for both that species and other species. For example, conifer range expansion on Mt. Rainier could positively increase carbon sequestration (by increasing woody cover in the park) while negatively encroaching on the alpine wildflower meadows, a major attraction for tourists. Widespread range contractions could potentially reduce the ability of those forests to sequester carbon from the atmosphere. Thus, with better predictions of tree shifts, we can make more informed management decisions for ecotourism and conservation.

With increasing temperatures and altered precipitation patterns over the past several decades, montane environments are undergoing rapid anthropogenic changes. Climate controlled treelines are moving up in elevation worldwide, with dramatic consequences for alpine ecosystems, however little is known about the local drivers of variation that influence the rate of tree line change, like small-scale topography. We conducted a repeat photography study to assess the rate of tree encroachment into the subalpine meadows of Mt. Rainier National Park in various local landforms (coves, ridges, and slopes) as well as quantify the rate of treeline change over the 20th and early 21st century. We did this by aligning historical and repeat oblique photographs in a Geographic Information System and manually delineating tree encroachment within the subalpine zone. We also verified our results by comparing them to per-pixel classifications of aerial imagery collected in 1951 and 2013. Tree encroachment has occurred in all sites on Mt. Rainier National Park over the last century. Oblique photographs showed an approximately thirty percent increase in tree cover from the 1930s to 2016. Local topography did have a significant influence on the amount of tree encroachment. Small-scale topographic ridges were predicted to show the greatest amount of tree encroachment. Our results contradict this prediction, showing that sloped sites have the greatest increase in trees, followed by ridges, then topographic depressions (coves). Analyses of aerial photos are ongoing, but are expected to show  similar results. Per-pixel classifications or aerial images will help us more easily distinguish changes in the various environments at Mt. Rainier, as well as correlate them to environmental drivers like snow duration. This study helps fill an important gap in knowledge about how topography is related to tree line change as well as raises further questions about what management practices can be implemented.

The link between climate and range distributions is a key area of study for ecologists interested in predicting how species will respond to anthropogenic climate change. However, most studies only focus on abiotic factors (like climate), and ignore biotic determinants of species performance. In this study, I seek to understand how competition influences the success of a common conifer species in the Pacific Northwest. Specifically, I ask whether interspecific responses to competition lead to a predictable competitive hierarchy across different locations or whether competitive hierarchies vary. To address this question, tree cores from six focal conifer species, Abies amabilis, Tsuga heterophylla, Tsuga mertensiana, Thuja plicata, Pseudotsuga menziesii, and Callitropsis nootkatensis were collected from 18 stands of varying elevation and climatic conditions within Mt. Rainier National Park, with 20 individual trees cored per species and stand. Annual tree growth was used as a proxy for performance as growth is known to be sensitive to competition and results can be extrapolated to overall population performance. The competitive neighborhood of each cored tree was determined by identifying all individuals within a 10-meter radius by species, and quantifying the distance of competitors to focal individuals as well as their size. Growth rates for each individual tree within the stand were compared to the sum of the basal area of neighbors around that tree, using analysis of variance to determine the relationship between the two variables. These relationships were then compared across species to see which species were competitively superior, and then compared across stands to determine whether competitive hierarchies varied. The importance of this study is to identify which species are more sensitive to competition which would expand our knowledge on the biotic factors that may limit their ability to shift their range.

With concern about global warming rising, assessing how increases in temperature impact plant performance is crucial in understanding how plant communities may alter. Genetic diversity, local adaptation to climate, and gene flow may interact with the direct effects of climate to determine how plants fare in a warmer world (e.g. through range shifts and shifts in phenology), but these interactions are poorly understood. Studying local adaptation is crucial for predicting how local populations will fare with warming, as a high degree of local adaptation may cause populations at the range center to be negatively influenced by warming. Conversely, populations in newly climatically unsuitable habitats may be able to cope with warming by adaption. This study measures how the climate of different elevations affects population growth of the high-elevation plant mountain monkeyflower, Mimulus tilingii, at Mt. Rainier National Park. Utilizing data from the past two years, we will parameterize matrix population models to explore how climate influences fitness and population growth rates of different populations, thereby assessing how local adaptation contributes to population dynamics (and potentially, the impacts of climate change). Matrix models will also allow us to determine how local adaptation affects particular life stages, which climatic factors drive local adaptation, and how genetic diversity promotes local adaptation. Previous results show that plants growing in different environments (high vs. low elevation) did not significantly differ in reproductive success whereas different genotypes (crossed between 2 elevations vs. 1 elevation) did, suggesting genetic diversity is an important primary driver of fitness. We predict that vital rates will be greater for populations grown in their native range (high elevation) with genotypes matched to their environment (within 1 elevation). Through these analyses, our understanding of population response due to warming will help us make better ecological inferences of what to expect in the future.

A seed’s successful growth into a seedling depends on it finding a suitable site for germination. This may be limited by the parent’s ability to distribute seeds over a large area (dispersal), or by the seed’s ability to grow in given environmental conditions (microsite characteristics). This study attempts to answer the question: how is tree germination limited by dispersal and by the soil moisture of germination sites? A forest in a 25.6 hectare plot located in the Gifford Pinchot National Forest is the subject of this study. This forest is dominated by Tsuga heterophylla and Pseudotsuga menziesii, but also includes Thuja plicata, Abies amabilis, and Acer macrophyllum. Past surveys on trees, seeds, and seedlings within the plot have been conducted since 2014. Specifically, the species, size, and location of all trees are known. Forty paired seed traps and seedling quadrats throughout the plot provide data on seed and seedling densities by species, as well as soil moisture readings. The role of dispersal in germination success will be assessed by comparing the number of germinants within each seedling quadrat to the abundance of nearby parent trees. Soil moisture’s role will be determined by comparing germination rates (calculated by seedling densities divided by seed densities) to soil moisture in seedling quadrats. Results will be separated for each species. The species composition of these communities may prove to be determined largely by differences in dispersal and moisture requirements between species. For example: we expect to find a high dispersal capacity for Tsuga heterophylla, which would explain its dominance in the stand. We also predict that most species will fail to germinate in areas of low moisture. This might suggest serious implications for how these trees will respond to the warmer and drier conditions expected to accompany anthropogenic climate change in the Pacific Northwest.

As our planet warms, it becomes increasingly important to study the impacts on biotic systems’ structure and function.  While there is consensus amongst the scientific community that changing climate will affect species’ ranges and distributions, there is a dearth of data on the intricacies of how such changes specifically alter the interactions among species. Notably, few studies simultaneously consider the effects of climate and competition on (current/existing) species performance, which is particularly important for generating a body of knowledge that relates climate change to shifts in species distributions.  This study seeks to investigate how competitive interactions vary across climate gradients for several conifer species common in the PNW and abundant in Mt. Rainier Park. Specifically, I am examining whether neighborhood effects on annual tree growth vary across elevational and climatic gradients. To accomplish this, tree cores were collected from Abies amabilis, Tsuga heterophylla, Tsuga mertensiana, Thuja plicata, Psuedosuga menziesii, and Callitropsis nootkatensis at 18 stands in Mt. Rainier Park, covering a large range of elevation (and thus climate). Additionally, the neighborhood of all cored trees, including the distance between cored trees and their neighbors, the species identity and diameter of neighbors was collected. Core samples were cross-dated by stand to ensure proper assignment of year of growth to each tree ring, and subsequently core and neighborhood data were analyzed using statistical models (multiple regressions) to extract relationship between competition, growth rates, elevational and climate variables. This is an ongoing project, whose growing body of data will be useful in predicting the way climate shifts may affect competition and thus growth of tree species in the PNW.

Plant phenology studies have shown that flowers are blooming earlier now than they did several decades ago. These shifts in bloom time are attributed to warming temperatures, earlier snowmelt, and other climatic factors related to temperature. One underutilized data source for phenology studies is herbarium specimens, which are plants that are collected, often during bloom, and preserved for the purpose of studying species traits and distributions. Since collection date and location is recorded, herbarium specimens can be used to study the relationship between climate change and long-term shifts in the timing of phenological stages such as flowering and fruiting. My goals for this study were to determine 1) What is the relationship between changes in climate over the past century and changes in bloom time? and 2) Do shifts in the timing of phenological stages in herbarium specimens match shifts reported by field studies? My study species is glacier lily (Erythronium grandiflorum), a subalpine plant native to the Pacific Northwest that is known from field studies to be sensitive to climate change because it blooms immediately after snowmelt. Specimen data from Pacific Northwest herbaria was accessed using the Consortium of Pacific Northwest Herbaria’s database. When not listed, phenological stages were classified using specimen images and geographic coordinates were determined using GEOLocate and descriptions of specimen collection locations. Historical climate information was gathered by entering specimen geographic coordinates and elevations into ClimateWNA, a spatial climate model. I hypothesized that warmer spring temperatures and less snowfall would result in earlier bloom times. I also expected that my results would support shifts in bloom times reported by field studies. My findings contribute to the collective understanding of the historical effects of climate change, which assists predictions and management strategies concerning the future effects of climate change on plant species and communities.

Seed masting occurs when seed production of a species is annually variable across large geographic regions, where years of extremely high seed production across individuals in a population is followed by one or more years of extremely low seed production. It occurs across large geographic regions and can occur within and amongst species. However, the drivers of masting and the implications of masting are often poorly known. Here, we use a long-term and spatially extensive data set of conifer seed production to A) test whether seed viability estimates are consistent with pollination efficiency (increased pollination success in high seed production years) driving masting; and B) explore the implications of masting for nitrogen dynamics. We found strong support for seed masting in six focal conifer species in Mt. Rainier national park from 7 years of data (2009-2015) collected from 18 locations. To further explore masting, we tested whether pollination efficiency is the driver of seed masting in Tsuga heterophylla, by testing whether seed viability (seed quality) is higher in mast years (using 4 years of data from 7 stands – which includes 2 mast years). Results indicate a higher proportion of filled seeds in mast years (i.e. higher viability), providing tentative support for pollination efficiency as the underlying cause of seed masting in these focal species. Germination tests of seeds in growth chambers will provide additional information. To determine the consequences of masting for nitrogen dynamics we measured the nitrogen content in seeds and leaf litter (across mast and non-mast years), and used these estimates to estimate total nitrogen content of leaf litter and seeds (i.e. nitrogen cycling). Understanding the mechanisms behind masting is important, as masting can control tree population demography, and influences ecosystem dynamics.

Retinol Binding Protein (RBP) is a protein used by the body to transport Vitamin A and is often used to monitor short-term nutritional changes. Traditionally, RBP has been measured via a nephelometric immunoassay with a high sample requirement. In an attempt to reduce turnaround time and to reduce the volume of sample needed for testing, an in-house assay using liquid chromatography tandem-mass spectrometry (LC-MS/MS) technology is being developed.The method being developed is based on the Vitamin D Binding Protein assay which sets a precedent for LC-MS/MS methods targeted toward high-abundance globulins. This method consists of denaturation of RBP using heat and denaturation agents, then digested by trypsin into peptides which are detected by LC-MS/MS. Currently there are three peptides with multiple transitions being monitored for each (FSGTWYAMAK, YWGVASFLQK, and LIVHNGYC[+57]DGR).The assay is being developed and validated according to the document CLSI C-62A and the assay is currently in its pre-validation phase. Experiments done include standard curve verification and comparison to development data, simple repeatability 5x5, carryover and various troubleshooting experiments.Data analysis has exposed high control and internal standard variability in the assay which are being resolved, but progress looks promising.The RBP assay is still far from completion and further testing and troubleshooting needs to be done to reduce variability before it can enter the validation phase. Once optimized, the LC-MS/MS assay, while less automated than nephelometric platforms, should allow UW Medical Center to have better control over an in-house assay in terms of turnaround time, while also using much smaller volumes of sample. LC-MS/MS also has improved sensitivity when compared to the nephelometric assay. This increased performance may lead to assay utility with other conditions such as diabetes, liver disease and kidney disease.
 

During embryogenesis, phosphorylation of serine and threonine residues on p120 catenin causes disassociation from cadherin transmembrane proteins and activation of downstream pathways leading to cell motility. We have mutated a single amino acid on p120 catenin, T310, to determine its involvement in cell migration of the dorsal axis during zebrafish embryogenesis in vivo. T310 is a target of glycogen synthase kinase 3 (GSK3) and phosphorylation of this site facilitates disassociation from membrane-bound N-cadherin in astrocytes, decreasing cell-cell adhesion and activating the cell motility pathway. We created two mutations of p120 catenin: T310E that mimics the negatively charged phosphate and T310A containing a neutral residue that cannot be phosphorylated. Either mutated or wild type mRNA was co-injected with morpholino, which knocks down endogenous p120 catenin, to determine the injected protein’s ability to rescue the short-tailed knockdown phenotype. As expected, co-injection of wild type p120 catenin mRNA and morpholino rescued the damaged phenotype resulting in embryos with normal tail extension. Co-injecting either T310A or E with morpholino resulted in partial rescue, suggesting that phosphorylation at this site may be variable during development or that the effects cannot be measured by phenotypic quantification methods. We are currently studying the effects of phosphorylation at other sites, S268 and S269, which may give insight on the downstream effects p120 catenin in cell motility, including morphological bone abnormalities and metastasizing cancer cells.

The Hille lab studies the movement of cells during embryogenesis using the model organism of zebrafish. Currently, we are focusing on how the phosphorylation of certain residues on the protein p120 catenin affects both cell movement and cell-cell adhesion during the early stages of embryonic development. Protein p120 catenin is imperative in the motility or adhesion of cell-cell junctions, and knocking it down results in wider somites and abnormal notochord protrusions as discussed in the Hsu et al paper published earlier by the lab. We are focused on how specific mutations in the genetic sequence of this protein affect cell migration and adhesion in the early development of zebrafish. Other members of the lab have mutated a phosphorylated tyrosine residue (Y228) within the DNA encoding for p120 to either phenylalanine and glutamic acid residues. We are then taking the mutated embryos and staining the somites and presumptive slow muscle cells using a myoD probe in order to visualize the resulting phenotype. This process involves a four-day protocol that is repeated weekly throughout the quarter in order to produce the best staining and results. After hybridization is complete, we then dissect the embryos under a dissecting scope and image them using a high-powered microscope. Using software, we analyze the images, measuring somite width as well as the number of somites in order to determine the developmental stage of the embryo. We then compare somite length and mesodermal formation between both mutated and uninjected, control embryos to gain a better understanding of which amino acid residues in the p120 protein are important in its function during embryogenesis.

Cell movement during development is crucial as the location of a cell determines what that cell will ultimately become. Cells require both adhesion and motility for movement. Adhesion is cells holding on to each other via proteins called cadherins. Motility is movement through projections called fillopodia and lamelliopodia. We are interested in the protein p120-catenin, which determines how cells move. We purpose that phosphorylation, or the addition of negative PO4- groups at specific amino acid residues changes p120 catenin’s function. Specifically we are interested in tyrosine (Y) residues 228 and 335 which likely effect p120 antagonistically, by mediating adhesion and motility, respectively. In order to mimic the process of phosphorylation we mutated each residue to either glutamate (E) or phenylalanine (F). Glutamate, which is charged, mimics phosphorylation, while phenylalanine cannot be phosphorylated, but is otherwise similar to tyrosine. To examine which pathway is activated, we quantified whether proteins associated with motility or adhesion were interacting with p120 catenin for each mutation. The primary technique we used to explore this is coimmunoprecipitation. Coimmunoprecipitation works by using antibodies to link p120 catenin to magnetic beads, as well as any proteins interacting with it. The beads can then be extracted and we can determine the amount of each protein interacting with p120 catenin through a process called western blot. So far we have verified that our mutations for Y228 and Y335 produce proteins, which guarantees that any later results are due to phosphorylation. Also preliminary coimmunoprecipitations suggest Y228 does mediate adhesion. Additionally injection of Y228E in embryos, results in embryos with shortened tails and poor yolk constriction, which are associated with excessive adhesion. Taken together these results strongly support our hypothesis for Y228 mediating adhesion. Based on these results, in the future we plan on examining Y335 and verifying our Y228 results.

Bees and flowers trade nectar for reproduction. Rhinos provide food to birds in exchange for removal of parasites. Mutually beneficial interactions like these pervade the natural world, but their impact on the process of evolution is not well understood. Here, we test whether each species in a microbial mutualism repeatedly adapts to the changes in their partners, or coevolves. In our model system 22 communities of the Methanococcus maripaludis (archaea) and Desulfovibrio vulgaris (sulfate reducing bacteria) evolved for over 3000 generations in the laboratory. To test whether coevolution occurred, we separated the D. vulgaris and the M. maripuludis in the mutualistic community at 1000 generations. We then paired these 1000 generation D. vulgaris populations with M. maripaludis at different evolutionary time points and measured the growth rate and yield of each population. If M. maripuludis coevolved with D. vulgaris then we would expect the 1000 generation D. vulgaris to perform better with future partners (0-1000 generations) than past partners (1000-2000 generations) because mutualists are expected to have positive effects on each other's fitness. A preliminary experiment with 6 communities indicated the opposite, that D. vulgaris grew faster with past generations compared to future generations. The yield of communities was similar across pairings. These results could indicate D. vulgaris has coevolved with M. maripaludis, and that there is an underlying antagonism between these species that has not been recognized. We present the results of a more extensive experiment, testing more mutualistic communities at more time points, and looking for patterns of coevolution in both species.  
 

We are studying the regulation of p120 catenin in zebrafish embryonic development. P120 catenin plays an essential role in the pathway for cell migration and normal embryonic development. Our research on cell migration and the extension of the dorsal axis can be applied to all vertebrates. These functions are also relevant to many clinical issues such as Spina Bifida in humans, wound healing, and cancer metastasis. The particular protein we're studying (p120 catenin) facilitates the activation of Rac1 GTPase when it is bound to both Vav2 and Rac1 GTPase. Vav2 is a guanine nucleotide exchange factor (GEF) that activates Rac1 by exchanging GTP for GDP. Rac1 GTPase activates the actin polymerization and lamellipodia. There has been research suggesting that the dephosphorylation of certain tyrosine residues in p120 catenin is necessary for this binding and subsequent Rac activation. Rac activation promotes cell migration in zebrafish embryonic development. This research has prompted us to ask how phosphorylation at Y217 and co-phosphorylation at Y228-Y217 of p120 catenin affect gastrulation of zebrafish embryos. Based on previous research, we expect embryonic injection of mRNA containing Y217E and Y228E-Y217E mutations to result in abnormal gastrulation. Abnormal phenotypes exhibit delays with cell migration and yoke constriction. In contrast, mutations Y217F and Y228F-Y217F should result in a rescue phenotype similar to the wild type, since the phenyalannine residues are not negatively charged. In order to test this, we designed primers for each mutation, and inserted them using PCR. We used gel electrophoresis to confirm the effectiveness of the PCR by comparing the size of the PCR product with its expected length. We amplify the DNA in E. coli, followed by DNA purification, and sequencing. With confirmed mutations, the next step is linearization of the DNA followed by production of mRNA. We are now in the process of synthesizing mRNA for the multiple DNA mutations we have created. We will then inject them into zebrafish embryos with knocked down wild type p120 catenin and look for resulting phenotypes.