This dissertation examines fungal genetics in two systems: Penicillium chrysogenum and Rhizopogon salebrosus. Penicillium chrysogenum holds historical and medical significance for the discovery and development of penicillin as an antibiotic. Rhizopogon salebrosus is an ecologically important mycorrhizal fungus that forms symbioses with both green plants and non-photosynthetic mycoheterotrophic plants. Mycoheterotrophs, plants that parasitize mycorrhizal fungi and cheat the classical mycorrhizal symbiosis, have been integral to the discovery and continuing study of mycorrhizal fungi.

The first chapter describes the utilization of assembly-free synteny analysis to identify rearrangements in wild P. chrysogenum strains. While traditional methods of mutagenesis and selection have been effective in improving production of compounds to commercial scale, the genetic changes behind the altered phenotypes have remained largely unclear. We utilized high-throughput Illumina short read sequencing of a wild Penicillium chrysogenum strain in order to make whole genome comparisons to a sequenced improved strain (WIS 54-1255). We developed an assembly-free method of identifying rearrangements and validated the in silico predictions with a PCR-based assay followed by Sanger sequencing. In addition to finding a previously published inversion in the penicillin biosynthesis cluster, we located several genes related to penicillin production associated with these rearrangements. By comparing the configuration of rearrangement events among several historically important strains known to be good penicillin producers to a collection of recently isolated wild strains, we suggest that wild strains with rearrangements similar to those in known good penicillin producers may be viable candidates for further improvement efforts.

The second chapter describes use of the phytohormone gibberellic acid (GA), a phytohormone which induces germination in many plants, to induce asymbiotic germination of two obligate mycoheterotrophic plants. These plants had previously only been seen to germinate in the presence of a symbiotic fungus, and such limitations prevented further study with these plants. We found that GA stimulates Pterospora andromedea germination at frequencies that can be far higher than that achieved with its fungal symbiont, R. salebrosus, alone. With constant exposure to 0.5M GA, we found nearly 80% germination, as opposed to only 15% with R. salebrosus. Even a short exposure to GA is sufficient for significantly enhanced germination. One day of exposure to 0.5mM GA induced ~55% stage 3 germination, and three days of exposure led to ~ 70% stage 3 germination. Exposing seeds to a lower dosage (0.1mM GA) for a longer time (2 weeks) produced a comparable germination rate. The closely related plant Sarcodes sanguinea was more resistant to GA stimulation. Exposure to GA is the only known method of inducing monotrope germination without the presence of a specific fungal symbiont. GA stimulation achieved workable germination rates for further studies, and GA may also be used as an improved assay for seed viability.

In the third chapter we utilize GA germination of P. andromedea seeds to examine transcriptional differences in R. salebrosus with partner and non-partner mycohterotroph seeds. The monotropes constitute a group of obligately dependent, non-photosynthetic mycoehterotrophs with extreme host specificity. Here we generate a de novo transcriptome assembly and examine differential expression when R. salebrosus interacts with seeds from partner (R. salebrosus type) and non-partner (R. arctostaphyli type) P. andromedea. Because P. andromedea seeds are very small, with few nutritional resources, germination in the presence of a suitable host fungus is a critical step in the establishment of symbiosis. We have identified putative genes in categories thought to be important for ectomycorrhizal symbiosis. These include stress response, host defense, cellular transport, secretion, carbohydrate metabolism and transport, and signal transduction. To our knowledge, this is the first study of gene expression with any mycorrhizal fungus involved with a mycoheterotrophic plant.

For my dissertation I focused on two key questions central to fungal diversity: the role of

dispersal in fungal community assembly, and the response of fungi to catastrophic disturbances. As we enter the Anthropocene and catastrophic disturbances are increasing in frequency, it is important to understand how fungi will respond to disturbances since they are major drivers of many ecological processes such as decomposition and forest function. To do this, I utilized large scale-sampling and ITS amplicon-based metagenomics to examine the environmental drivers of ectomycorrhizal fungal spore bank diversity across the North American continent. Two of my study plots were then burned in a catastrophic wildfire. Thus I was able to examine ectomycorrhizal fungal spore bank recovery after the fire, and I made a case for a group of fire fungi, adapted to wildfires.

To examine the role of dispersal in fungal community assembly, I used a high elevation

subalpine basin located in Yosemite National Park where isolated congeneric pine trees have established at varying distances from each other and the forest edge. Since ectomycorrhizal fungi are obligate symbionts of trees, isolated trees are islands from the perspective of the fungi. I used these well-replicated “islands” to test the theory of island biography in host-associated fungi. Moreover, because two distantly related pine species co-occurred in varying distances from each other and the forest edge, I could disentangle host from distance and other environmental factors to determine the drivers of community structure and beta-diversity in the ectomycorrhizal fungi associated with the trees. To do so, I determined fungal community composition with Illumina MiSeq sequencing of ITS amplicons, and applied generalized dissimilarity modeling (GDM), a non-linear form of matrix regression, to test whether total and ectomycorrhizal fungal (EMF)

communities were primarily structured by distance decay, host, seasonality, or edaphic

environmental filtering. I identified soil nutrient environment as a stronger predictor of EMF

community composition than host identity. I found pH and organic matter to be the strongest predictors of EMF, and total fungal composition. This result fits an emerging paradigm that soil pH is a major driver of microbial community composition.

The original inspiration for this dissertation was to resolve the over two hundred year-old debate regarding the trophic nature of the leafless and non-photosynthetic species of Pyrola, Pyrola aphylla Sm. (Ericaceae). To do this, I integrated the use of molecular methods and the analysis of stable isotopes to examine the role of mycorrhizal fungi in the carbon acquisition strategies of P. aphylla as well as its closely related and leafy-green sister taxa P. picta Sm. and Chimaphila umbellata (L.) W. Bartram. My goals were to: 1) determine if P. aphylla was fully mycoheterotrophic and therefore dependent on mycorrhizal fungi to meet all of its carbon and nutrient demands (Chapter 1); 2) identify the mycorrhizal fungi associated with P. aphylla and P. picta (Chapter 2); 3) determine if P. picta and C. umbellata were partially mycoheterotrophic meeting their carbon demands both through photosynthesis and mycorrhizal fungi (Chapters 2 and 3) and; 4) examine the role of increased shade on the degree of mycoheterotrophy in P. picta and C. umbellata (Chapter 3).

The novel findings of this work include a new example of full mycoheterotrophy in Ericaceae based on the similarity of the carbon and nitrogen stable isotope values of P. aphylla to other full mycoheterotrophs that associate with ectomycorrhizal fungi. Surprisingly, unlike all other fully mycoheterotrophic plants within Ericaceae based on the DNA sequence analysis of the fungi associated with P. aphylla, this species has not specialized on a particular lineage of ectomycorrhizal fungi. Rather, P. aphylla associates with a phylogenetically broad suite ectomycorrhizal fungi and there is little to no overlap in fungal symbionts of this species across its geographic range. Pyrola picta was also found to associate with a diversity of ectomycorrhizal fungi indicating that fungal specificity is not necessarily a precursor to the loss of photosynthesis among mycoheterotrophic plants. Furthermore, both P. picta and C. umbellata have nitrogen stable isotope values that are indistinguishable from fully mycoheterotrophic species in Ericaceae, while their bulk leaf carbon stable isotope values are most similar to autotrophic plants in the forest understory. These findings led to the final experiment of this dissertation where leaf soluble sugars of P. picta and C. umbellata were extracted and analyzed for their carbon stable isotope values. This experiment was conducted on populations of P. picta and C. umbellata in the foothills of the Sierra Nevada Mountains in California. In the field, light availability and access to mycorrhizal networks were manipulated to determine if the degree of dependency on fungal carbon gains in the two test species increased under decreased light availability. This study revealed that P. picta and C. umbellata have disparate ecophysiologies that are potentially related to their C-acquisition strategies. Based on the out-put of an isotope mixing model C. umbellata showed no indications of mycoheterotrophy while P. picta is partially mycoheterotrophic, but a decrease in light availability did not significantly increase the fungal-C sink strength of this species.

Based on the outcomes of this dissertation, future work on mycoheterotrophic plants should include: examining within an coevolutionary framework the significance of fungal specificity for mycoheterotrophy, determining what factors-environmental or physiological, lead to the nitrogen stable isotope values of these plants, and gaining a better understanding of the biochemical pathways and transformations of carbon and nutrients in this symbiosis. The tribe Pyroleae provides a useful group with which to explore these lines of research, as it is the only group of eudicots found thus far that contains species across the spectrum from autotrophy to full mycoheterotrophy.

This dissertation explores the diversity, ecology and functional diversity of saprotrophic fungal across different plant community types. Saprotrophic fungi are responsible for recycling the majority of carbon from dead organic matter and have the unique ability to break down and release nutrients that can then be readily available for other organisms. A number of different biotic and abiotic factors affect the structure of saprotrophic fungal communities. Here, we investigated the effects of nutrient addition, substrate availability, and dominant plant community type on the structure and diversity of saprotrophic fungal communities. In addition, we investigated the diversity and structure of functional diversity of saprotrophic fungal communities.

The first chapter explores saprotrophic fungal diversity found in leaf litter in a diverse lowland tropical forest. We investigated the affect of nutrient addition on the saptrotrophic fungal community using a replicated factorial N, P, K, micronutrient fertilization experiment. We found that the control plots had the lowest alpha diversity compared to treatments that received fertilization. The long-term addition of nutrients increased species richness relative to controls and had an effect on taxonomic composition of the leaf litter fungal communities at lower taxonomic levels, but not at higher taxonomic levels.

The second chapter characterizes saprotrophic fungal communities on straw and wood substrates across forest and grassland plant community types and characterized how these communities changed over time as substrate quality changed. The goal was to determine if substrate, space, time or plant community were the major determinants of fungal saprotrophic community composition. We found that the wood substrates collected in the grassland and forest had the highest richness, estimated richness and phylogenetic diversity. Overall, there was a decrease in richness on the substrates in both plant communities over time, and plant community type appears to have a greater influence on saprotrophic fungal community structure than substrate type or substrate quality.

In the third chapter, we investigated the diversity of saprotrophic basidiomycete laccase genes in grassland and forest plant community types and on straw and wood substrates. Functional diversity was assessed by the functional gene encoding laccase. Laccases play an important role in soil organic matter turnover and are able to completely degrade lignin. We found that the wood substrates had significantly higher richness of estimated OTUs as compared to the straw substrates. Laccase gene diversity was compared to basidiomycete diversity. We also found that both the laccase genes and basidiomycete assemblages associated strongly with plant community type.

This dissertation explores symbiotic microbial community ecology. Symbiosis is a prominent, and yet relatively under-studied phenomenon between two or more organisms. The scope of symbiosis is wide ranging, occurring between macro-macro organisms, micro-micro organisms, macro-micro organisms and everything in between. The span of symbiosis is great, from parasitic to mutually beneficial relationships between the organisms involved. The three chapters presented here comprise an initial look into symbiotic ecosystems using cutting edge sequencing technology. The studies aimed to discover interactions between bacterial microbes and their eukaryotic partners.

Chapter 1 describes the microbes that live in the gut of the passalid beetle, Odontotaenius disjuctus. This beetle feeds only on wood, which is low in nutrients, particularly nitrogen. Parallel to the termite-microbe system, I hypothesized that these microbes assist the beetles in digestion of the wood. PhyloChip microarray technology was used to characterize the microbial communities. I found that each gut region (there are 4) of the beetle contained a different microbial community, and that the anterior hindgut of the beetle contained strong anaerobic signals whereas other parts of the gut were more aerobic. The microbes that live in each of these gut regions reflect the oxygen availability of that environment. There was also a signal of potential nitrogen fixation in the anaerobic anterior hindgut.

In chapter 2 and 3, I explored fungal-bacterial interactions. Fungal-bacterial interactions have been sparsely characterized, appearing sporadically in the literature. Some studies describe bacteria found on the outside of the fungal hyphae, other times on the inside. One remarkable study found that a pathogenic fungus was not pathogenic without its toxin-producing endosymbiont. Other studies focused on the interaction between leaf-cutting ants, fungi, and bacteria associates. Of the estimated 1.5 million species of fungi, this interaction must be tremendously widespread and waiting to be characterized.

In chapter 2, I explored the microbial communities that live with various ectomycorrhizal fungi. Mycorrhizal fungi exchange mineral nutrients with plants for photosynthetic carbon. The experiments were designed to test for species and/or community specificity of bacteria to their fungal hosts. Several studies have aimed to answer similar questions, but the difference between the experiments presented in this chapter and others is that this involved temporal component, many more samples, and 454 sequencing that produced many orders of magnitude more sequences. The results showed that fungal species strongly determines the bacterial community. Burkholderia and members of the Rhizobiales were the most commonly encountered bacteria. Some of these have been found by other researchers, indicating a tight relationship between them.

And finally, in chapter three, I explored the interactions between saprobic fungi that form fungal mats in the soil and the bacteria that live with them. I tested for differences in bacterial community between fungal mat and non-mat leaf litter, whether fungal species has an influence on the bacterial community, and whether any distinct taxonomic group of bacteria were associated with the fungi. 454 pyrosequencing technology was employed for this purpose. The results showed that there is a strong difference in bacterial community composition between mat and non-mat litter. The Actinobacteria, particularly a Pseudonocardia species, showed strong preference for fungal mats. Pseudonocardia species are known for their antibiotic production and have been reported growing with other organisms, the most famous being the interactions between leaf-cutting ants, fungi, and Pseudonocardia.

Together, these three chapters provide different windows in which to peer into the world of microbial symbiosis, particularly those of bacteria with animal, plants, and fungi. It can be concluded that different symbiotic environments will select for different communities of bacteria, such as different gut sections and the presence of different species of fungi. When high throughput sequences of these environments are examined carefully, they can reveal clues into the important organisms that persist and participate in the ecology of said environments, priming for more extensive studies in symbiosis.

Abstract

Testing the role of arbuscular mycorrhizal fungi in plant adaptation to serpentine soil

by

Shannon Peters Schechter

Doctor of Philosophy in Microbiology

University of California, Berkeley

Professor Thomas D. Bruns, Chair

This dissertation explores a new theoretical and experimental framework in which plant edaphic adaptation is mediated through arbuscular mycorrhizal fungi (AMF). The first chapter describes the primary ecological relationship between adapted plants and AMF by examining the AMF assemblages associated with field populations of serpentine and non-serpentine adapted ecotypes of California native plant Collinsia sparsiflora. The second chapter tests for plant-fungal specificity between C. sparsiflora ecotypes and serpentine and non-serpentine AMF using a common garden greenhouse experiment. Chapter three tests if soil edaphic factors alone could shape distinct serpentine and non-serpentine AMF assemblages by sampling non-C. sparsiflora root AMF assemblages from adjacent serpentine and non-serpentine sites. The final chapter addresses the functional role of AMF in serpentine adaptation with a greenhouse experiment using serpentine and non-serpentine AMF and C. sparsiflora ecotypes grown in sterilized serpentine soil.

I found that serpentine and non-serpentine adapted ecotypes of C. sparsiflora associate with distinct AMF assemblages- an Acaulospora 1OTU- dominated serpentine, and a Glomus 1 OTU-dominated non-serpentine plant ecotype AMF assemblage (Chapter 1). However, I also found a relationship between plant ecotype AMF assemblage and soil nutrients. Thus, this distinction between plant ecotype AMF assemblages might be explained two ways: 1) the plant ecotypes have a high specificity for particular AM fungi within a ubiquitous soil assemblage or 2) the plant ecotypes were tapping non-specifically into AMF assemblages shaped by edaphic factors.

I tested the first scenario in Chapter 2 by growing C. sparsiflora serpentine and non-serpentine ecotypes in a common pool of serpentine and non-serpentine AMF and then identified the root AMF of each plant ecotype. I found that the mixing of serpentine and non-serpentine AMF soil inoculum as the source of the common garden resulted in a non-serpentine soil type. Consequently, while the C. sparsiflora ecotypes associated with distinct AMF assemblages within the common garden, overall the ecotype AMF assemblages resembled that of a non-serpentine soil (i.e. Glomus 1 dominated). Therefore, I found no evidence of host-specificity between C. sparsiflora serpentine and non-serpentine ecotypes and serpentine and non-serpentine AMF. However, these results do indicate that the soil may select the AM fungi and potential for host choice of AMF based on soil type is present.

I tested the second scenario in Chapter 3. I found that serpentine and non-serpentine AMF assemblages are distinct from each other. Variance partitioning analysis showed that both soil edaphic factors (33.5%), and plant assemblages (25.6%) drove the distinction between serpentine and non-serpentine AMF assemblages. This study confirms that there is a strong ecological relationship between AMF and plant tolerance to serpentine soil - plants growing in serpentine soil associate with serpentine-tolerant AMF taxa.

Finally, I tested for a functional difference between serpentine and non-serpentine AMF assemblages that directly impact C. sparsiflora growth and fitness on serpentine (Chapter 4). Only shoot dry weight showed a significant response to AMF source. I found that serpentine AMF significantly increased growth of hosts over non-serpentine AMF and AMF-free controls. This indicates that serpentine AMF have a specialized adaptation to serpentine conferring growth enhancement to hosts, but it is still unclear what this adaptation is or which function is contributing to growth enhancement. I also found trends that imply that C. sparsiflora serpentine adapted ecotypes have a greater response to AMF than non-serpentine ecotypes, but these trends were not significant.

Ectomycorrhizal communities may play a major role in preventing decreases in forest productivity associated with the depletion of nutrients caused by anthropogenic nitrogen and in facilitating increased productivity in response to elevated atmospheric CO2 concentrations. In my doctoral research I attempted to shed some light on the following question: As nutrient demand by forest trees is altered by human induced global change how will the functioning of ectomycorrhizal communities respond?

In chapter 1, I present the results of a study of the effects of nitrogen fertilization on ectomycorrhizal communities in an Eastern US hardwood forest. I found the ectomycorrhizal communities of the organic and mineral horizon to be quite distinct, and that high, but not moderate levels of nitrogen fertilization altered the ectomycorrhizal community composition and decreased ectomycorrhizal species richness. I also found that ectomycorrhizal colonization intensity increased in the mineral soil, and when considered in conjunction with other studies conducted in the same research forest, this may indicate that the ectomycorrrhizal community is shifting in accordance with the shifting nutrient demands of the forest. In Chapter 2, I attempted to elucidate the role of soil heterogeneity in shaping fungal community composition in the mineral soil. Depth and soil carbon content were consistently correlated with fungal community composition. The parent material from which the overlying soil is derived and soil calcium content may be important in determining fungal community composition but our sampling scheme did not allow us to isolate these chemical factors from the potential influence of geographic location on fungal community. In chapter 3, I present a literature review that endeavored to determine whether there is potential for ectomycorrhizal communities or individuals to alter their mineral weathering capabilities and nutrient provision to host plants in response to altered nutrient demand from their host plants. Knowledge of the mechanisms that control belowground carbon allocation by plants in response to nutrient demand is limited, but there is a potential for plants to respond to increased demand of phosphorous and potassium by allocating more carbon to the fungal symbionts most adept at providing these nutrients. Future studies on ectomycorrhizal weathering should explicitly test the role of host nutrient demand in stimulating fungal weathering. In chapter 4, I investigated the role of plants, ectomycorrhizae, and low molecular weight organic acids in stimulating mineral weathering, as well as the potential for elevated carbon dioxide to affect biotic weathering. We found that plants, but not their associated ectomycorrhizae, stimulated weathering. Elevated CO2 did not affect weathering rates. The lack of an effect of ectomycorrhizal colonization may have been due to low levels of mycorrhizal colonization. The biotic weathering observed in this study was driven by the uptake of nutrient cations, and not by substrate acidification or root exudation. My doctoral research suggests that ectomycorrhizae may play an important role in mineral nutrient provision.