Master of Science / Department of Biology / Carolyn J. Ferguson / The genus Phlox L. presents intriguing opportunities for systematics research, and P. nana is of particular interest. Phlox nana occurs chiefly in mountains of the Chihuahuan desert to northern New Mexico, and it exhibits much morphological variation across its range. Historically, this taxon has been recognized as a single species (sometimes with infraspecific taxa), or as several species. Perhaps most interesting, variation in ploidy level (cytotypic variation) has been evidenced for P. nana. This research employed flow cytometry methods in conjunction with chromosome counts to document patterns of cytotypic variation. Intensive fieldwork in Arizona, New Mexico and Texas enabled excellent sampling, and evaluation of ploidy level for 76 populations was achieved. Diploid and tetraploid chromosome counts were made (four diploid counts; five tetraploid counts), and flow cytometry was conducted on all populations, providing evidence for diploid, tetraploid and hexaploid populations. Polyploids were found to occur in many geographical areas, and in some regions, diploids and polyploids occur in close geographical proximity (e.g., within both the Davis Mountains and the Chisos Mountains of west Texas). Genome size data are presented (with discussion of unusual populations), and geographic patterns of cytotypic variation are presented and discussed. Patterns are also briefly considered with respect to morphology and taxonomy: cytotypic variation does not readily align with historical recognition of taxonomic variation, and this work sets the stage for ongoing, detailed morphometric study. Research on particular species of Phlox benefits from an understanding of a broad phylogenetic context, and low copy nuclear DNA regions are an important resource for phylogeny development. This research further evaluated part of the NADP-dependent isocitrate dehydrogenase gene (idhB) for its usefulness in inferring relationships in Phlox. Samples were PCR amplified for idhB and cloned, and resulting sequences were added to a larger set of idhB sequence data previously developed in the lab. A total of 163 samples were included, and Bayesian Inference and Maximum Parsimony analyses were conducted for complete data sets. Phylogenetic findings are discussed in light of previous work based on chloroplast and high copy nuclear DNA regions, and challenges and utility of using idhB are discussed.

Master of Science / Division of Biology / Martha E. Mather / In contrast to well documented adverse impacts of large dams, little is known about how smaller low-head dams affect fish biodiversity. Over 2,000,000 low-head dams fragment United States streams and rivers and can alter biodiversity. The spatial impacts of low-head dams on geomorphology and ecology are largely untested despite how numerous they are. A select review of how intact low-head dams affect fish species identified four methodological inconsistencies that impede our ability to generalize about the ecological impacts of low-head dams on fish biodiversity. We tested the effect of low-head dams on fish biodiversity (1) upstream vs. downstream at dams and (2) downstream of dammed vs. undammed sites. Fish assemblages for both approaches were evaluated using three summary metrics and habitat guilds based on species occurrence in pools, riffles, and runs. Downstream of dams vs. undammed sites, we tested if (a) spatial extent of dam disturbance, (b) reference site choice, and (c) site variability altered fish biodiversity at dams. Based on information from geomorphic literature, we quantified the spatial extent of low-head dam impacts using width, depth, and substrate. Sites up- and downstream of dams had different fish assemblages regardless of the measure of fish biodiversity. Richness, abundance and Shannon’s index were significantly lower upstream compared to downstream of dams. In addition, only three of seven habitat guilds were present upstream of dams. Methodological decisions about spatial extent, and reference choice affected observed fish assemblage responses between dammed and undammed sites. For example, species richness was significantly different when comparing transects within the spatial extent of dam impact but not when transects outside the dam footprint were included. Site variability did not significantly influence fish response. These small but ubiquitous disturbances may have large ecological impacts because of their potential cumulative effects. Therefore, low-head dams need to be examined using a contextual riverscape approach. How low-head dam studies are designed has important ecological insights for scientific generalizations and methodological consequences for interpretations about low-head dam effects. My research provides a template on which to build this approach that will benefit both ecology and conservation.

Doctor of Philosophy / Biology / Walter K. Dodds / Ari M. Jumpponen / Due to agricultural practices and urbanization, tallgrass prairie ecosystems have become threatened as < 5% of its historical coverage exists today. The small remainder of praire that does exist is further threatened by the encroachment of woody plant species. Woody plant encroachment may not only alter prairie ecosystem function, but also prairie microbial communities responsible for these functional processes. Further, prairies are high disturbance ecosystems, especially prairie streams which are hydrologically harsh. They support communities that frequently undergo succession due to recurring flood and drought conditions, yet little is known about the response of microbial communities to these disturbances. In my dissertation, I first address the degree of woody vegetation expansion in riparian corridors (parallel to streams) in watersheds with variable fire frequency and grazing. I found that the rate of riparian woody expansion declines with higher fire intervals and is not affected by grazing, but even annual burns may not prevent woody plant expansion in riparian zones from occurring. Second, I quantified the effect of using restorations of riparian corridors, through removal of woody plants, on physical, chemical, and microbial community (bacteria and fungi) dynamics across stream to upslope soils. Removal restoration causes a decrease in NH₄⁺ and soil water content, and causes streams and upslope soils to become similar in fungal community richness unlike forested landscapes. Bacterial communities were minimally impacted by removals, but were highly structured among stream to upslope soils due to multiple environmental gradients (i.e., pH, NO₃⁻, soil moisture). Lastly, I examined the successional development of biofilm-associated microbial communities in a prairie stream from both a functional and structural perspective. I found that biofilm microbes exhibited strong successional trajectories, with communities developing towards net autotrophy and therefore becoming reliant upon in-stream derived carbon. Further, bacterial communities displayed spatial differences, but much stronger temporal patterns in community composition were detected. These studies highlight how woody plant encroachment may influence stream ecosystems in addition to spatiotemporal trends in microbial community assembly.

Master of Science / Department of Geology / Sambhudas Chaudhuri / Rare earth element (REE) concentrations were measured in a source (reference) clay Ca-smectite standard and in the roots, stems, and leaves of a species of poplar plant (Populus eugenei). The poplar plant was grown in the clay standard under controlled laboratory conditions during a period of about three months. REEs were shown to fractionate in the clay mineral and plant materials with greater fractionation observed in plant materials. The REE data provide insight into the process of weathering of clay minerals such as a Ca-bearing smectite and provide insight into the degradation of and the composition of clay minerals in the plant environment. The degradation process is not followed by significant interlayer ion exchange effect on remaining clay minerals in the root environment. REEs were found to be transported into complex forms, potentially as REE-carboxylic anion pair complexes. The plant materials in this study were in general heavy REE (HREE) enriched relative to the source clay minerals due to the complexation effect. The REE anomalies observed in this study, in addition to the Ce and Eu anomalies, may be explained by the selective uptake by the plant by an enzyme effect rather than due to the influence of oxidation-reduction. The enzyme influence was more evident in the REE distribution when compared among the plant organs. These REE characteristics described for the plants may eventually be incorporated with data from numerous other studies and also used as a guide in the assessment of the contribution of plant materials to dissolved REE content in surface water and groundwater.

Master of Science / Department of Entomology / Srinivas Kambhampati / Termites (Isoptera) are separated into morphologically and behaviorally specialized castes of sterile workers and soldiers, and the reproductive alates. Previous research on eusocial insects has indicated that caste differentiation has a genetic basis. Although much has been studied about the genetic basis of caste differentiation and behavior in the honey bee, Apis mellifera, termites remain comparatively understudied. Therefore, my objective was to compare the gene expression patterns of different castes of the termite Reticulitermes flavipes based on EST analyses and a microarray. Soldier, worker, and alate caste and two larval life stage cDNA libraries were constructed, and ~15,000 randomly chosen clones were sequenced to compile an EST database. Putative gene functions were assigned based on a BLASTX Swissprot search. Categorical expression patterns for each library were compared using the in silico methods of BLAST2GO and r-statistics. I chose 2,240 unique-ESTs based on their putative function and sequence quality, which I used to fabricate a Combimatrix microarray. I used the microarray to compare expression levels between workers and soldiers from Kansas and Florida populations. Seventy to ninety percent of the sequences from the ESTs of each caste and life stages had no significant similarity to those in existing databases. All libraries contained sequences with putative reproductive functions, which was unexpected in the non-reproductive soldier and worker castes. Sequences of interest that showed a putative bias among castes include a viral protein in soldiers and a possible chemosensory protein in alates, which may be involved in termite reproductive functionality or communication. The microarray showed increased expression in the soldier caste of a sequence that matched tropomyosin and an increased expression level of a sequence that matched a PDZ-domain containing protein in some worker samples. This study leads to several candidate genes of potential caste specific function, which can be further tested using functional analysis and between the different castes and life stages of R. flavipes. These genes include the sequences similar to pebIII and RGS-GAIP. I have also expanded upon the available sequences for this termite and utilized the r-statistic in silico method for the first time to putatively compare gene expression in the different castes of a eusocial insect. The in silico analysis allowed us to identify several genes which may show biased expression patterns in the different cDNA libraries and which may reveal caste-specific expression controlling important functions after further analysis. These candidates include: an alate-biased gene, which had a predicted function of neurotransmitter secretion and cholesterol absorption as well as a late larval-biased gene which was predicted to be involved in protein biosynthesis and ligase activity.

Doctor of Philosophy / Department of Biology / Mark Ungerer / Elucidating the molecular basis of adaptive phenotypic variation represents a central aim in evolutionary biology. Using the model plant species Arabidopsis thaliana, I studied the intra-specific variation in freezing tolerance among natural accessions across its native range. Considerable variation in freezing tolerance among 71 selected accessions was observed both with and without a prior cold acclimation treatment, suggesting that both differences in cold-acclimation capacity and in intrinsic physiology contribute to this variation. A highly significant positive relationship was observed between freezing tolerance and latitude of origin of these accessions. This clinal pattern of variation is found to be attributable, at least in part, to relaxed purifying selection on CBF/DREB1 genes in the species’ southern range. These CBF/DREB1 genes encode transcriptional activators that play a critical role in the ability of A. thaliana plants to undergo cold acclimation and thereby achieve maximum freezing tolerance. Relative to accessions from northern regions, accessions of A. thaliana from the southern part of their geographic range exhibit significantly higher levels of nonsynonymous polymorphisms in coding regions of CBF/DREB1 genes. Relaxed selection on the CBF/DREB1s in southern accessions also has resulted in mutations in regulatory regions that lead to abrogated expression. These mutations in coding and regulatory regions compromise the function of CBF/DREB1 transcriptional activators during the cold acclimation process, as determined by reductions in rates of induction and maximum levels of expression in the downstream genes they regulate. These mutations could be selective neutral or beneficial in southern accessions depending on whether there is an allocation cost associated with cold acclimation. The fitness benefit and possible allocation cost of cold acclimation was examined in freezing and freezing-free environments using natural accessions exhibiting contrasting abilities of cold acclimation as well as transgenic CBF gene over-expression or knockdown/knockout lines. The extent to which cold acclimation benefits the plant in presence of freezing temperature is revealed, but a cost of cold acclimation wasn’t detected in the absence of freezing temperature under our experimental design, which suggests that these mutations in CBF genes in southern accessions might be neutral to natural selection.

Master of Science / Division of Biology / Stella Y Lee / Neuronal Ceroid Lipofuscinoses (NCLs) are a group of lysosomal storage diseases that are characterized by accumulating autofluorescent lipopigments in cells. NCLs are a form of progressive neurodegenerative diseases with symptoms ranging from blindness, loss of speech and motor activities to ataxia and seizures. Patients do not live to adulthood in most cases, making it prevalent in children. Among the many genes that cause NCL, CLN5 leads to different forms of NCL (infantile, late infantile, juvenile, and adult). CLN5 protein resides in the lysosomes but its function has not been established. It is predicted to contain eight N-glycosylation sites, but the role of N-glycosylation on its function and trafficking has not been assessed. We analyzed the role of N-glycosylation on the transport and stability of human CLN5. We created N-glycosylation mutants of each site by changing the Asn to Gln and our analysis of these mutants show that all the eight N-glycosylation sites are used in vivo. We also report effects of abolishing individual N-glycosylation sites on the trafficking of CLN5. While the lack of glycosylation at some sites results in CLN5 being retained in the ER or Golgi, others do not affect CLN5 trafficking. Cycloheximide chase experiments show that one of the mutants (N401Q) in CLN5 leads to lower protein levels in cell pellets with an increased secretion compared to CLN5 wild type, while other mutations show differential stability in cell pellets. These results demonstrate that each N-glycosylation site plays a different role(s) in the stability, transport and/or function of CLN5.

Master of Science / Department of Biology / Loretta Johnson / Plant epicuticular wax (ECW) isolates internal tissues from harsh external conditions increasing drought tolerance. Beta-diketone-rich ECW reflect light and result in a glaucous phenotype that may ameliorate the thermal environment of the leaf. The overall goal is to characterize the form and function of ECW in two closely related, but phenotypically divergent grasses. [italicized]Andropogon gerardii ssp. [italicized]gerardii, big bluestem, is a non-glaucous, agronomically and ecologically dominant grass in the United States while [italicized]Andropogon gerardii ssp. [italicized]hallii, sand bluestem, is a glaucous subspecies restricted to dry, sandy soils. The objectives are to contrast sand and big bluestem ECW chemistry, morphology, and physiology to determine the distinctions in ECW resulting in the glaucous phenotype and determine the effect this has on leaf optical qualities and permeability. Gas chromatography mass spectrometry (GC-MS) and scanning electron microscopy (SEM) were used to examine ECW chemistry and micromorphology. It was hypothesized that beta-diketones and beta-diketone tubules where present only in leaves of sand bluestem and that the ECW was more reflective and abundant and the cuticle was less permeable. Beta-diketones and tubular ECW were absent in big bluestem and common on sand bluestem’s surface, although less than 20% of ECW was beta-diketones. Functional implications of ECW phenotypes were investigated by comparing minimum conductance (G[subscript]min), wax load, reflectance, and transmittance. Reflectance, with and without ECW, and G [subscript]min were measured with an infrared gas analyzer and a spectroradiometer, respectively. Sand bluestem had twice the ECW in mg cm[superscript]2 (P=.01) and three times lower G [subscript]min in ms[superscript]-1 10[superscript]-5 (P=.02). Partial least squares (PLS) models were trained to predict subspecies from reflectance spectra and were able to distinguish the subspecies. These experiments indicate that in comparison to big bluestem, increased reflectance is a property uniquely imparted to sand bluestem by ECW and the presence of beta-diketones determines the distinction. Glaucous crop species have shown higher yield under drought and extreme weather, including drought, is expected to become more common. Therefore, this study of glaucous waxes, may be applied in engineering drought tolerance.

Doctor of Philosophy / Department of Biology / Michael A. Herman / All multicellular organisms consist of a variety of cell types. One of the mechanisms to generate this cellular diversity is the asymmetric cell division, which requires the establishment of cell polarity. In Caenorhabditis elegans hermaphrodites, 807 of 949 somatic cell divisions are asymmetric. The centrosome and the Wnt signaling pathway both have been shown to regulate cell polarity and subsequently asymmetric divisions in many model organisms. However, it is not clear whether the Wnt signaling pathway manipulates the cell polarity through specific cellular organelles, such as the centrosome. To address this question, we examined a centrosomal component, SPD-5, to see whether it cooperates with the Wnt signaling pathway to regulate certain asymmetric cell divisions. We showed that SPD-5, which was originally found to be critical for the embryonic development, also played a role during certain post-embryonic cell divisions in C. elegans. Specifically the asymmetric divisions of seam cells that required SPD-5 function were also known to be regulated by the Wnt signaling pathway. Thus the stem-cell like seam cell divisions could be an intriguing system to study the interaction of centrosomes and the Wnt pathway. We found that SPD-5 was required for a successful cell division, similar to other centrosomal components. This suggests that SPD-5 still functions as a centrosomal component during C. elegans post-embryonic development. It has been shown that establishment of seam cell polarity relies on the asymmetric localization of certain Wnt pathway components. Interestingly, we found that SPD-5 was required for the proper localization of several Wnt components in a way that was independent of a key MTOC (microtubule-organizing center) member γ-tubulin. In addition, SPD-5 genetically interacted with the Wnt pathway components APR-1/APC and POP-1/Tcf to regulate asymmetric divisions of seam cells. These data suggest that SPD-5 interacts with the Wnt signaling pathway in controlling the polarity of seam cells. Overall, our results suggest a novel role of SPD-5 in cooperating with the Wnt signaling pathway to regulate cell polarity and asymmetric cell division, in addition to its function as a centrosomal component.

Doctor of Philosophy / Department of Biology / Keith B. Gido / Habitat fragmentation and loss threaten global biodiversity, but organism responses to changing habitat availability are mediated by structural properties of their habitats. In particular, organisms inhabiting dendritic landscapes with hierarchically arranged branches of habitat tend to have limited access to some patches even in the absence of fragmentation. Consequently, organisms inhabiting dendritic landscapes such as streams respond strongly to fragmentation. Using a combination of meta-analysis, field observations, and ecological network modeling I show that stream fishes respond to fragmentation in predictable ways. First, I addressed how dams and stream dewatering have created a mosaic of large river fragments throughout the Great Plains. Using a geographic information system and literature accounts of population status (i.e., stable, declining, extirpated) for eight “pelagic-spawning” fishes, I found stream fragment length predicted population status (ANOVA, F2,21 = 30.14, P < 0.01) and explained 71% of reported extirpations. In a second study, I applied a new measure of habitat connectivity (the Dendritic Connectivity Index; DCI) to 12 stream networks in Kansas to test the DCI as a predictor of fish response to fragmentation by road crossings. Results indicated fish communities in stream segments isolated by road crossings had reduced species richness (alpha diversity) and greater dissimilarity (beta diversity) to segments that maintained connectivity with the network, and the DCI predicted patterns in community similarity among networks (n = 12; F1,10 = 19.05, r2 = 0.66, P < 0.01). Finally, I modeled fish distributions in theoretical riverscapes to test for mechanistic linkages between fragmentation and local extirpations. Results suggested the number of small fragments predicted declines in patch occupancy, and the magnitude of change in occupancy varied with dispersal ability (“high” dispersers responded more strongly than “low” dispersers). Taken together, these works show context-dependencies in fish responses to fragmentation, but a unifying theme is that small fragments contribute to attenuated biodiversity. Moreover, the predictable manner in which stream fish react to fragmentation will aid in biodiversity conservation by revealing potential responses to future scenarios regarding changes to habitat connectivity.