Host and Environmental Drivers of Foliar Fungal Abundance and Composition in Coastal Douglas-fir

Relative to the land area they cover, forests account for an outsized proportion of the world’s terrestrial biomass. Across biomass-dense temperate forests, anthropogenically induced climate change is predicted to shift the ranges of several tree species, including the widely distributed Pseudotsuga menziesii (Mirbel) Franco. While abiotic factors drive these current predictions, P. menziesii forests are also increasingly affected by foliar fungal disease as winters become warmer. However, little is known about how the broader community of P. menziesii foliar fungi, which may include mutualists, is being impacted by a changing climate. Study of foliar fungal communities in this context is also complicated by the propensity of needles to grow and age variably within different sections of tree crowns, creating the conditions for community assembly processes to operate at multiple spatial and temporal scales. Additionally, plant populations coevolve with their communities, resisting or tolerating fungi to varying degrees, and emphasizing or counteracting the selective forces of the surrounding environment. This dissertation uses both observational and experimental studies to investigate how P. menziesii var. menziesii foliar fungal communities are shaped by host genetic and environmental factors. Old-growth P. menziesii var. menziesii forest in the U.S. Pacific-Northwest is characterized as having large trees with complex crown structures, created from repeated crown disturbance and subsequent infill by epicormic branches and reiterated trunks. Proceeding from the treetop to lower heights deeper in the crown, the amount of penetrating sunlight decreases, air and leaf temperatures become cooler and more stable, leaves stay wet or dry for longer, and needle physiology and morphology diverge. In addition to creating a complex environmental gradient along which foliar fungal taxa can sort, the vertical structure of P. menziesii crowns also provides an axis along which dispersal can occur. At the same time, P. menziesii needles occurring at each position in the crown are aging, potentially allowing deterministic community assembly processes to exert their influences over time. While studies of the vertical stratification of communities within tree crowns has precedent, the factors correlated with within-crown changes in foliar fungal communities are still poorly understood. Additionally, given the complexity of old-growth crowns, uncertainty exists around whether the vertical position measured in the tree crown adequately reflects aspects of the crown microenvironment that may influence community assembly. As described in Chapter 2 of this dissertation, needles from four age classes were sampled at incremented heights within the crowns of trees from different sites across an old-growth forest. To better define the crown microenvironment, airborne light detection and ranging (LIDAR) data were used to obtain an estimate of the crown closure occurring above each sampled point in the crown. Needle communities were characterized by sequencing the ITS2 subregion amplified from DNA extracts, and the relationships between community properties (alpha-diversity, composition, and structure between needle ages) were tested. These results showed that diversity and richness increased with increasing crown closure and that older needles were also richer. Compositions differed along gradients in crown closure, but these results depended on the age of the needle. Portions of crowns with lower crown closure were dominated by Nothophaeocryptopus gaeumannii and did not converge on a stable community structure over four years of needle cohorts. Conversely, portions of the crown with greater closure were dominated by Rhabdocline parkeri and converged on a stable community structure as needles got older. The natural distribution of P. menziesii var. menziesii spans approximately 20 degrees of latitude and nearly two kilometers in elevation. Although temperatures gradually increase north to south, the temperature gradient west to east is more pronounced, with temperatures decreasing and seasonally varying with increasing elevation and distance from the coast. Accessions of P. menziesii var. menziesii collected from across its range show genetically differentiated phenotypes for traits related to phenology and growth, reflecting signatures of local adaptation. Although recent research has found a genotypic basis for resistance and tolerance to disease caused by foliar fungal pathogens, it is still unclear how foliar fungal communities more broadly respond to trees with varying genotypic backgrounds across fluctuating environments, and whether these observed communities reflect local adaptation in their hosts. Contributing additional complexity to the delimitation of host and environmental influences on foliar fungal communities, a growing body of literature recognizes the utility of abundance data for identifying differences between communities that would otherwise go undetected with analyses of compositional data acquired from standard high-throughput amplicon sequencing approaches. Whether this utility can be achieved for foliar fungal communities of P. menziesii var. menziesii remains to be seen. In order to control for the effects of the sampling environment and host genotypic background on foliar fungal communities, Chapters 3 and 4 leverage a network of six P. menziesii var. menziesii common gardens, from which second-year needles were sampled. For Chapter 3, needles from two P. menziesii var. menziesii common gardens were sampled three years in a row from the same trees, and a recently described metabarcoding technique was adapted for use with the fungal ITS2 subregion to obtain fungal load estimates for each fungal taxon occurring in each needle sample. These results showed that communities were dominated by four major foliar fungal taxa that differed in their abundances across sampling years, gardens, and host ecotypes. Joint species distribution modeling revealed that sampling year and host ecotype terms did not interact, rejecting the hypothesis that fluctuating environments affect how hosts influence the assembly of their foliar fungal communities. Instead, the communities at the two gardens showed different patterns in fungal occurrence and abundance over the sampling period, with montane gardens experiencing the greatest turnover. Using the same common garden network and load-based metabarcoding technique in Chapter 4, needles from six P. menziesii var. menziesii common gardens were sampled in a single year and processed using the same metabarcoding technique to characterize needle communities. These results rejected the hypothesis that local populations host different communities compared to those of foreign populations grown at the same gardens. Instead, results indicated that ecotypic differences existed between coastal populations and montane populations. This ecotype effect operated independently from the effect of the garden environment. Further, dominant fungal taxa demonstrated distinct responses to different ecotypes.
KEYWORDS: Epiphytes, Endophytes, Phyllosphere, Mycology, Pinaceae, Ecology, Fungi, Metabarcoding