Fungi are ubiquitous members of any natural community, whether it be a forest, a grassland, a coral reef, or the gut of a beetle. Where species are found tells us more than just the limits of their distribution but also gives us important clues about what those species are doing in their environment and the evolutionary journey they took to get there. This is especially true for species that have evolved closely knit, symbiotic relationships with other organisms such as plants, invertebrates, mammals, bacteria, and other fungi. Especially in these symbiotic relationships, fungi must access carbon and other nutrients in very different ways that interact with their host at a genetic level to either circumvent host-defenses in the case of antagonism or to coordinate mutual benefit in the case of mutualisms (Quiza et al., 2015). Mycorrhizae, one of the six key traits of fungi, are a model system for studying and understanding symbiosis (Nagy, 2018). My research interests are broadly focused on understanding patterns of distribution, diversification, and functionality of ectomycorrhizal species using molecular approaches. These interests can be broken down into three main focuses for my proposed research:
1) How does geographic distribution, life history, morphological adaptation, and host coevolution drive diversification in ectomycorrhizal fungi?
2) What are the functional differences at a genomic, transcriptomic, and metabolomic scale within and between independent lineages of ectomycorrhizal fungi?
3) How is host selection and specificity mediated differently at a molecular level by species?
Systematics, biogeography, and diversification of ectomycorrhizal fungi
The ectomycorrhizal habit has been identified as a key innovation that has promoted a higher diversification rate in mushroom-forming fungi (Sánchez-García & Matheny, 2017). Among the most species rich genera of mushroom-forming fungi are the iconic ectomycorrhizal lineages Cortinarius, Russula/Lactarius, Tricholoma, Amanita, and Inocybe. The ecological and evolutionary processes that have contributed to diversification in these groups is poorly understood, primarily due to the difficulty of obtaining well-populated datasets. As part of my dissertation research at the University of Tennessee, we used a megaphylogeny approach to infer a global phylogeny of the genus Russula and we found that an overall higher net diversification rate driven by host switching and not by long distance dispersal has driven the diversification of this lineage in the temperate zone (Looney et al., 2016). While this study was able to capitalize on global amplicon data, taxon sampling was still patchy. To more closely examine the evolutionary drivers of diversification in Russula, a subgroup called subsection Roseinae was selected to test whether reproductive isolation in glacial refugia acted as a species pump in the eastern United States (Looney et al., in prep.). It is my objective to expand this approach to other clades within Russula as well as other groups to test evolutionary hypotheses about diversification of ectomycorrhizal fungi in relation to their traits and functions.
I will use a multiplexing long-read amplicon sequencing approach using the Pacbio Sequel technology for MLST (multilocus sequence typing) to efficiently and cost-effectively produce large datasets for phylogenetic analyses (Chen et al., 2015). These datasets will be ideal for systematics and can be used for novel biodiversity discovery using coalescent-based approaches and the description of new taxa to science. They will also be ideal for inferring the past, current, and future distribution of these taxa in response to environmental factors and global climate change. This research will rely heavily on the use of collections housed in herbaria as well as citizen science projects such as the North American Mycoflora Project (NAMP) and the NAMA voucher project, to which I am contributing.
An evolutionary approach to functional diversity of ectomycorrhizal fungi
The age of genome-enabled mycology has been ushered in thanks to the efforts of the one thousand fungal genomes project (1KFG) and the Joint Genome Institute (JGI) of the DOE. Through the 1KFG and in collaboration with Oak Ridge National lab where I spent a year of my PhD researching as an SCGSR fellow, I produced a novel dataset of standard draft genomes for Russulaceae that represents the most extensively sampled lineage of mycorrhizal fungi to date (Looney et al., 2018). This dataset was proposed to answer whether we can detect functional diversity within an ectomycorrhizal lineage. To do this, I performed comparative genomic analyses of this dataset comparing it to closely related saprotrophic lineages within Russulales to test whether genomic architecture and gene content differed between the ectomycorrhizal and saprotrophic members (Looney et al., in prep.). For this analysis, we focused on secreted proteins, including CAzymes, proteases, lipases, and small secreted proteins and found an enrichment in specific orthologous clusters, indicating a conserved function for the ectomycorrhizal Russulaceae. We also detected extensive genome reorganization in ectomycorrhizal genomes, including increase of synteny, increased repeated content, and an association between transposable elements and genes for secreted proteins, all of which are inferred to have occurred before the actual evolution of the ectomycorrhizal habit.
Building on this dataset through PMI are a number of studies where we have leveraged an associated culture collection to test hypotheses generated by this genomic analysis, including the isolation and identification of novel sesquiterpenoid compounds and lipo-chitin oligosaccharides that are key for establishment of the symbiotic interaction. For the Populus system, we are currently sequencing major ectomycorrhizal associates to inform metatranscriptomic data from a GWAS population of P. trichocarpa. The associates are also in culture and we are planning to use them to investigate stable ectomycorrhizal community function in association with different plant genotypes.
Host selection and specificity in ectomycorrhizal associates of pine and poplars
In collaboration with PMI and the Vilgalys Lab at Duke I am currently working on two complimentary systems for looking at plant-microbe interactions between ectomycorrhizal communities and their host. Over the past three years we have visited natural stands of Populus trichocarpa in WA and OR as well as P. nigra in France to collect soil, fine roots, and sporocarps of associated fungi to develop a culture collection and for amplicon sequencing. We have compared ectomycorrhizal communities and find a restricted set of species from a few lineages that are commonly associated with these trees, with sister taxa being shared across continents. To leverage the culture collection we have established a split-root system to look at mycorrhization and plant growth effects on numerous plant genotypes as well as a gnotobiotic system for mycorrhizal synthesis and transcriptome profiling. For the pine system I am working with a network of collaborators on an NSF-funded project that seeks to understand the co-evolutionary dynamics between pine and the host-specific genus Suillus. To exploit this system we are looking at pine plantation invasions in Australia, New Zealand, and South America to track the origins of the introduced species and differences in ecosystem function between introduced and native sites. These communities are great systems because they have a reduced community, where these invasive species have succeeded in colonizing and competing with the native community. The aim is to understand the mechanisms that facilitated this invasion and the adaptation of these ECM to these environments.