Effects of Chemical Deicers on Amphibian Communites

Roads and highways present a unique challenge to wildlife as they exhibit substantial impacts on the surrounding ecosystem through the interruption of a number of ecological processes. With new roads added to the national highway system every year, an understanding of these impacts is required for effective mitigation of potential environmental impacts. A major contributor to these negative effects is the deposition of chemicals used in winter deicing activities to nearby surface waters. These chemicals often vary in composition and may affect freshwater species differently. The negative impacts of widespread deposition of sodium chloride (NaCl) have prompted a search for an `environmentally friendly' alternative. However, little research has investigated the potential environmental effects of widespread use of these alternatives. Herein, I detail the results of laboratory tests and field surveys designed to determine the impacts of road salt (NaCl) and other chemical deicers on amphibian communities in Michigan's Upper Peninsula. Using larval amphibians I demonstrate the lethal impacts of a suite of chemical deicers on this sensitive, freshwater species. Larval wood frogs (Lithobates sylvatica) were tolerant of short-term (96 hours) exposure to urea (CH4N2O), sodium chloride (NaCl), and magnesium chloride (MgCl2). However, these larvae were very sensitive to acetate products (C8H12CaMgO8, CH3COOK) and calcium chloride (CaCl2). These differences in tolerance suggest that certain deicers may be more harmful to amphibians than others. Secondly, I expanded this analysis to include an experiment designed to determine the sublethal effects of chronic exposure to environmentally realistic concentrations of NaCl on two unique amphibian species, L. sylvatica and green frogs (L. clamitans). L. sylvatica tend to breed in small, ephemeral wetlands and metamorphose within a single season. However, L. clamitans breed primarily in more permanent wetlands and often remain as tadpoles for one year or more. These species employ different life history strategies in this region which may influence their response to chronic NaCl exposure. Both species demonstrated potentially harmful effects on individual fitness. L. sylvatica larvae had a high incidence of edema suggesting the NaCl exposure was a significant physiologic stressor to these larvae. L. clamitans larvae reduced tail length during their exposure which may affect adult fitness of these individuals. In order to determine the risk local amphibians face when using these roadside pools, I conducted a survey of the spatial distribution of chloride in the three northernmost counties of Michigan. This area receives a relatively low amount of NaCl which is confined to state and federal highways. The chloride concentrations in this region were much lower than those in urban systems; however, amphibians breeding in the local area may encounter harmful chloride levels arising from temporal variations in hydroperiods. Spatial variation of chloride levels suggests the road-effect zone for amphibians may be as large as 1000 m from a salt-treated highway. Lastly, I performed an analysis of the use of specific conductance to predict chloride concentrations in natural surface water bodies. A number of studies have used this regression to predict chloride concentrations from measurements of specific conductance. This method is often chosen in the place of ion chromatography due to budget and time constraints. However, using a regression method to characterize this relationship does not result in accurate chloride ion concentration estimates.

IMPACTS OF CLIMATE CHANGE ON SOIL MICROORGANISMS IN NORTHERN HARDWOOD FORESTS

As global climate continues to change, it becomes more important to understand possible feedbacks from soils to the climate system. This dissertation focuses on soil microbial community responses to climate change factors in northern hardwood forests. Two soil warming experiments at Harvard Forest in Massachusetts, and a climate change manipulation experiment with both elevated temperature and increased moisture inputs in Michigan were sampled. The hyphal in-growth bag method was to understand how soil fungal biomass and respiration respond to climate change factors. Our results from phospholipid fatty acid (PLFA) analyses suggest that the hyphal in-growth bag method allows relatively pure samples of fungal hyphae to be partitioned from bacteria in the soil. The contribution of fungal hyphal respiration to soil respiration was examined in climate change manipulation experiments in Massachusetts and Michigan. The Harvard Forest soil warming experiments in Massachusetts are long-term studies with 8 and 18 years of +5 °C warming treatment. Hyphal respiration and biomass production tended to decrease with soil warming at Harvard Forest. This suggests that fungal hyphae adjust to higher temperatures by decreasing the amount of carbon respired and the amount of carbon stored in biomass. The Ford Forestry Center experiment in Michigan has a 2 x 2 fully factorial design with warming (+4-5 °C) and moisture addition (+30% average ambient growing season precipitation). This experiment was used to examine hyphal growth and respiration of arbuscular mycorrhizal fungi (AMF), soil enzymatic capacity, microbial biomass and microbial community structure in the soil over two years of experimental treatment. Results from the hyphal in-growth bag study indicate that AMF hyphal growth and respiration respond negatively to drought. Soil enzyme activities tend to be higher in heated versus unheated soils. There were significant temporal variations in enzyme activity and microbial biomass estimates. When microbial biomass was estimated using chloroform fumigation extractions there were no differences between experimental treatments and the control. When PLFA analyses were used to estimate microbial biomass we found that biomass responds negatively to higher temperatures and positively to moisture addition. This pattern was present for both bacteria and fungi. More information on the quality and composition of the organic matter and nutrients in soils from climate change manipulation experiments will allow us to gain a more thorough understanding of the mechanisms driving the patterns reported here. The information presented here will improve current soil carbon and nitrogen cycling models.

Plant-mediated effects on microbial diversity in mesocosms of an oligotrophic bog

Globally, peatlands occupy a small portion of terrestrial land area but contain up to one-third of all soil organic carbon. This carbon pool is vulnerable to increased decomposition under projected climate change scenarios but little is known about how plant functional groups will influence microbial communities responsible for regulating carbon cycling processes. Here we examined initial shifts in microbial community structure within two sampling depths under plant functional group manipulations in mesocosms of an oligotrophic bog. Microbial community composition for bacteria and archaea was characterized using targeted 16S rRNA Illumina gene sequencing. We found statistically distinct spatial patterns between the more shallow 10-20 cm sampling depth and the deeper 30-40 cm depth. Significant effects by plant functional groups were found only within the 10-20 cm depth, indicating plant-mediated microbial community shifts respond more quickly near the peat surface. Specifically, the relative abundance of Acidobacteria decreased under ericaceous shrub treatments in the 10-20 cm depth and was replaced by increased abundance of Gammaproteobacteria and Bacteroidetes. In contrast, the sedge rhizosphere continued to be dominated by Acidobacteria but also promoted an increase in the relative recovery of Alphaproteobacteria and Verrucomicrobia. These initial results suggest microbial communities under ericaceous shrubs may be limited by anaerobic soil conditions accompanying high water table conditions, while sedge aerenchyma may be promoting aerobic taxa in the upper peat rhizosphere regardless of ambient soil oxygen limitations.

USING INDICATORS OF BIOTIC INTEGRITY FOR ASSESSMENT OF STREAM CONDITION

Multiple indices of biotic integrity and biological condition gradient models have been developed and validated to assess ecological integrity in the Laurentian Great Lakes Region. With multiple groups such as Tribal, Federal, and State agencies as well as scientists and local watershed management or river-focused volunteer groups collecting data for bioassessment it is important that we determine the comparability of data and the effectiveness of indices applied to these data for assessment of natural systems. We evaluated the applicability of macroinvertebrate and fish community indices for assessing site integrity. Site quality (i.e., habitat condition) could be classified differently depending on which index was applied. This highlights the need to better understand the metrics driving index variation as well as reference conditions for effective communication and use of indices of biotic integrity in the Upper Midwest. We found the macroinvertebrate benthic community index for the Northern Lakes and Forests Ecoregion and a coldwater fish index of biotic integrity for the Upper Midwest were most appropriate for use in the Big Manistee River watershed based on replicate sampling, ability to track trends over time and overall performance. We evaluated three sites where improper road stream crossings (culverts) were improved by replacing them with modern full-span structures using the most appropriate fish and macroinvertebrate IBIs. We used a before-after-control-impact paired series analytical design and found mixed results, with evidence of improvement in biotic integrity based on macroinvertebrate indices at some of the sites while most sites indicated no response in index score. Culvert replacements are often developed based on the potential, or the perception, that they will restore ecological integrity. As restoration practitioners, researchers and managers, we need to be transparent in our goals and objectives and monitor for those results specifically. The results of this research serve as an important model for the broader field of ecosystem restoration and support the argument that while biotic communities can respond to actions undertaken with the goal of overall restoration, practitioners should be realistic in their expectations and claims of predicted benefit, and then effectively evaluate the true impacts of the restoration activities.

EVALUATION AND MONITORING OF ASH (FRAXINUS SPP.) TOLERANT TO LONG-TERM EMERALD ASH BORER (AGRILUS PLANIPENNIS [COLEOPTERA: BUPRESTIDAE]) EXPOSURE

Invasive insects that successfully establish in introduced areas can significantly alter natural communities. These pests require specific establishment criteria (e.g. host suitability) that, when known, can help quantify potential damage to infested areas. Emerald ash borer (Agrilus planipennis [Coleoptera: Buprestidae]) is an invasive phloem-feeding pest which is responsible for the death of millions of ash trees (Fraxinus spp. L.). Over 200 surviving ash trees were previously identified in the Huron-Clinton Metroparks located in southeast Michigan. Trees were assessed over a four year period and a hierarchical cluster analysis was performed on dieback, vigor, and presence of signs and symptoms, in order to place trees into one of three tolerance groups. The clustering of trees with different responses to emerald ash borer attack suggests that there are different tolerance levels in North American ash trees in southeastern Michigan, and these groups were designated as apparently tolerant, not tolerant and intermediate tolerance. Adult landing rates and evidence of adult emergence were significantly lower in the apparently tolerant group compared with the not tolerant group, but larval survival from eggs placed on trees did not differ between tolerance groups. Therefore, it appears that apparently tolerant trees survive because they are less attractive to adult beetles which results in fewer eggs being laid on them. Trees in the apparently tolerant group remained of higher vigor over the four years of the study. North American ash may survive the emerald ash borer epidemic due to natural variation and inherent resistance regardless of the lack of co-evolutionary history with emerald ash borer.

ANALYSIS OF POPULUS TREMULOIDES CLONAL VARIATION AND DELINEATION IN THE OTTAWA NATIONAL FOREST

The technique of delineating Populus tremuloides (Michx.) clonal colonies based on morphology and phenology has been utilized in many studies and forestry applications since the 1950s. Recently, the availability and robustness of molecular markers has challenged the validity of such approaches for accurate clonal identification. However, genetically sampling an entire stand is largely impractical or impossible. For that reason, it is often necessary to delineate putative genet boundaries for a more selective approach when genetically analyzing a clonal population. Here I re-evaluated the usefulness of phenotypic delineation by: (1) genetically identifying clonal colonies using nuclear microsatellite markers, (2) assessing phenotypic inter- and intraclonal agreement, and (3) determining the accuracy of visible characters to correctly assign ramets to their respective genets. The long-term soil productivity study plot 28 was chosen for analysis and is located in the Ottawa National Forest, MI (46° 37'60.0" N, 89° 12'42.7" W). In total, 32 genets were identified from 181 stems using seven microsatellite markers. The average genet size was 5.5 ramets and six of the largest were selected for phenotypic analyses. Phenotypic analyses included budbreak timing, DBH, bark thickness, bark color or brightness, leaf senescence, leaf serrations, and leaf length ratio. All phenotypic characters, except for DBH, were useful for the analysis of inter- and intraclonal variation and phenotypic delineation. Generally, phenotypic expression was related to genotype with multiple response permutation procedure (MRPP) intraclonal distance values ranging from 0.148 and 0.427 and an observed MRPP delta value=0.221 when the expected delta=0.5. The phenotypic traits, though, overlapped significantly among some clones. When stems were assigned into phenotypic groups, six phenotypic groups were identified with each group containing a dominant genotype or clonal colony. All phenotypic groups contained stems from at least two clonal colonies and no clonal colony was entirely contained within one phenotypic group. These results demonstrate that phenotype varies with genotype and stand clonality can be determined using phenotypic characters, but phenotypic delineation is less precise. I therefore recommend that some genetic identification follow any phenotypic delineation. The amount of genetic identification required for clonal confirmation is likely to vary based on stand and environmental conditions. Further analysis, however, is needed to test these findings in other forest stands and populations.

IONIC LIQUID EXTRACTION UNVEILS PREVIOUSLY OCCLUDED HUMICBOUND IRON IN PEAT POREWATER

Extracellular iron reduction has been suggested as a candidate metabolic pathway that may explain a large proportion of carbon respiration in temperate peatlands. However, the o-phenanthroline colorimetric method commonly employed to quantitate iron and partition between redox species is known to be unreliable in the presence of humic and fulvic acids, both of which represent a considerable proportion of peatland dissolved organic matter. We propose ionic liquid extraction as a more accurate iron quantitation and redox speciation method in humic-rich peat porewater. We evaluated both o-phenanthroline and ionic liquid extraction in four distinct peatland systems spanning a gradient of physico-chemical conditions to compare total iron recovery and Fe2+:Fe3+ ratios determined by each method. Ionic liquid extraction was found to provide more accurate iron quantitation and speciation in the presence of dissolved organic matter. A multivariate approach utilizing fluorescence- and UV-Vis spectroscopy was used to identify dissolved organic matter characteristics in peat porewater that lead to poor performance of the o-phenanthroline method. Where these interferences are present, we offer an empirical correction factor for total iron quantitation by o-phenanthroline, as verified by ionic liquid extraction. The written work presented in this thesis is in preparation for submission to Soil Biology and Biochemisrty by T.J. Veverica, E.S. Kane, A.M. Marcarelli, and S.A. Green.

Big Manistee River Tributaries as Potential Arctic Grayling Habitat

The Big Manistee River was one of the most well known Michigan rivers to historically support a population of Arctic grayling (Thymallus arctics). Overfishing, competition with introduced fish, and habitat loss due to logging are believed to have caused their decline and ultimate extirpation from the Big Manistee River around 1900 and from the State of Michigan by 1936. Grayling are a species of great cultural importance to Little River Band of Ottawa Indian tribal heritage and although past attempts to reintroduce Arctic grayling have been unsuccessful, a continued interest in their return led to the assessment of environmental conditions of tributaries within a 21 kilometer section of the Big Manistee River to determine if suitable habitat exists. Although data describing historical conditions in the Big Manistee River is limited, we reviewed the literature to determine abiotic conditions prior to Arctic grayling disappearance and the habitat conditions in rivers in western and northwestern North America where they currently exist. We assessed abiotic habitat metrics from 23 sites distributed across 8 tributaries within the Manistee River watershed. Data collected included basic water parameters, streambed substrate composition, channel profile and areal measurements of channel geomorphic unit, and stream velocity and discharge measurements. These environmental condition values were compared to literature values, habitat suitability thresholds, and current conditions of rivers with Arctic grayling populations to assess the feasibility of the abiotic habitat in Big Manistee River tributaries to support Arctic grayling. Although the historic grayling habitat in the region was disturbed during the era of major logging around the turn of the 20th century, our results indicate that some important abiotic conditions within Big Manistee River tributaries are within the range of conditions that support current and past populations of Arctic grayling. Seven tributaries contained between 20-30% pools by area, used by grayling for refuge. All but two tributaries were composed primarily of pebbles, with the remaining two dominated by fine substrates (sand, silt, clay). Basic water parameters and channel depth were within the ranges of those found for populations of Arctic grayling persisting in Montana, Alaska, and Canada for all tributaries. Based on the metrics analyzed in this study, suitable abiotic grayling habitat does exist in Big Manistee River tributaries.

Influence of Vegetation Removal and Altering Water Levels on CO2 Flux Rates of a Northern Bog

Boreal peatlands contain approximately one third of the global soil carbon and are considered net sinks of atmospheric CO2. Water level position is one of the main regulators of CO2 fluxes in northern peatlands because it controls both the thickness of the aerobic layer in peat and plant communities. However, little is known about the role of different plant functional groups and their possible interaction with changing water level in boreal peatlands with regard to CO2 cycling. Climate change may also accelerate changes in hydrological conditions, changing both aerobic conditions and plant communities. To help answer these questions, this study was conducted at a mesocosm facility in Northern Michigan where the aim was to experimentally study the effects of water levels, plant functional groups (sedges, shrubs and mosses) and the possible interaction of these on the CO2 cycle of a boreal peatland ecosystem. The results indicate that Ericaceous shrubs are important in the boreal peatland CO2 cycle. The removal of these plants decreased ecosystem respiration, gross ecosystem production and net ecosystem exchange rates, whereas removing sedges did not show any significant differences in the flux rates. The water level did not significantly affect the flux rates. The amount of aboveground sedge biomass was higher in the low water level sedge treatment plots compared to the high water level sedge plots, possibly because the lowered water level and the removal of Ericaceae released nutrients for sedges to use up.

INTERACTIVE EFFECTS OF CLIMATE CHANGE AND FUNGAL COMMUNITIES ON THE DECOMPOSITION OF WOOD-DERIVED CARBON IN FOREST SOILS

Soils are the largest sinks of carbon in terrestrial ecosystems. Soil organic carbon is important for ecosystem balance as it supplies plants with nutrients, maintains soil structure, and helps control the exchange of CO2 with the atmosphere. The processes in which wood carbon is stabilized and destabilized in forest soils is still not understood completely. This study attempts to measure early wood decomposition by different fungal communities (inoculation with pure colonies of brown or white rot, or the original microbial community) under various interacting treatments: wood quality (wood from +CO2, +CO2+O3, or ambient atmosphere Aspen-FACE treatments from Rhinelander, WI), temperature (ambient or warmed), soil texture (loamy or sandy textured soil), and wood location (plot surface or buried 15cm below surface). Control plots with no wood chips added were also monitored throughout the study. By using isotopically-labelled wood chips from the Aspen-FACE experiment, we are able to track wood-derived carbon losses as soil CO2 efflux and as leached dissolved organic carbon (DOC). We analyzed soil water for chemical characteristics such as, total phenolics, SUVA254, humification, and molecular size. Wood chip samples were also analyzed for their proportion of lignin:carbohydrates using FTIR analysis at three time intervals throughout 12 months of decomposition. After two years of measurements, the average total soil CO2 efflux rates were significantly different depending on wood location, temperature, and wood quality. The wood-derived portion soil CO2 efflux also varied significantly by wood location, temperature, and wood quality. The average total DOC and the wood-derived portion of DOC differed between inoculation treatments, wood location, and temperature. Soil water chemical characteristics varied significantly by inoculation treatments, temperature, and wood quality. After 12 months of decomposition the proportion of lignin:carbohydrates varied significantly by inoculation treatment, with white rot having the only average proportional decrease in lignin:carbohydrates. Both soil CO2 efflux and DOC losses indicate that wood location is important. Carbon losses were greater from surface wood chips compared with buried wood chips, implying the importance of buried wood for total ecosystem carbon stabilization. Treatments associated with climate change also had an effect on the level of decomposition. DOC losses, soil water characteristics, and FTIR data demonstrate the importance of fungal community on the degree of decomposition and the resulting byproducts found throughout the soil.