The classification of repeat elements generated by RepeatMasker is shown in Table 3.Our assembly is the first sequenced genome in the genus Arctostaphylos, representing the initial step toward understanding genetics and adaptation in this highly diversified genus. In addition to enhancing ongoing phylogenetic and conservation research, this assembly will enable investigations of the genetic basis of adaptations including drought tolerance and fire resilience. These traits, which are ecological hallmarks of manzanitas, are of growing importance in the context of the increased drought and fire frequency and intensity that are occurring in California as a result of climate change. This assembly can also serve as a reference for studying the diversification and population genetics of Arctostaphylos, which may shed light on aspects of diversification in other complex groups of the CFP. Arctostaphylos is the third genus with a genome assembly in the heath family, Ericaceae, following release of assembled genome sequences of Rhododendron and Vaccinium . These two genera are of significant economic importance: many species, hybrids, and cultivars of Rhododendron, including rhododendron and azalea, are important landscape and ornamental plants, dutch buckets for sale and the fruits of many Vaccinium species, which include cranberry, blueberry, and huckleberry, are consumed by humans and other animals.

Species in the Ericaceae are also notable for their ability to tolerate acidic and nutrient-poor soils that often characterize boreal forests and bogs, allowing them to thrive in habitats that are inaccessible to most species. Their tolerance for these conditions is due in part to the formation of mutualistic associations between the roots of the plants and soil fungi of a type unique to the heath family known as ericoid mycorrhizae. Ericoid mycorrhizae are distinct from common mycorrhizal associations found in most angiosperms, and are far less well understood . Complete genome sequences from three genera in this family will provide a strong foundation for investigating the basis of this unique mutualism and its ability to promote survival in inhospitable soils. The size of the A. glauca assembly is 547Mb, which is similar to the two Rhododendron genomes and half that of V corymbosum, which is tetraploid . The tetraploid nature of V. corymbosum also explains the vastly greater number of duplicated genes in its assembly compared to the two diploid assemblies. The scaffold N50 of the A. glauca assembly is longer than R. williamsianum, and close to R. simsii and V. corymbosum, suggesting that the contiguity of Arctostaphylos is comparable to the other taxa . Analysis using RepeatModeler indicated that 57.71% of the A. glauca genome is composed of different categories of repetitive elements . In contrast, analysis using RepeatModeler identified only 26%, 47.5%, and 44.3% of the genome comprising repeat elements in R. williamsianum, R. simsii, and V. corymbosum respectively.

The BUSCO completeness assessment of the A. glauca assembly is higher than R. williamsianum and close to the V. corymbosum , indicating that our final assembly is high quality . Overall, the A. glauca, R. simsii and V. corymbosum genomes are of comparably high contiguity and completeness. The lower contiguity and completeness of the R. williamsianum genome may be due to the lack of HiFi or other longread data in the assembly. This explanation is consistent with other studies demonstrating improved assembly with the inclusion of longer reads . Although the big berry manzanita is a common and widespread species, nearly half of the 60+ manzanita species are rare or threatened. Many are now represented by only one or two populations, and are thus vulnerable to complete eradication by the increasingly common and intense wildfires experienced across California each year. Our manzanita genome sequence will help fulfill the overall goal of the CCGP, serving as a key resource to assess genetic diversity in these threatened endemics and move forward with coordinated conservation programs. According to the most conservative estimates, global mean annual temperatures are now outside the historic range of the last 1,300 years . Simultaneously, mean annual precipitation has declined in many parts of the Northern Hemisphere, resulting in increased drought events . Extreme climatic shifts are predicted to affect, both directly and indirectly, bio-geochemical cycling, energy fluxes, wildlife habitat, and ecosystem goods and services on a global scale . An important component in preparing for the effects of these events is to understand how communities will change in response to them, making this a critical topic for ecological research . For species to survive in dry climates, they must have evolved drought tolerance mechanisms . However, extreme climate events can expose species that are typically capable of withstanding regular drought stress to conditions outside of their normal range. Furthermore, physiological responses to extreme drought can also have a negative feedback on plants’ defensive abilities, rendering them susceptible to biotic attack including by insects or disease agents .

Consequently, synergies between extreme climatic events and biotic attack will likely lead to more dramatic changes than would otherwise occur in historically “drought tolerant” plant communities . Future climate change is expected to exacerbate these interactions worldwide. . Widespread tree mortality from drought has been documented in forested systems around the world , and biotic attack has been associated with many of these events . However, much less focus has been given historically to understanding the consequences of extreme drought on shrubland communities like chaparral , particularly in conjunction with biotic influences. Therefore, as we face predictions of hotter, longer, and more frequent drought , it is becoming increasingly critical to hone in on the mechanisms, tipping points, and ecosystem impacts of these events. Furthermore, identifying plant mortality thresholds is of upmost importance for predicting susceptibility to extreme drought events of the future . California recently experienced a record-breaking, multi-year drought from 2012- 2018, estimated to be the most severe event in the last 1,000 years , with the 2013-2014 winter season being one of the driest on record . Drought tolerance has long been considered a common trait of shrub species in California chaparral communities where hot, rainless summers are the norm . However, in the Santa Ynez mountain range in Santa Barbara County, the dominant and widespread big berry manzanita exhibited dramatic dieback related to multi multi-year drought along with infection by opportunistic fungal pathogens in the Botryosphaeriaceae . These observations indicate that this species may be reaching a threshold in its drought resistance capabilities. Studies have reported Arctostaphylos spp. to exhibit unusual scales of dieback during periods of extreme drought stress, , however this could be the most severe dieback event in recent history, both in terms of scale and severity. Manzanita are important members of the chaparral ecosystem, providing habitat for wildlife and food through their nectar and berries . Additionally, their structure makes them important components of historical chaparral fire regimes, hydroponic net pots and their fire-induced germination strategies contribute to post-fire successional trajectories . Large-scale mortality of this species could reduce resource availability for wildlife, as well as alter fuel composition and structure in the region, resulting in an increased risk of more intense, faster burning fires. Therefore, the potential continued dieback of A. glauca is of great concern for both ecosystem functions and human populations alike. Significant dieback of A. glauca in Santa Barbara county, California, was first observed in winter, 2014 . Preliminary observations revealed patterns of dieback occurring along an elevational gradient, with effects being most pronounced at lower elevations than at higher elevations. It was also observed that dieback was most prevalent in stands located on steep, exposed southerly-facing slopes. These observations are consistent with findings by previous studies , Since A. glauca is classically drought-tolerant and able to function at very low water potentials , it raises the question of what is driving this extreme dieback event? Could A. glauca be reaching a tipping point as a result of extreme drought stress, presence of a fungal pathogen, or both?My dissertation research focuses broadly on the influence of drought and fungal pathogens on this classic, drought tolerant chaparral shrub species. Through a combination of methods, I explore the individual and interacting roles of water stress and opportunistic fungal pathogens in A. glauca in a major dieback event, and track the fate of individual shrubs through the progression of an historic drought.

My findings are organized into three chapters based on the following questions: What are the identities and distribution of fungal pathogens associated with A. glauca dieback ; How do drought stress and fungal infection interact to promote dieback and mortality in A. glauca ; and How does A. glauca dieback progress over time during drought, and how do landscape variables and drought stress correlate with dieback ? In Chapter 2, I identify fungal pathogens in A. glauca, and discuss their distribution across the landscape in the Santa Barbara county front country region. Based on preliminary findings showing significant levels of N. australe in the field, I expected to find high incidence of this opportunistic pathogen in A. glauca across the landscape, suggesting their role in drought-related dieback. The data support this prediction, as over half of the pathogens isolated were members of the Bot. family, and the majority of these were identified as N. australe, a novel pathogen in the region. Furthermore, Bot. infection was highly correlated with dieback severity, which was greatest at lower elevations. Taken together, the results show that opportunistic Bot. pathogens, particularly N. australe, are highly associated with A. glauca dieback across the landscape, and that lower elevations may be particularly vulnerable. In Chapter 3, I address the hypothesis that extreme drought and N. australe function synergistically to promote faster and greater mortality than either factor alone. I designed a full-factorial greenhouse experiment to identify whether A. glauca dieback is driven by extreme drought, infection by the fungal pathogens, or both. The results of this experiment support my hypothesis. Young A. glauca inoculated with N. australe while simultaneously exposed to extreme water stress exhibited faster stress symptom onset, faster mortality, and overall higher morality than those subjected to either factor alone. These results provide strong evidence that the severe A. glauca dieback event observed during the 2012-2018 drought was the result of synergistic interactions between extreme drought and opportunistic pathogens, rather than the nature of the drought or particularly virulent pathogens. In Chapter 4, I explore factors that are associated with climatic stress in order to draw correlations between A. glauca stress and dieback severity. Identifying such relationships can be useful in making predictions on dieback and mortality across the landscape. By analyzing data on predawn xylem pressure potentials and net photosynthesis in shrubs along an elevational gradient, I found that patterns of water availability and physiological function both varied greatly across the landscape, and only weakly correlate with dieback severity, suggesting factors other than elevation and aspect must also be important in driving plant stress and dieback. Extreme heterogeneity across this landscape likely confounded my results, yet may also play an important role in supporting the resiliency of A. glauca populations as a whole. By measuring the progression of dieback in these same shrubs over time, I found that dieback severity throughout the drought increased most at lower elevations compared to high, providing evidence that shrubs at lower elevations may be particularly vulnerable. Unexpectedly, no new mortality was observed in surveyed shrubs as the drought progressed, even though many plants exhibited severe levelsof dieback throughout the study. This result shows that high levels of dieback severity do not necessarily predict morality in A. glauca. In summary, my dissertation provides strong evidence that A. glauca dieback during the recent California drought was caused by synergistic interactions between extreme drought stress and infection by widely distributed opportunistic fungal pathogen N. australe. Infection and dieback severity varied considerably across the landscape, however, there is some evidence to suggest that populations at lower elevations may be at highest risk for severe dieback, either due to increased water stress, close proximity to fungal inoculum sources, or both. Additionally, shrubs located on southwest-facing slopes may also be more vulnerable due to increased sun exposure and thus environmental stress. Management efforts may want to focus on these areas when this region experiences future drought. Finally, although extreme dieback was recorded throughout the study, none of the observed shrubs succumbed to mortality. This may be the result of overall physiological resiliency, and the ability of adult shrubs to allocate resources to keep portions of the canopy alive.