Thesis(M.S.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references.

Understanding the impacts of explosive contamination on vegetation is key to understanding explosives behavior in the environment. I quantified shrub growth responses to 1,3,5-trinitroperhydro-1,35-triazine (RDX) soil contamination across three life stages and I hypothesized that RDX would have the greatest impact on seed germination. Morella cerifera seeds were germinated on soils amended with RDX up to 1500 mg RDX kg-1 dry soil. Juvenile and adult individuals were exposed for 6 weeks to soil amended with RDX up to 750 and 1500 mg RDX kg-1 dry soil, respectively. Morphological responses were quantified for juveniles while physiological measurements were quantified for adults. RDX induced a significant response in all age groups and, in accordance with the hypothesis, germination was the most impacted of the three stages. Impacts varied by concentration in addition to life stage, showing that many variables influence plant response to RDX.

Philosophiae Doctor - PhD / Frankia is one of two partners in the globally distributed N2-fixing actinorhizal symbiosis between this filamentous soil-dwelling actinomycete and almost 300 species of host plants from eight diverse angiosperm families. The actinorhizal symbiosis is a major contributor to nitrogen reservoirs in terrestrial ecosystems, and allows actinorhizal plants to perform the role of pioneers in newly formed and nitrogen-poor soils. Frankia are differentiated into four main host-infection groups (1: Alnus/Comptonia/Myrica-infective, 2: Rosaceae/Datisca/Coriaria-infective, 3: Elaeagnaceae/Gymnostoma-infective and 4: Casuarina-infective), and there is a large degree of phylogenetic clustering within these HIGs. Of these host lineages, species from the genus Morella, from the family Myricaceae, are notable as they have the ability to establish effective partnerships with Frankia from more than one host-infection group. Africa houses 16 of the world’s 33 currently accepted Morella species, and Morella is the continents only genus containing endemic actinorhizal species. Despite this, the diversity of Frankia in symbiosis with African Morella has never been explored. To address this lack of knowledge I investigated Frankia in root nodules of six Morella species from the Cape flora of Southern Africa, as well as in rhizosphere soils from selected hosts. Partial nif H gene fragments recovered from 202 root nodules yielded 26 unique sequences, which phylogenetic analysis assigned to Frankia Cluster I (the Alnus host infection group) and Frankia Cluster III (the Elaeagnus host infection group)1. Nineteen nif H sequences were assigned to three sub-clusters within Frankia Cluster III (CC-3, CC-4 and CC-5), and the remaining seven sequences to two sub-clusters within Cluster I (CC-1 and CC-2), one of which (CC-1) is novel to the current study. Identical sequences were recovered from nodules collected at geographically distant locations, suggesting a cosmopolitan distribution within the region for some subgroups from both clusters, but more localized distribution (or tighter host-specificity) for others. Soil pH correlated with strain presence in nodules, with Cluster I sequences being associated with hosts growing in acidic soils exclusively. Furthermore, three Morella species from the Cape flora of southern Africa are promiscuous in their natural habitats, with host infection group influenced by habitat edaphic conditions. In order to explore the correlation between soil characteristics and Frankia presence in nodules, nif H soil libraries were created from selected host rhizospheres. While Cluster III sequences from these libraries corresponded closely to sequences found in nodules from the same sites, the dominant Cape Cluster I group (CC-1) was absent from all six libraries, even when present in nodules recovered from the same soils. Whether this was due to low abundance of -but strong selection for- these strains by hosts under particular conditions, or due to the absence in soil of hyphal forms of these strains could not be determined. Cluster III strains are known to be better able to persist saprophytically than their relatives from other host-infection groups. A second group of Cluster I strains, detected at only one sampling site, was present in that site's corresponding soil library. An Alnus-infective subgroup, cluster AI, which has been detected in soils collected on five continents, was also detected in the of the Cape soil libraries but never in nodules, raising questions as to this group’s ability to persist in soil in the absence of known suitable hosts. Ten Frankia strains representing all three of the numerically dominant subgroups (CC-1, CC-3 and CC-4, found in 186 of 202 root nodules) were isolated from four Morella species. These isolates represent six of the most abundant unique nodular nif H sequences found in the field survey, and display morphological and cultural characteristics typical of Frankia. Phylogenetic analysis confirmed their identity as Frankia, and multilocus analysis revealed that the isolates belong to three genospecies. Two of these genospecies fall into existing groups within the Elaeagnus-infective Cluster III, while the remaining genospecies is a novel addition to the otherwise well-described Alnus-infective Cluster I. Whole genome sequencing of a representative from each of the Cape genospecies allowed for basic annotation and genome descriptions, which agreed in each case with what has been previously found for strains from the Elaeagnus and Alnus host-infection groups, respectively. Similarly, the organization of nitrogenase gene clusters in each of the sequenced strains mirrors that found in other strains from their respective host-infection groups, indicating that this gene cluster is highly conserved in different Frankia lineages. For the first time the diversity of Frankia nodulating endemic African Morella, and present in root-associated soils of these species, has been explored. This is also the first study to report isolation and description of Frankia strains from actinorhizal plants endemic to Africa.

Causes and consequences of woody encroachment into grass dominated systems have been widely studied, however functional mechanisms which promote encroachment are largely unknown. Many expansive woody species are shrubs with rhizobial or actinorhizal N-fixing symbiotic associations. Morella cerifera L. (Myricaceae) is an actinorhizal N-fixing shrub which rapidly expands into grasslands on the barrier islands off the coast of Virginia, USA. The objective of this research was to determine physiological drivers of woody encroachment resulting in increased woody cover of M. cerifera on Southeastern, US barrier islands. Variations in physiology and resource use efficiencies (RUE) of M. cerifera and co-occurring shrubs were determined, and edaphic characteristics beneath shrub thicket canopies and in open areas were quantified as indications of resource availability. Analysis of dune vegetation and soils showed severe freshwater limitation and reduced plant height of dune forbs suggesting dunes represent an upper elevational boundary for M. cerifera distribution. Soil N availability was higher beneath shrubs compared to open areas, and both physiology and isotope effects showed facilitation of the non-fixing shrub, Baccharis halimifolia, by M. cerifera which may lead to increased rates of woody encroachment as B. halimifolia colonizes expanding thicket edges. Morella cerifera and other N-fixers had higher %refixation within stems which resulted in higher carbon use efficiency (CUE) and water use efficiency of N-fixing shrubs compared to non-fixers. Results of an N-fertilization experiment suggest B. halimifolia has higher dependence on and demand for soil nutrients compared to M. cerifera. Morella cerifera showed no signs of resource deficiency or reduced physiological capacity even at 0 ppm total Nsoil. Morella cerifera transitioned from utilizing solely fixation derived N to soil N as N concentrations increased providing another mechanism leading to increased CUE and, indirectly, overall RUE. In summary greater RUE, lower resource demand, and greater resource availability for M. cerifera compared to co-occurring shrubs may result from symbiotic root associations with bacteria and fungi. While expansion of M. cerifera thickets is limited to lower elevational interdunal depressions, expansion may continue and result in increased rates of woody encroachment through facilitation of co-occurring shrubs.

Patterns of the expansion of woody cover into grasslands on barrier islands of the Virginia coast were investigated. Seed dispersal of the dominant shrub Morella spp., was sampled deploying seed traps (n = 82) throughout a landscape under shrub encroachment pressure on Hog Island, VA. Traps were placed underneath: fruiting Morella, non-fruiting Morella, co-occurring species (Iva frutescens and Baccharis halimifolia) and in grass land, (no shrub cover). Environmental filters that act upon dispersed seeds and subsequently determine establishment patterns were also investigated. Dispersal distribution throughout the encroachment zone was leptokurtic and dispersal among cover types suggest co-occurring shrub species facilitate dispersal by functioning as bird perches. Interaction of biotic and abiotic factors mediate a complex process of establishment by influencing dispersal, germination and seedling survival to ultimately determine distribution patterns of woody plants in coastal environments.

In the nutrient-poor soils characteristic of coastal environments, symbiotic association with the nitrogen-fixing root endosymbiont Frankia is essential to establishment and survival of the woody shrub Morella cerifera. Nutrient deficiency quickly becomes severe unless seedlings are infected by Frankia soon after germination. However, the means of arrival of Frankia prior to shrub establishment has not been determined. Using sterilized lab-grown M. cerifera seedlings and fecal samples collected from passerine birds on the Eastern Shore of Virginia, viability of avian dispersal of the bacteria was tested. Although passerine fecal samples did produce nodules on some sterilized M. cerifera seedlings, these experimental inoculations did not lead to significantly higher likelihood of nodulation, relative to sterilized reference seedlings. Non-sterilized seedlings displayed greatest percent nodulation; results suggest that passerines contribute to Frankia dispersal, but also that the actinomycete is contained on or within viable seeds or fruits of M. cerifera, and therefore may be co-dispersed directly from the parent plant.

It is important to understand mechanisms that facilitate expansion of two common shrubs, Morella cerifera and Baccharis halimifolia in coastal environments. The purpose of my study was to investigate the physiological and structural changes that occur as leaves age. Photosynthesis, incident light, chlorophyll, and leaf C:N ratios were quantified for young, intermediate, and old leaves (distal, central and proximal leaves, respectively). Leaf structural differences were also compared. Leaves did not change morphologically with age. Light decreased with leaf age and during winter months. Photosynthesis showed no seasonal or age related patterns. Chlorophyll increased initially and then declined with age due to self shading. Nitrogen content was highest during spring. Seasonality and leaf age had unique effects on the two study species. Understanding senescence adaptations of these two shrubs can help explain their abundance in coastal ecosystems.

Morella cerifera is a rapidly expanding native shrub on the Virginia barrier islands which displaces other native coastal species and may interrupt normal sediment dynamics. Barrier islands are considered stressful environments with low nutrients, high solar load, and frequent drought and salt exposure; facilitation often dominates in stressful environments according to the Stress Gradient Hypothesis. The objective of this project was to understand the importance of species interactions with grasses on the growth and physiology of M. cerifera at the seedling life stage through both field and lab experiments. Grasses provided ~1.3°C insulation to shrubs during winter freeze events and a freezing threshold for M. cerifera seedlings was experimentally found between -6°C and -11°C. Seedlings competed for light with grasses during warm months and grew more where grasses were clipped, revealing a tradeoff between winter insulation and summer light competition. M. cerifera shows evidence of ecosystem engineering at the seedling stage by significantly reducing summer maximum temperatures. This enables rapid expansion of M. cerifera across the landscape. As M. cerifera expands, island migration is altered, leading to decreased island stability and increased erosion. Although seedlings are small and relatively vulnerable, this life stage appears to have significant implications for the ecosystem trajectory and stability of the Virginia barrier islands.