Amazonian droughts are becoming more frequent and intense, having a profound effect on water availability for plants. However, we still have limited abilities to predict the effect of climate change on plant species survival and distribution in mega-diverse systems like the Amazon. Plant hydraulic traits coupled with the assessment of environmental characteristics emerge as an important tool to assess species physiological performance and resilience, especially during extreme climatic events. Here we investigate plant hydraulic strategies and physiological performance of tropical tree species (i) across contrasting environments, (ii) during an extreme drought event and (iii) during the course of a day to understand how water availability may shape species distribution and plant vulnerability to drought in the Amazon Basin.

In the first chapter, I investigate if the current patterns of species’ distribution in the main Amazonian habitats can be explained by species hydraulic strategies and how variation in the hydraulic properties of Amazonian forests influences species-specific vulnerability to drought across different habitat types. I found strong segregation between species from wet vs. dry environments in relation to their functional traits, suggesting that water availability could be a strong predictor of species functional composition in the different Amazonian environments. Also, the xylem of dry-habitat species are more embolism resistant, but it may not be correct to assume that these species will be the ones performing better under a warmer and drier climate.

The second chapter assesses plant physiological performance during a strong El Niño. This is the first study in the Amazon to measure in situ tree physiological stress before, during and after a natural drought event. I show that the warmer and drier conditions imposed by the El Niño greatly amplified trees’ physiological stress and could affect growth, phenology and potentially lead to tree mortality. Given the extreme nature of the this El Niño and that temperatures are predicted to increase in the tropics, this work can serve as a case study of the possible impact climate warming can have on tropical trees.

In the third chapter, I analyzed the diurnal patterns of stomatal conductance, water potential, and sap velocity during the dry season and investigate how they change with canopy temperature and vapor pressure deficit. I found that unlike preview reported, plants have a re-hydration period during the hottest time of the day probably due to an imbalance between stem sap-velocity and leaf transpiration rates. This study helps to elucidate the array of processes influencing diurnal patterns of plant-water balance in tropical trees.

Flowers have long been considered one of the hallmarks of angiosperm evolution. They are morphologically complex structures that both promote efficient pollination and protect the developing embryo. When it was championed in 1793 by Christian Konrad Sprengel, this view of the role of flowers in reproduction, however, was highly controversial: how could a form so beautiful and pure as a flower ever be involved in something as vulgar as reproduction? Sprengel and his predecessor, Josef Köhlreuter, are considered the founders of pollination biology, and their work set the stage for that of Charles Darwin nearly a century later. Darwin saw the interaction between flowers and their pollinators as a prime example of the power of natural selection. This approach to studying the evolution of flowers-of focusing on the biotic drivers of floral morphological change-has dominated our understanding and interpretation of floral evolution. Yet, new evidence suggests that extrinsic, abiotic factors and the costs of producing and maintaining flowers may also have influenced the evolution of floral form. These non-pollinator agents of selection could represent another major shift in our understanding of how flowers have evolved.

The series of studies presented in this dissertation takes one important resource, water, and examines how the requirements of providing water to flowers may influence their functioning and evolution. Two complementary approaches are used in these studies: (1) physiological measurements of the dynamics of water use on a few species and (2) comparisons of hydraulic traits for diverse sets of species. Together, these two approaches show the variability of flower water use, the anatomical traits associated with the flux of water through flowers, and how these physiological traits-and, by extension, the water requirements of flowers-vary among extant species. Together, these studies support the conclusion that maintaining flower water balance has been an important factor influencing floral evolution and, more generally, angiosperm ecology.

Three studies are presented that seek to measure, using different approaches, how the water flux to flowers and the hydraulic efficiency of flowers varies among species (Chapters 1-3), within species throughout floral development (Chapters 1 and 2), and diurnally with changing environmental conditions (Chapter 3). Using a new implementation of the heat ratio method for measuring sap flow (Chapter 1), I found that sap flow velocities to flowers and inflorescences vary diurnally, throughout floral development, and among species and microhabitats. Such high variability suggested that a better approach to comparing the hydraulic architecture of flowers would be to measure the maximum efficiency of the floral hydraulic system. In Chapter 2, I quantified for a phylogenetically diverse set of species the maximum hydraulic conductance of whole flowers. This, too, was highly variable among species, as were other hydraulic traits, and the variation in all traits was driven by just two genera of early-divergent angiosperm lineages. Variation in these traits highlighted the existence of two seemingly discrete hydraulic strategies: one strategy is to maintain a high hydraulic conductance and continuously import water via the xylem while the other strategy is to have a low hydraulic conductance with long water turnover times, slow desiccation rates, and presumably high hydraulic capacitance. Investigating the tradeoffs among these strategies further, Chapter 3 focused on characterizing the water relations of flowers of two Calycanthus species, which had among the highest hydraulic conductances measured in Chapter 2. Consistent with my predictions, high hydraulic capacitance in flowers mitigates the reliance on continuous xylem delivery of water. As a result, despite maintaining a high maximum hydraulic conductance (Chapter 2), Calycanthus flowers hydraulically underperform most of the time, reaching their maximum hydraulic conductance only when turgor loss is already inevitable. The results from Chapters 2 and 3 together suggest that the monocots and eudicots, compared to the ANITA grade and magnoliids, developed thicker cuticles and reduced their stomatal abundances, which together reduce rates of water loss from flowers and prolonged the time that these flowers can remain turgid without the import of new water.

Having characterized in Chapter 2 some of the anatomical traits that correlate with the hydraulic capacity of flowers, I sought in Chapters 4 and 5 to examine for a large set of species how these traits have evolved and vary among species. Specifically, I asked three questions: (1) Has there been coordinated evolution of water balance traits within flowers, which would suggest that maintaining water balance has been an important component in floral evolution? (2) Is there modularity in hydraulic trait evolution, such that flower and leaf traits have evolved independently? (3) Have hydraulic traits been under natural selection? The results from these two chapters strongly support the conclusions that floral hydraulic traits are under selection, that maintaining water balance has been an important component of floral trait evolution, and that hydraulic traits have evolved independently in flowers and leaves. These results show, for the first time, the importance of water balance in floral evolution and highlight that the physiological demands of and constraints on flowers may provide a strong counterbalance to selection by animal pollinators.

As yet, studies of the physiology of flowers have received little attention and have been ignored in physiological trait databases. As a result, there has been no overarching theory describing or predicting patterns of variance in floral physiological traits. This series of studies is a first attempt at providing such a framework for predicting how floral physiological traits may vary among species and how this may differ between reproductive and vegetative traits. Although it focuses only on traits associated with the movement of water, the results show that there may be consistent trait associations and syndromes among flowers, regardless of morphology. This should be a first step in understanding how flowers function physiologically and how their functioning may vary with a variety of ecological factors and over evolutionary timescales.

Seasonal changes in water availability have been demonstrated to play a fundamental role in determining plant survival-mortality dynamics in a number of different ecosystems. Tropical montane cloud forests are often considered to be aseasonal environments that do not experience significant water deficits; however, there is growing recognition that many of these rare ecosystems experience one or more dry seasons annually. Moreover, many tropical montane cloud forests are projected to experience decreases in dry season precipitation and increases in dry season temperature as a function of climate change. While the regular presence of clouds may mitigate plant water stress occurring during the dry season, clouds are also projected to decrease in frequency, intensity and cover. At present, little is known about the plant-climate interactions in tropical montane cloud forests.

In order to improve our understanding of the effects of climate change on tropical montane cloud forests, I studied how seasonal changes in water availability affect plant functioning using observational and experimental approaches at a number of different scales:

In Chapter 1, I present the results from a study on tropical montane cloud forest ecohydrology. Despite longstanding recognition of the unique nature of hydrologic cycling in tropical montane forests, comprehensive and comparative studies remain limited. I studied the intra- and inter-annual variation in the inputs, pools and fluxes of water in a seasonally dry tropical montane cloud forest near Veracruz, Mexico using hydrogen and oxygen stable isotope ratios in water. There was significant seasonal variation in the hydrogen and oxygen stable isotope ratios of precipitation inputs driven by differences in the origin and size of storm events. This variation facilitated the separation of different pools of water, revealing the presence of two separate soil water pools, one highly mobile pool contributing to streams and a second less mobile pool being used by plants. At the peak of the dry season, the predominant deciduous and evergreen tree species were accessing shallow soil water from this second, less mobile pool. The results provide a foundation upon which to better understand the coupling between the hydrology and ecology of tropical montane cloud forests now and given projected scenarios of climate change.

In Chapter 2, I present the results from a study on the effects of a sustained decrease in water availability on the growth and physiology of tropical montane cloud forest plant seedlings. Research on the functional response of tropical plants to seasonal changes in water availability has largely focused on tropical lowland ecosystems. I conducted an experimental dry-down of seedlings from four common tropical montane plant species in the genus Ocotea at a common site near Monteverde, Costa Rica. Despite only a small decrease in soil water availability, plants subjected to the experimental dry down demonstrated species-specific reductions in both physiology and growth. The results indicate that water is likely to

play a strong role in tropical montane cloud forest plant functioning.

In Chapter 3, I present the results from a study on the interactions between clouds and tropical montane cloud forest plants, with a focus on the prevalence and significance of foliar water uptake. Foliar water uptake, the direct uptake of water accumulated on leaf surfaces into leaves, is a common phenomenon in ecosystems that experience frequent fog or cloud immersion, but has not been studied in tropical montane cloud forests. I quantified cloud cover patterns in two neighboring, seasonally dry tropical montane cloud forests near Monteverde, Costa Rica using remote sensing data. I then correlated these patterns with ground-based observations of leaf wetting occurring due to the physical impaction of cloud moisture on leaf surfaces. During the dry season, when rainfall is reduced and leaf wetting due to clouds is the primary source of water in the ecosystem, leaf wetting events resulted in foliar water uptake in all species studied. While all the species demonstrated the capacity to improve their leaf water potential as a result of foliar water uptake, this capacity differed between the two forests and among the species studied within a forest. The results indicate that changes in the frequency, intensity, and duration of cloud cover projected to occur as a function of regional warming will not affect all species or forests equally.

In Chapter 4, I present the results from a study on the effects of leaf wetting events and the resultant foliar water uptake on leaf water pressure-volume relations. Research on foliar water uptake has almost exclusively focused on the implications of additional water for leaf water potential and its impacts on photosynthetic performance. However, the study of pressure-volume relations can provide important insights into whether foliar water uptake alters traits responsible for the movement and conservation of water in leaves. I compared the pressure-volume relations of leaves rehydrated through both xylem and foliar water uptake compared to xylem alone in four tropical montane plant species near Monteverde, Costa Rica. Rehydration through both pathways caused inconsistent differences among species; however, one species demonstrated a decrease in the modulus of elasticity and an increase in capacitance that may be a function of differences in leaf anatomy. The results indicate that changes in pressure-volume traits associated with leaf wetting and foliar water uptake may have consequences for plant functioning and suggest that future research should more explicitly consider the role of leaf wetting in plant-water relations.

Taken together, these studies suggest that the annual dry season experienced by the tropical montane cloud forests studied herein result in plant water deficits that impact plant functioning. However, these plant water deficits can be mitigated by the presence of clouds. The species-specific nature of many of the results indicate that projected changes in dry season water availability may alter plant survival-mortality dynamics and thus affect the species composition of tropical montane cloud forests in the future.

It is widely recognized that many aspects of plant form and function are coupled to variation in water availability. This is because plant persistence is ultimately dependent upon the process of carbon fixation and it is physically impossible for a plant to transport CO2 to the sites of photosynthetic metabolism inside the leaf without, at the same time, loosing water to the surrounding atmosphere. How the efficiency of water use changes as leaves differ in size and longevity, and where leaves acquire their water from, are often times not well defined. In general, the water lost to the atmosphere by leaves is thought to originate from the soil via uptake by roots. However, previous research has shown that water deposited on leaf surfaces is often available for use via direct foliar uptake. Using field observations and a greenhouse experiment I show that leaf water interception can represent an overlooked water source for leaves that temporarily, but significantly, decouples leaf-level water and carbon relations from variation in soil water availability (Chapter 1). Additionally, within a particular environment water loss per unit leaf area is expected to increase with leaf size. Recent research suggests the construction cost of a leaf also increases with size and/or longevity. If leaves have maximized the ability to transport water to surfaces for energy and gas exchange in order to maximize CO2 uptake from the atmosphere, then vascular network efficiency (Leaf hydraulic conductance) should be size invariant. Using a survey of 60 angiosperm species I show that leaf hydraulic conductance is maximized for a given surface area (Chapter 2). By extension, if the lifetime return (carbon gain) on dry-mass invested in leaf area (construction cost plus maintenance respiration per unit leaf area) is maximized, then leaf hydraulic conductance per unit leaf dry mass should scale isometrically with leaf lifespan. Using plants from a common garden and previously published values of leaf lifespan and leaf hydraulic conductance for species inhabiting a broad range of vegetation types and climate, I explored the relationship between leaf longevity and leaf hydraulic conductance per unit leaf mass. I observed a negative correlation between leaf hydraulic conductance per unit leaf mass and leaf lifespan. Further, the slope of the relationship describing the covariation between leaf hydraulic conductance per unit mass and leaf lifespan is not significantly different from one. Isometric scaling (slope = 1) provides strong support for a constant net carbon gain per leaf despite significant variation in leaf size, longevity and environment. Therefore, variation in gross primary productivity is a function of the number of leaves a plant maintains over a given unit of time (Chapter 3).

The Earth’s tallest trees tend to grow in geographic regions where water is abundant. An abundance of water in the environment, however, does not by itself enable trees to overcome several inherent challenges with being tall. First, water must be transported over very long distances through tiny conduits, and it adheres to their inside walls. Thus, the progress of water flow is impeded by hydraulic resistance that may accumulate with longer transport distances. Second, gravity pulling down on the water imparts a tension that increases with height in tree at a rate of ‒0.01 MPa m-1. Therefore, water inside foliage and branches at the top of a 100 m tall tree is under far more tension than foliage emerging from the base, yet normal physiological processes still occur. Third, environmental conditions become increasingly desiccating with height in forest canopy. Relative humidity decreases while sunlight, wind speed, and vapor pressure deficit increase along this vertical gradient. The strategies trees use to overcome these three challenges in part enable them to grow so tall. In order to improve our understanding of the limits to tree height growth, I studied the mechanisms by which tall trees cope with these height-related constraints, using the conifers coast redwood (Sequoia sempervirens (D. Don) Endlicher) and giant sequoia (Sequoiadendron giganteum (Lindley) J. Buchholz) as well as the angiosperm mountain ash (Eucalyptus regnans F. von Mueller).

In Chapter 1, I explored two mechanisms that trees use to compensate for the accumulation of hydraulic resistance with height growth. The first mechanism is varying the diameters of the conduits with height in tree according to a theoretical model, wherein a series of conduits from tree top to base widens at a specific minimum rate that is maintained throughout height growth. The premise is based in fluid dynamics. An elongating series of cylindrical conduits with constant diameter is hydraulically less efficient than the same elongating series that also widens. I tested this model in exceptionally tall individuals of coast redwood, giant sequoia, and mountain ash. Data matched the theoretical predictions. However, a decelerating rate of conduit widening was observed in the bottom one-third of the trees suggesting a limit to this hydraulic compensation mechanism that I interpreted as an optimization of carbon investment for a given hydraulic benefit. The second mechanism is a whole-tree increase in the amount of sapwood that provisions the leaves. Across a large range of tree sizes, I quantified the rate of accumulation of sapwood relative to leaf area using data available from the literature. Sapwood accumulated at a faster rate than leaf area in the conifers but the rates were equivalent in mountain ash. Sapwood and leaves serve water supply and demand roles, but they are also carbon sinks and sources, respectively. I therefore interpreted these results as hydraulic compensation in the conifers that may limit height growth due to carbon balance constraints, whereas the mountain ash appeared to have additional height growth potential. Limits to these two compensation mechanisms may thus be imposed by carbon balance constraints that limit tree height growth.

Chapter 2 investigates water relations of the foliage of giant sequoia, to determine how foliage remains adequately hydrated against height-related constraints. Together with coauthors Rikke Reese Næsborg and Todd E. Dawson, I generated pressure-volume curves on foliage collected crown-wide from 12 large giant sequoia trees up to 95 m tall, to identify the tissue-level drivers responsible for maintenance of sufficient turgor pressures and water contents both with height tree and over time as the dry season progressed. Hydraulic capacitance was about twice as large as reported for other tree species. Maintenance of turgor pressure in all parts of the trees was accomplished by increases in tissue osmotica with height that depressed the turgor loss point at a rate equivalent to the gravitational potential gradient. High relative water contents were sustained with height by building structurally stiffer tissues as well as carrying an increased proportion of water in the symplasm versus apoplasm. Seasonal increases in the fraction of apoplastic water were important for maintaining physiological function when water in the environment may have been more limiting. This suite of foliar water relations traits permits minimum midday water potentials to operate close to the turgor loss point and may also enable the Earth’s largest tree species to survive short-term drought.

In Chapter 3, I quantify the importance of stem water storage in tall giant sequoia. Water storage in trees is known to contribute substantially to daily transpiration to extend physiological function, but does gravity dampen the dynamics of water storage with height? In the top 5 to 6 m of tall giant sequoia trees I collected detailed architectural information and installed automated sensors that monitored diurnal fluxes in sap flow, stem diameter, and water potential. To provide context for water use at the tree tops, I also installed sap flow gauges at the tree bases. Unsurprisingly, larger stems released larger volumes of stored water. However, tree top water storage contributed a tiny fraction of daily transpired water, and hydraulic capacitance was similarly low, supporting the hypothesis that chronically low water potentials dampen the water storage dynamics with height in tall trees. Lag times among the sensors indicated that a large portion of the stored water was expressed from live secondary phloem tissues of the inner bark. Despite the reliance on seemingly small volumes of stored water, whole-tree sap flow exceeded 3000 L d-1, which is the highest daily water budget reported for any tree on Earth.

These studies together underscore how water use and management in giant trees is truly dynamic over space and time. Over a tree’s lifetime, modifications to sapwood anatomy and volume foster efficient water movement along the entire flow path. Seasonal shifts in the compartments where foliar water is held may extend physiological processes during dry periods. The physiological consequences of daily swings in atmospheric conditions are controlled by tissue osmotic and elastic properties, as well as the release of stored water to the transpiration stream. Each of these dynamics varies along a tree’s vertical profile to compensate for height-related constraints and enables the tallest trees to support functional leaves well beyond 100 m above the ground.

In a world of changing climate, changes in the magnitude and timing of fluctuations in environmental conditions are expected to alter plant functioning. Plants are already responding to anthropogenic climate change and in the next hundred years they will face an increase in the severity, duration, and frequency of extreme climatic events, such as heatwaves. To understand the impacts of climatic extremes on plants we need to study the exposure and vulnerability of natural systems in the context of natural climate variability. Few studies have examined plant responses to heatwaves - short-term, intense increases in temperature. Here, the ecophysiology of the California native shrub Heteromeles arbutifolia is explored, with the goal of determining the extent of within species variation in ecophysiological responses to heatwaves.

Chapter 1 examines if the ability to tolerate heatwaves varies among distant populations of H. arbutifolia. Vulnerability to heatwaves may depend on genetic variation in phenology, growth, reproduction and physiological tolerances, and these factors may vary within a species among different populations. This study showed that two populations of a widespread California native shrub responded differently to an artificial heatwave. In the greenhouse, regardless of treatment, plants from the northern site had higher stomatal conductance and plants from the southern site had higher nighttime respiration. An artificial spring heatwave treatment, when water availability was high but air temperature suddenly increased 5°C, resulted in increased transpiration by plants from both sites but the magnitude of the increase was greater in plants from the northern site. However, our results also show that an artificial spring heatwave did not result in lower photosynthetic rates or higher respiration rates, indicating that high water availability may have allowed plants from both sites to cope with extreme temperatures.

Chapter 2 examines the responses of two growth forms of H. arbutifolia to naturally occurring heatwaves in the field. . H. arbutifolia is a resprouter, with plants regenerating after a fire from shoots produced by belowground meristems fuelled by non-structural carbohydrate reserves. There is strong evidence that resprouting individuals have higher photosynthetic and transpiration rates than mature non-sprouting individuals. Therefore, vulnerability to heatwaves may depend on plant growth form. This study shows that when seasonal water availability was lower during a fall heatwave, plants reduced stomatal conductance, but when water availability was higher during a spring heatwave stomatal conductance was unaffected by the heatwave. Resprouts in this study did have higher electron transport rates and higher transpiration rates, yet these differences did not result in mature shrubs and resprouts responding differently to either a spring or fall heatwave. Detecting a strong heatwave response in the field for drought tolerant shrub species such as H. arbutifolia may require heatwaves of larger magnitude or heatwaves coupled with severe drought. These extreme heatwaves or combinations of extreme heatwaves and drought are rare and therefore difficult to capture in the field; however, it is just such events that are likely to be the most important to predict plant vulnerabilities as extreme events become more common in the future.

Chapter 3 examines the seasonal patterns of water and carbon use in the two growth forms of H. arbutifolia. Most of the research comparing resprouting and mature individuals of a species happens immediately following a disturbance. To assess the relative risks to an obligate resprouting species it is important to understand the long-term performance of both forms of the plant. This study focuses on differences 4-years and 5-years post-disturbance. We found that many aspects of the physiology of two types of had converged but that resprouts had higher photosynthetic rates at the end of the dry season. If differences between mature and resprouting shrubs are more pronounced when leaves are experiencing water limited conditions, this could have important implications during drought years or under projected climate change if the precipitation regime changes.

This research is some of the first to examine the influence of ecotypic variation in response to heatwaves. Overall, these studies show that H. arbutifolia is likely to be robust to heatwaves of the magnitude studied. H. arbutifolia exhibits within species variation despite growing in a wide range of environments throughout its range. Understanding the magnitude of within species variation between different populations and different forms of the plant (i.e. resprouting and mature individuals) will be important to determine the vulnerability of the species to future climate change.

Abstract

Beyond Soil: Nitrogen Cycling and Plant Uptake in Weathered Bedrock Rhizospheres of Deeply Rooted Californian Forests

By

Kelsey Lee Crutchfield-Peters

Doctor of Philosophy in Integrative Biology

University of California, Berkeley

Professor Todd E. Dawson, Chair

Across diverse ecosystems worldwide, plant roots and their associated microbiome (rhizospheres) are known to extend beyond soil horizons and into weathered bedrock where they drive water and carbon cycling meters below the base of soil. Little is known however about how deep rhizospheres drive nutrient cycling in weathered bedrock horizons. In my dissertation research, I combine biogeochemical, plant ecophysiological and genetic techniques to explore how nitrogen (N)–the most limiting nutrient to plant growth in terrestrial ecosystems–is cycled throughout a deep rooting profile. My research is based at the Eel River Critical Zone Observatory in northern CA, where more than a decade of research has demonstrated the importance of the weathered bedrock in forest and watershed function. I build upon this work to understand the role of the weathered bedrock vadose (unsaturated) zone in deep N cycling and explore how dominant plant species acquire and cycle nitrogen in soil versus weathered bedrock. In chapter one, I investigate N cycling dynamics in the weathered bedrock vadose zone (WBVZ), asking: How do the chemical forms and concentrations of biologically available dissolved N change year-round? And what is the source and fate of this dissolved N? I take advantage of a novel field instrument called the Vadose Zone Monitoring System (VMS) to sample water every 1.5 meters throughout a WBVZ, from the base of soil down to groundwater (~16 m depth). Alongside my colleagues, I have sampled water approximately every two weeks for more than 2 years for Total N, Total Carbon (TIC/TOC), inorganic N (ammonium, NH4+, and nitrate, NO3-) as well as CO2 and O2 gasses across the entire VMS depth profile. From these data I demonstrate that (1) biologically available N is on the same order of magnitude as temperate forest soils and is seasonally-cycled in the WBVZ, (2) the dominant form of that N is organic N and it is strongly linked to dissolved organic carbon (DOC) dynamics, and (3) decreases in N concentration in the WBVZ is both dependent on incoming precipitation and linked to periods of high forest-level metabolic activity, suggesting plant use of N from the WBVZ. Additionally, ?13C-DOC values from the VMS suggest that dissolved organic matter in the vadose zone is derived from fresh plant sources and not lithologically derived from parent material at the site. I conclude that deep N cycles are driven by rhizospheres meters below the base of soil and represent a major gap in our current understanding of deeply rooted ecosystems. In chapter two, I build upon my findings in chapter 1 and explore the uptake of plant-available N in Douglas fir (Pseudotsuga menziesii), a dominant tree species at the Eel River CZO that can extend dense root networks into fractured rock horizons. Using an isotopic labeling experiment, I address the following questions: How do soil-grown versus rock-grown fine roots differ in their N uptake capacity? And what sources of nitrogen are preferred between soil and rock grown roots? I dug seven 1 m deep pits at the Eel River CZO and sampled terminal fine root tips from mature P. menziesii trees for a dual 15N and 13C tracer experiment. I incubated fine roots from either soil or weathered bedrock fractures in one of three labeled nutrient solutions (15NH4Cl, K15NO3- or 15N-glycine) at 10, 40 or 100 micromolar concentrations. After incubation, I calculate N uptake capacity for soil and rock grown roots for each treatment based on the degree of 15N isotopic enrichment. For the glycine treatments, I also estimated 13C uptake to assess in-tact amino acid uptake. I found that rock-grown roots have a similar N uptake capacity to soil-grown roots, but that rock grown roots have slightly lower uptake rates. I also found that both soil- and rock-grown roots prefer NH4+, which is the dominant form of inorganic N available at our site. Second to NH4+, all roots took up glycine at higher rates than NO3-––which had consistently low uptake across all concentrations. However, there was no significant uptake of 13C in any of the glycine treatments, indicating that the ectomycorrhizal symbionts which associate with P. menziesii rapidly decarboxylated the glycine to acquire the N. Ultimately, I found that despite drastic differences in the soil and saprolite environments, the mycorrhizal fine roots of P. menziesii are equally capable of acquiring N. Furthermore, they are primed to take advantage of the most available forms of N at the site: both inorganic-N in the form of NH4+ as well as organic N through the action of rapid depolymerization of more complex organic molecules. These findings support the potential of deep roots in the WBVZ to take advantage of the two most common forms of N that are dynamically cycled: NH4+ and organic N. In my third and final chapter I use RNA-seq to explore differential gene expression in P. menziesii fine roots sampled from soil and weathered rock, asking: How does differential gene expression change broadly in soil versus bedrock environments? And, more specifically, how does expression of N uptake genes (e.g., Ammonium transporter (AMT) and nitrate transporter (NRT) gene families) in P. menziesii fine roots differ between roots grown in these two environments? To address these questions, I collected mycorrhizal fine root tips of P. menziesii from soil and saprolite horizons from 6 trees in summer 2020 and 7 trees from summer 2021 and extracted total plant RNA for Illumina 150 PE Sequencing. From this work I identify the P. menziesii ortholog of the MADs-box gene, DAL20 as the singular gene that is consistently upregulated in rock-grown roots between summers 2020 and 2021. In other conifers, DAL20 is exclusively expressed in root tissue and its close homolog in Arabidopsis thaliana increases root apical meristem size and results in increased primary root growth when upregulated. I found that expansion genes, involved in loosening the cell wall to allow for cell expansion, were also upregulated in rock environments. While there were no major differences in N uptake genes (AMT or NRT families) between soil and saprolite environments, there were consistent annual patterns in expression in AMT and NRT expression regardless of year or substrate (e.g., soil or rock). There was one AMT gene that is consistently highly expressed above all others, and it is the 4th most highly expressed of the 23,397 genes analyzed for differential gene expression. In other study systems, patterns like this in AMT expression were linked to transfer of N from mycorrhizal symbionts. Together, these patterns suggest a rock environment ‘syndrome’ in genes that enhance root growth and flexibility and point toward a unique gene possibly involved in N transfer to the plant from the mycorrhizal symbiont.

Anthropogenic climate change is expected to increase the frequency, intensity, and duration of extreme drought events around the world, with one of the consequences being widespread episodes of tree mortality. Drought-induced tree mortality is now widely documented across diverse ecosystems. Consequently, understanding the physiological causes and ecological consequences of drought-induced tree mortality is critically important to effectively predict and manage the consequences of future drought events around the world. However, accurately predicting drought-induced mortality risk for different species across broad climatic, topographic, and environmental conditions is challenging, because drought exposure can vary substantially both within and across species’ geographic ranges. For plant communities facing increased likelihoods of exposure to extreme drought, acclimation and survival of individual trees will require morphological and physiological modifications that offset the negative impacts of drought. Therefore, investigating such responses when historic, multi-year drought conditions arise is essential to advance our understanding of the physiological capacities of different plant species, and will improve modelling efforts geared towards predicting vulnerability to future extreme drought events. This work examines how variation in climate, topography, and drought exposure affected the in-situ responses of two coexisting tree species to a historic five-year drought event at several sites spanning a climatic gradient.

This work focuses on two long-lived, drought-adapted tree species in California: blue oak (Quercus douglasii, Hook. & Arn., Fagaceae) and valley oak (Quercus lobata, Née, Fagaceae). These oaks are iconic endemic tree species that coexist with each other in deciduous woodlands in the Mediterranean-climate region of California. In response to the onset of California’s historic 2012 - 2016 drought (the region’s most severe drought in over 160 years of meteorological records and 1200 years of dendro-climatological reconstructions), I established three study sites across a climate gradient that captured a wide range of variation in historical climate and overall drought exposure. Within these sites, I quantified the differential extent of seasonal water stress and water status recovery until the drought subsided in spring 2016. I found that water stress though each growing season was associated with the restriction of roots to seasonal precipitation in shallow soil zones compared to deeper soil moisture reserves. Seasonal water status recovery was driven by the extent of water stress experienced during the previous growing season, likely due to promotion of xylem embolism. This lack of recovery was most pronounced in the blue oaks at the most xeric and drought exposed study site, where the greatest extent of canopy damage and mortality was also documented.

To place these results involving water status, canopy damage, and mortality into more of an ecophysiological context, I quantified seasonal changes in the stable isotope composition of leaves and xylem water, and how these changes reflect differential photosynthetic performance of the two species as a function of their capacity to uptake water through the drought. I found that these species had different water acquisition strategies within and across my study sites, likely reflecting differences in root architecture or placement in relation to soil water profiles spread throughout the landscape. These differences translated into differing degrees of carbon fixation through the drought, such that seasonal gas exchange was most inhibited in blue oaks at the most xeric and drought exposed site. Across sites, seasonal reductions in transpiration and water potential in both species were all directly associated with a lack of root access to stable groundwater resources in deeper soils.

To investigate how morphological modifications of the canopy architectures and of the leaves of each species may have allowed for acclimation to the high evaporative demands imposed by the drought, I quantified seasonal changes in leaf size, leaf mass per area, and leaf area-to-sapwood area ratios. I found that large reductions in the number and size of leaves were exhibited by blue oaks at the most xeric and drought exposed site that had roots which were restricted to shallow rooting zones. These reductions consequently reduced the effective leaf area per unit of sapwood area throughout their canopies, which acts to reduce vulnerability to hydraulic failure and drought-induced mortality. Conversely, valley oaks maintained larger leaves and larger leaf area-to-sapwood area ratios through the drought in response to more stable access to deeper groundwater resources. Seasonal changes in leaf mass per area were not observed in either species.

Taken together, these results indicate that the maintenance of physiological water-carbon balance for these species under multiple years of extreme drought is driven by variation in functional rooting depth and its effects on the seasonal trajectories of plant water status, seasonal water status recovery, and canopy architecture modifications, which control the efficiency of gas exchange through growing seasons. Despite the resilience of these oak species to such extreme drought conditions demonstrated in this work under natural, in-situ field conditions, their inability to sufficiently acclimate and maintain water-carbon balance can still lead to significant degrees of canopy damage and mortality. With the greatest drought impacts observed in individuals of both species that had more restricted capacities for water acquisition through this historic drought event, this work demonstrates the ecophysiological conditions under which these species are most vulnerable to damage and mortality.

Our ability to forecast future climate scenarios and manage our water resources requires an understanding of the fluxes of transpired water in forested areas. In addition to quantifying the amounts of water moving through trees, we also need to know where different species of trees get their water and how responsive species are in changing their source water to drought or other climatic factors. The critical zone (CZ), which on hillslopes can be defined as the area between the tree tops and the top of underlying fresh bedrock, has emerged as an important multidisciplinary framework by which to study the flux of water up into trees, or down into runoff generating pathways. A common approach to defining the water sources used by the vegetation employs the use of different stable isotope ratios of water (2H/H and 18O/16O) in both the various reservoirs and in the trees; such information can also be exploited as a hydrologic tracer for any given catchment or in the CZ. This study was conducted at “Rivendell”, an intensively instrumented site at the South Fork Eel Critical Zone Observatory. Rivendell spans a north-south divide. The North Slope is an unchanneled 32° hillslope draining to Elder Creek, upstream from the confluence with the South Fork Eel. The South Slope is 25°, and is a large head scarp of a deep-seated landslide draining directly to the South Fork Eel. Soils are thin (10-70 cm), and are underlain by saprolite (a friable, soil-like material of variable thickness with relict rock properties), vertically dipping weathered bedrock of argillite and sandstone above fresh bedrock. The depth of the transition from weathered bedrock to fresh bedrock varied from 6 m near Elder Creek to approximately 26 m at the hillslope divide. The site is within the Coastal Belt of the Franciscan Formation. The climate is Mediterranean, and the bulk of the 1535 mm of annual rainfall that occurred during the study period fell between October and May.

I used nearly 2000 samples of natural abundance water isotopes to quantify the variation in potential sources of water used by the vegetation and to trace precipitation inputs through the different materials of the critical zone: soil, saprolite, and weathered bedrock and to follow subsurface mobile waters through the weathered zone to a seasonally dynamic groundwater that then drained to Elder Creek. All runoff occurred as groundwater discharge. Most samples were collected bi-weekly from June 2011-January 2013. The isotopic composition of the shallow soil showed strong seasonally dynamics, shifting from evaporatively enriched (heavier than “average” rainfall) in the summer (detected by having more positive H and O stable isotope ratios) to depleted (lighter than “average” rainfall or the mobile water fraction collected with lysimeters) in the winter (detected by having more negative H and O stable isotope ratios). In contrast, the isotopic composition of the saprolite behaved oppositely: it was lightest in the summer, and shifted toward the average annual rainfall values during the winter, only to return to very light values progressively through the summer. Deep weathered bedrock was always distinct in being isotopically light (negative). Mobile water sampled from lysimeters passed through the soil in winter but due to mixing within the saprolite and weathered bedrock of the critical zone, by the time the water reached the groundwater it was isotopically invariant and appears to have become homogenized such that its value was equal to the average annual precipitation isotope values. As a consequence, the groundwater and resulting Elder Creek flow were also isotopcially nearly invariant. Surprisingly, water held more tightly within the interior of argillite (but not sandstone) is very negative isotopically, falling well to the left of the Local Meteoric Water Line (LMWL)–the precipitation inputs that fell on to the site during the study period. The persistently negative isotopic compositions of critical zone materials suggest the possibility of either inherited paleo-meteoric water or post-precipitation isotope effects could be influencing the isotope values of water at depth and that these post-precipitation isotope effects may be common, yet are largely unaccounted for, in eco-hydrologic applications. Despite a transient isotopic response due to precipitation inputs, the soil moisture, rock moisture, and mobile water have distinct isotopic compositions that lead to a structured heterogeneity that persist over multiple years. Thus, the material properties in the critical zone impart an isotopic ‘fingerprint’ on their waters that should be traceable into the xylem of vegetation growing on the site.

During the study period (2011-2013), I continually sampled tree xylem from 33 Douglas-fir (Pseudotsuga menziesii), 8 madrone (Arbutus menziesii), 16 live oak (Quercus wislizenii), and 5 tanoak (Notholithocarpus densiflora) trees to determine the source water of different species of vegetation growing across the north-south divide. The δD of the Douglas-fir matches that of the saprolite and weathered bedrock moisture, but not the soil.

In order to determine the source of Douglas-fir water, we conduced a targeted tracer experiment in which D2O enriched water was slowly added via 12 pipes to the saprolite in early June, 2014. I measured the isotope composition of xylem water of 17 trees and found the “spiked” isotope tracer in two Douglas–fir but not in the hardwoods. This tracer experiment showed that Douglas-firs do use rock moisture. However, the long term monitoring shows that the δ18O composition of Douglas-fir xylem water is commonly more positive than rock moisture and no mixing line of observed sources can explain this seemingly deviant heavy δ18O. Based on these combined results I hypothesize that the dominant water source used by Douglas-fir xylem may have experienced fractionation in the subsurface before being taken up by the trees. This fractionation may occur in the critical zone due to uptake of rock moisture through mycorrizhae (that are symbiotically connected to Douglas-fir roots), or (less likely) there may be mixing within the bole of the Douglas-fir that leads to a positive δ18O offset. If true, the isotope composition of Douglas fir xylem water would not be a reliable indicator of source water.

In contrast to the Douglas fir, hardwoods on this site appear to be opportunistic water users. Hardwoods use mobile moisture and bulk soil water in the winter, and the evaporatively- enriched bulk soil moisture, and, to a lesser extent, non-evaporatively enriched saprolite moisture, in the summer. An analysis of an adjacent Douglar fir and madrone tree showed that even the roots from the different trees that crossed each other in the subsurface and the adjacent boles of the trees both in summer and after winter rains held isotopically distinct water. The roots of an individual tree, especially in summer, are isotopically diverse and size dependent, indicating that the bole values record a mixture of sources. South Slope hardwoods may show signs of greater reliance on deeper, non-evaporatively enriched saprolite moisture in late summer. If this finding is supported with further observations it would suggest that these trees compete with Douglas-firs for rock moisture.

The use of mobile water by the hardwoods on our study site, the strong summer diurnal oscillations in stream flow in Elder Creek, and the documented rise in stream runoff (including summer baseflow) after clear cut logging in nearby experimental watersheds all demonstrate that forests use water that would otherwise contribute to streamflow. Subsurface isotopic dynamics of the critical zone moisture causes the retained moisture to be isotopically distinct from streamflow, but the mobile that drains to depth and mixes enroute results in a groundwater (and resulting streamflow) that is nearly invariant isotopically (and similar to annual rainfall values). Thus, forests do use water that contributes to runoff and yet can be isotopically distinct from streamflow. These observations challenge the recently proposed concept of only ‘two water worlds’: a hypothesis that argues that plant available moisture is hydrologically disconnected from stream flow.

The forest canopy at our study site and in much of the northern California landscape is mixed hardwoods and Douglas-fir. The opportunistic hardwoods can extract moisture to lower water potentials, giving them access to different water sources than conifers like Douglas-fir can use and thus, in severe drought, may effectively compete with Douglas-fir that prefer, and may require, using water sources that are at a higher water potential. Such a competition needs to be investigated as it matters to future forest composition under drier and warmer future climates in the Northern California Coast Ranges.

Collectively, the results of my investigations point toward the importance of material properties in influencing the isotopic composition of the waters they hold, and to vegetative water-source use that is species-specific. The observed isotope effects require future study to determine the mechanisms responsible for the chronically negative rock moisture of the soil and weathered bedrock, the cause of the invariant Douglas-fir xylem water that lacks similarity with any subsurface reservoir, and the possibility of hardwood use of rock moisture and subsequent competition with Douglas-fir trees at some places and at some times in the watershed.