Genome-wide association identifies candidate genes for drought tolerance in coast redwood and giant sequoia.

Drought is a major limitation for survival and growth in plants. With more frequent and severe drought episodes occurring due to climate change, it is imperative to understand the genomic and physiological basis of drought tolerance to be able to predict how species will respond in the future. In this study, univariate and multitrait multivariate genome-wide association study methods were used to identify candidate genes in two iconic and ecosystem-dominating species of the western USA, coast redwood and giant sequoia, using 10 drought-related physiological and anatomical traits and genome-wide sequence-capture single nucleotide polymorphisms. Population-level phenotypic variation was found in carbon isotope discrimination, osmotic pressure at full turgor, xylem hydraulic diameter, and total area of transporting fibers in both species. Our study identified new 78 new marker × trait associations in coast redwood and six in giant sequoia, with genes involved in a range of metabolic, stress, and signaling pathways, among other functions. This study contributes to a better understanding of the genomic basis of drought tolerance in long-generation conifers and helps guide current and future conservation efforts in the species.

Tracheid buckling buys time, foliar water uptake pays it back: Coordination of leaf structure and function in tall redwood trees.

Tracheid buckling may protect leaves in the dynamic environments of forest canopies, where rapid intensifications of evaporative demand, such as those brought on by changes in light availability, can result in sudden increases in transpiration rate. While treetop leaves function in reliably direct light, leaves below the upper crown must tolerate rapid, thermally driven increases in evaporative demand. Using synchrotron-based X-ray microtomography, we visualized impacts of experimentally induced water stress and subsequent fogging on living cells in redwood leaves, adding ecological and functional context through crown-wide explorations of variation in leaf physiology and microclimate. Under drought, leaf transfusion tracheids buckle, releasing water that supplies sufficient temporal reserves for leaves to reduce stomatal conductance safely while stopping the further rise of tension. Tracheid buckling fraction decreases with height and is closely coordinated with transfusion tissue capacity and stomatal conductance to provide temporal reserves optimized for local variation in microclimate. Foliar water uptake fully restores collapsed and air-filled transfusion tracheids in leaves on excised shoots, suggesting that trees may use aerial water sources for recovery. In the intensely variable deep-crown environment, foliar water uptake can allow for repetitive cycles of tracheid buckling and unbuckling, protecting the tree from damaging levels of hydraulic tension and supporting leaf survival.

Shoot dimorphism enables Sequoia sempervirens to separate requirements for foliar water uptake and photosynthesis.

PREMISE: Trees in wet forests often have features that prevent water films from covering stomata and inhibiting gas exchange, while many trees in drier environments use foliar water uptake to reduce water stress. In forests with both wet and dry seasons, evergreen trees would benefit from producing leaves capable of balancing rainy-season photosynthesis with summertime water absorption. METHODS: Using samples collected from across the vertical gradient in tall redwood (Sequoia sempervirens) crowns, we estimated tree-level foliar water uptake and employed physics-based causative modeling to identify key functional traits that determine uptake potential by setting hydraulic resistance. RESULTS: We showed that Sequoia has two functionally distinct shoot morphotypes. While most shoots specialize in photosynthesis, the axial shoot type is capable of much greater foliar water uptake, and its within-crown distribution varies with latitude. A suite of leaf surface traits cause hydraulic resistance, leading to variation in uptake capacity among samples. CONCLUSIONS: Shoot dimorphism gives tall Sequoia trees the capacity to absorb up to 48 kg H2 O h-1 during the first hour of leaf wetting, ameliorating water stress while presumably maintaining high photosynthetic capacity year round. Geographic variation in shoot dimorphism suggests that plasticity in shoot-type distribution and leaf surface traits helps Sequoia maintain a dominate presence in both wet and dry forests.

Ray fractions and carbohydrate dynamics of tree species along a 2750 m elevation gradient indicate climate response, not spatial storage limitation.

Parenchyma cells in the xylem store nonstructural carbohydrates (NSC), providing reserves of energy that fuel woody perennials through periods of stress and/or limitations to photosynthesis. If the capacity for storage is subject to selection, then the fraction of wood occupied by living parenchyma should increase towards stressful environments. Ray parenchyma fraction (RPF) and seasonal NSC dynamics were quantified for 12 conifers and three oaks along a transect spanning warm dry foothills (500 m above sea level) to cold wet treeline (3250 m asl) in California's central Sierra Nevada. Mean RPF was lower for both conifer and oak species with warmer dryer ranges. RPF variability increased with elevation or in relation to associated climatic variables in conifers - treeline-dominant Pinus albicaulis had the lowest mean RPF measured (c. 3.7%), but the highest environmentally standardized variability index. Conifer RPF variability was explained by environment, increasing predominantly towards cooler wetter range edges. In oaks, NSC was explained by environment - values increasing for evergreen and decreasing for deciduous oaks with elevation. Lastly, all species surveyed appear to prioritize filling available RPF with sugar to achieve molarities that balance reasonable tensions over starch to maximize stored carbon. RPF responds to environment but is unlikely to spatially constrain NSC storage.

Within-crown plasticity in leaf traits among the tallest conifers.

PREMISE OF THE STUDY: Leaves are the sites of greatest water stress in trees and a key means of acclimation to the environment. We considered phenotypic plasticity of Pseudotsuga menziesii leaves in their ecological context, exploring responsiveness to natural gradients in water stress (indicated by sample height) and light availability (measured from hemispherical photos) to understand how leaf structure is controlled by abiotic factors in tall tree crowns. METHODS: After measuring anatomy, morphology, and carbon isotope composition (δ13 C) of leaves throughout crowns of P. menziesii >90 m tall, we compared structural plasticity of leaves among the three tallest conifer species using equivalent data from past work on Sequoia sempervirens and Picea sitchensis. KEY RESULTS: Leaf mass per projected area (LMA) and δ13 C increased and mesoporosity (airspace/area) decreased along the water-stress gradient, while light did not play a detectable role in leaf development. Overall, leaves of P. menziesii were far less phenotypically responsive to within-crown abiotic gradients than either P. sitchensis, whose leaves responded strongly to light availability, or S. sempervirens, whose leaves responded equally strongly to water stress. CONCLUSIONS: P. menziesii maintain remarkably consistent leaf structure despite pronounced vertical gradients in abiotic factors. Contrasting patterns of leaf structural plasticity underlie divergent ecological strategies of the three tallest conifer species, which coexist in Californian rainforests.

Phenotypic plasticity of leaves enhances water-stress tolerance and promotes hydraulic conductivity in a tall conifer.

PREMISE OF THE STUDY: Leaves respond to environmental signals and acclimate to local conditions until their ecological limits are reached. Understanding the relationships between anatomical variation in leaves and the availability of water and light improves our ability to predict ecosystem-level impacts of foliar response to climate change, as it expands our knowledge of tree physiology. METHODS: We examined foliar anatomy and morphology of the largest plant species, Sequoiadendron giganteum, from leafy shoot samples collected throughout crowns of trees up to 95 m tall and assessed the functionality of within-crown variation with a novel drought/recovery experiment. KEY RESULTS: We found phenotypic variation in response to water availability in 13 anatomical traits of Sequoiadendron leaves. Shoot expansion was constrained by the hydrostatic gradient of maximum water potential, while functional traits supporting succulence and toughness were associated with sites of peak hydraulic limitation. Water-stress tolerance in experimental shoots increased dramatically with height. CONCLUSION: We propose a heat-sink function for transfusion tissue and uncover a suite of traits suggesting rapid hydraulic throughput and flexibility in water-stress tolerance investments as strategies that help this montane species reach such enormous size. Responses to water stress alter the amount of carbon stored in foliage and the rate of the eventual release of carbon.

Seed classification with random forest models.

Premise: To improve forest conservation monitoring, we developed a protocol to automatically count and identify the seeds of plant species with minimal resource requirements, making the process more efficient and less dependent on human operators. Methods and Results: Seeds from six North American conifer tree species were separated from leaf litter and imaged on a flatbed scanner. In the most successful species-classification approach, an ImageJ macro automatically extracted measurements for random forest classification in the software R. The method allows for good classification accuracy, and the same process can be used to train the model on other species. Conclusions: This protocol is an adaptable tool for efficient and consistent identification of seed species or potentially other objects. Automated seed classification is efficient and inexpensive, making it a practical solution that enhances the feasibility of large-scale monitoring projects in conservation biology.

Moving ecological tree-ring big data forwards: Limitations, data integration, and multidisciplinarity.

In recent years, tree-ring databases have emerged as a remarkable resource for ecological research, allowing us to address ecological questions at unprecedented temporal and spatial scales. However, concerns regarding big tree-ring data limitations and risks have also surfaced, leading to questions about their potential to be representative of long-term forest responses. Here, we highlight three paths of action to improve on tree-ring databases in ecology: 1) Implementing consistent bias analyses in large dendroecological databases and promoting community-driven data to address data limitations, 2) Encouraging the integration of tree-ring data with other ecological datasets, and 3) Promoting theory-driven, mechanistic dendroecological research. These issues are increasingly important for tackling pressing cross-disciplinary research questions. Finally, although we focus here on tree ring databases, these points apply broadly across many aggregative databases in ecology.

Towards multivariate functional trait syndromes: Predicting foliar water uptake in trees.

Analysis of functional traits is a cornerstone of ecology, yet individual traits seldom explain useful amounts of variation in species distribution or climatic tolerance, and their functional significance is rarely validated experimentally. Multivariate suites of interacting traits could build an understanding of ecological processes and improve our ability to make sound predictions of species success in our rapidly changing world. We use foliar water uptake capacity as a case study because it is increasingly considered to be a key functional trait in plant ecology due to its importance for stress-tolerance physiology. However, the traits behind the trait, that is, the features of leaves that determine variation in foliar water uptake rates, have not been assembled into a widely applicable framework for uptake prediction. Focusing on trees, we investigated relationships among 25 structural traits, leaf osmotic potential (a source of free energy to draw water into leaves), and foliar water uptake in 10 diverse angiosperm and conifer species. We identified consistent, multitrait "uptake syndromes" for both angiosperm and conifer trees, with differences in key traits revealing suspected differences in the water entry route between these two clades and an evolutionarily significant divergence in the function of homologous structures. A literature review of uptake-associated functional traits, which largely documents similar univariate relationships, provides additional support for our proposed "uptake syndrome." Importantly, more than half of shared traits had opposite-direction influences on the capacity of leaves to absorb water in angiosperms and conifers. Taxonomically targeted multivariate trait syndromes provide a useful tool for trait selection in ecological research, while highlighting the importance of micro-traits and the physiological verification of their function for advancing trait-based ecology.

Acclimation of interacting leaf surface traits affects foliar water uptake.

Absorption of water across the surfaces of leaves is an ecologically important aspect of tree physiology. Variation in foliar water uptake capacity depends on environmental conditions when traits associated with the uptake pathway respond to climatic signals. Using a series of experiments, we verify that water enters Sequoia sempervirens (D. Don) Endl. leaves by crossing the cuticle, and show that surface-trait acclimation alters the kinetic parameters of foliar water uptake. Under our experimental conditions, the cuticle was the primary pathway for water entry into the leaf. Exposure to climatic variation may induce surface acclimations, such as increased waxiness, that reduce water-film formation over stomata at the expense of dry-season foliar uptake rates. We found that water uptake is negatively associated with the interaction of leaf-surface wax coverage and stomatal density, and provide an accessible protocol to measure these key traits in Sequoia. Linking absorptive pathways and trait acclimation to physiological performance can provide a foundation for range-wide or genomic investigations of forest interactions with water and a mechanism-centered means to monitor canopy hydraulic parameters over time.

Leaf acclimation to light availability supports rapid growth in tall Picea sitchensis trees.

Leaf-level anatomical variation is readily apparent within tall tree crowns, yet the relative importance of water and light availability in controlling this variation remains unclear. Sitka spruce (Picea sitchensis, (Bong.) Carr.) thrives in temperate rainforests of the Pacific Northwest, where it has historically reached heights >100 m, despite rarely living more than 400 years alongside redwoods that are five times older. We examined leaves of trees up to 97 m tall using a combination of transverse sections, longitudinal sections, epidermal imprints and whole-leaf measurements to explore the combined effects of water stress and light availability on leaf development in P. sitchensis. In contrast to the situation in tall Cupressaceae, light availability-not hydraulic limitation-is the primary ecological driver of leaf-level anatomical variation in P. sitchensis. While height-associated decreases in leaf length and mesoporosity are best explained by hydrostatic constraints on leaf elongation, the majority of anatomical traits we measured reflect acclimation to light availability, including increases in leaf width and vascular tissue areas in the brightest parts of the crown. Along with these changes, the appearance of abaxial stomata in the bright upper crown, and the arrangement of mesophyll in uniseriate, transverse plates-with radially arranged apoplastic pathways leading directly to stomata before bridging them with a V-shaped cell-may enhance gas exchange and hydraulic conductivity. This suite of leaf traits suggests an adaptive strategy that maximizes photosynthesis at the expense of water-stress tolerance. Anatomical investigations spanning the height gradient in tall tree crowns build our understanding of mechanisms underlying among-species variation in growth rates, life spans, and potential responses to climate change.