No cell is an island: characterising the leaf epidermis using epidermalmorph, a new R package.

The leaf epidermis is the interface between a plant and its environment. The epidermis is highly variable in morphology, with links to both phylogeny and environment, and this diversity is relevant to several fields, including physiology, functional traits, palaeobotany, taxonomy and developmental biology. Describing and measuring leaf epidermal traits remains challenging. Current approaches are either extremely labour-intensive and not feasible for large studies or limited to measurements of individual cells. Here, we present a method to characterise individual cell size, shape (including the effect of neighbouring cells) and arrangement from light microscope images. We provide the first automated characterisation of cell arrangement (from traced images) as well as multiple new shape characteristics. We have implemented this method in an R package, epidermalmorph, and provide an example workflow using this package, which includes functions to evaluate trait reliability and optimal sampling effort for any given group of plants. We demonstrate that our new metrics of cell shape are independent of gross cell shape, unlike existing metrics. epidermalmorph provides a broadly applicable method for quantifying epidermal traits that we hope can be used to disentangle the fundamental relationships between form and function in the leaf epidermis.

Distinctive Arbutin-Containing Markers: Chemotaxonomic Significance and Insights into the Evolution of Proteaceae Phytochemistry.

Natural products isolation studies of eight endemic Tasmanian Proteaceae species - Agastachys odorata, Persoonia juniperina, Hakea megadenia, Hakea epiglottis, Orites diversifolius, Orites acicularis, Orites revolutus, and Telopea truncata - and three endemic Australian Proteaceae species Banksia serrata, Banksia praemorsa, and Banksia marginata were undertaken. Two previously unreported glycoside-derived natural products were identified, in addition to four other tremendously rare arbutin esters. The results of this study provide further evidence consistent with the proposal that these distinctive arbutin esters represent markers that can provide valuable insights into the chemical evolution of plant species within the family Proteaceae.

Convergent tip-to-base widening of water-conducting conduits in the tallest bryophytes.

PREMISE: Tip-to-base conduit widening is considered a key mechanism that enables vascular plants to grow tall by decreasing the hydraulic resistance imposed by increasing height. Widening of hydraulic anatomy (larger conducting elements toward the base of the vascular system) minimizes gradients in leaf-specific hydraulic conductance with plant height, allowing uniform photosynthesis across the crown of trees. Tip-to-base conduit widening has also been associated with changes in conduit number. However, in bryophytes, despite having representatives with internal water-conducting tissue, conduit widening has been scarcely investigated. METHODS: Here, we examined the changes in hydroid diameter and number with distance from plant tip in Dawsonia superba and D. polytrichoides, two representatives of the genus containing the tallest extant bryophytes. RESULTS: The position of these moss species on the global scale of conduit size and plant size was consistent with a general scaling among plants with internal water transport. Within plants, patterns of conduit widening and number with distance from plant tip in endohydric mosses were similar to those observed in vascular plants. CONCLUSIONS: This study demonstrated that land plants growing upward in the atmosphere show analogous conduit widening of hydraulic structures, suggesting that efficient internal water transport is a convergent adaptation for photosynthesis on land.

Diversity and abundance of soil microbial communities decline, and community compositions change with severity of post-logging fire.

Understanding the effects of logging and fire on forest soil communities is integral to our knowledge of forest ecology and effective resource management. The resulting changes in soil biota have substantial impacts on forest succession and associated ecosystem processes. We quantified bacterial and fungal abundance, diversity and community composition across a logging and burn severity gradient, approximately one month after fire, in temperate wet eucalypt forests in Tasmania, Australia. Using amplicon sequencing and real-time quantitative PCR of the bacterial 16S rRNA gene and fungal ITS1 region, we demonstrate that (i) burn severity is a strong driver of soil microbial community composition, (ii) logging and high severity burning substantially reduce the biomass and diversity of soil bacteria and fungi, and (iii) the impacts of logging and burning on soil microbial communities are largely restricted to the top 10 cm of soil, with weak impacts on the subsoil. The impacts of disturbance on microbial community composition are greater than the effects of site-to-site edaphic differences. Fire also drives more divergence in community composition than logging alone. Key microbial taxa driving differences in severely burnt soils include bacterial genera implicated in plant-growth promotion and producing antifungal compounds as well as saprotrophic fungi that are also capable of forming ectomycorrhizal associations. Our research suggests that low-moderate severity burns are important for maintaining diversity and biomass in soil microbial communities but having a range of burn severities across a site contributes to the overall diversity of habitat conditions providing for both microbial and plant diversity.

A Novel Patient Values Tab for the Electronic Health Record: A User-Centered Design Approach.

BACKGROUND: The COVID-19 pandemic has shined a harsh light on a critical deficiency in our health care system: our inability to access important information about patients' values, goals, and preferences in the electronic health record (EHR). At Memorial Sloan Kettering Cancer Center (MSK), we have integrated and systematized health-related values discussions led by oncology nurses for newly diagnosed cancer patients as part of routine comprehensive cancer care. Such conversations include not only the patient's wishes for care at the end of life but also more holistic personal values, including sources of strength, concerns, hopes, and their definition of an acceptable quality of life. In addition, health care providers use a structured template to document their discussions of patient goals of care. OBJECTIVE: To provide ready access to key information about the patient as a person with individual values, goals, and preferences, we undertook the creation of the Patient Values Tab in our center's EHR to display this information in a single, central location. Here, we describe the interprofessional, interdisciplinary, iterative process and user-centered design methodology that we applied to build this novel functionality as well as our initial implementation experience and plans for evaluation. METHODS: We first convened a working group of experts from multiple departments, including medical oncology, health informatics, information systems, nursing informatics, nursing education, and supportive care, and a user experience designer. We conducted in-depth, semistructured, audiorecorded interviews of over 100 key stakeholders. The working group sought consensus on the tab's main content, homing in on high-priority areas identified by the stakeholders. The core content was mapped to various EHR data sources. We established a set of high-level design principles to guide our process. Our user experience designer then created wireframes of the tab design. The designer conducted usability testing with physicians, nurses, and other health professionals. Data validation testing was conducted. RESULTS: We have already deployed the Patient Values Tab to a pilot sample of users in the MSK Gastrointestinal Medical Oncology Service, including physicians, advanced practice providers, nurses, and administrative staff. We have early evidence of the positive impact of this EHR innovation. Audit logs show increasing use. Many of the initial user comments have been enthusiastically positive, while others have provided constructive suggestions for additional tab refinements with respect to format and content. CONCLUSIONS: It is our challenge and obligation to enrich the EHR with information about the patient as a person. Realization of this capability is a pressing public health need requiring the collaboration of technological experts with a broad range of clinical leaders, users, patients, and families to achieve solutions that are both principled and practical. Our new Patient Values Tab represents a step forward in this important direction.

Links between environment and stomatal size through evolutionary time in Proteaceae.

The size of plant stomata (adjustable pores that determine the uptake of CO2 and loss of water from leaves) is considered to be evolutionarily important. This study uses fossils from the major Southern Hemisphere family Proteaceae to test whether stomatal cell size responded to Cenozoic climate change. We measured the length and abundance of guard cells (the cells forming stomata), the area of epidermal pavement cells, stomatal index and maximum stomatal conductance from a comprehensive sample of fossil cuticles of Proteaceae, and extracted published estimates of past temperature and atmospheric CO2. We developed a novel test based on stochastic modelling of trait evolution to test correlations among traits. Guard cell length increased, and stomatal density decreased significantly with decreasing palaeotemperature. However, contrary to expectations, stomata tended to be smaller and more densely packed at higher atmospheric CO2. Thus, associations between stomatal traits and palaeoclimate over the last 70 million years in Proteaceae suggest that stomatal size is significantly affected by environmental factors other than atmospheric CO2. Guard cell length, pavement cell area, stomatal density and stomatal index covaried in ways consistent with coordinated development of leaf tissues.

Leaf hydraulic conductance is linked to leaf symmetry in bifacial, amphistomatic leaves of sunflower.

The hydraulic implications of stomatal positioning across leaf surfaces and the impact on internal water flow through amphistomatic leaves are not currently well understood. Amphistomaty potentially provides hydraulic efficiencies if the majority of hydraulic resistance in the leaf exists outside the xylem in the mesophyll. Such a scenario would mean that the same xylem network could equally supply a hypostomatic or amphistomatic leaf. Here we examine leaves of Helianthus annuus to determine whether amphistomaty in this species is associated with higher hydraulic efficiency compared with hypostomatic leaves. We identified asymmetry in the positioning of minor veins which were significantly closer to the abaxial than the adaxial leaf surface, combined with lower Kleaf when transpiration was driven through the adaxial rather than the abaxial surface. We also identified a degree of coordination in stomatal behaviour driven by leaf hydraulics, where the hydraulic conditions experienced by an individual leaf surface affected the stomatal behaviour on the opposite surface. We found no advantage to amphistomaty based on efficiencies in construction costs of the venous system, represented by vein density:stomatal density, only limited hydraulic independence between leaf surfaces. These results suggest that amphistomaty does not substantially increase whole-leaf hydraulic efficiency.

Pliocene reversal of late Neogene aridification.

The Pliocene epoch (5.3-2.6 Ma) represents the most recent geological interval in which global temperatures were several degrees warmer than today and is therefore considered our best analog for a future anthropogenic greenhouse world. However, our understanding of Pliocene climates is limited by poor age control on existing terrestrial climate archives, especially in the Southern Hemisphere, and by persistent disagreement between paleo-data and models concerning the magnitude of regional warming and/or wetting that occurred in response to increased greenhouse forcing. To address these problems, here we document the evolution of Southern Hemisphere hydroclimate from the latest Miocene to the middle Pliocene using radiometrically-dated fossil pollen records preserved in speleothems from semiarid southern Australia. These data reveal an abrupt onset of warm and wet climates early within the Pliocene, driving complete biome turnover. Pliocene warmth thus clearly represents a discrete interval which reversed a long-term trend of late Neogene cooling and aridification, rather than being simply the most recent period of greater-than-modern warmth within a continuously cooling trajectory. These findings demonstrate the importance of high-resolution chronologies to accompany paleoclimate data and also highlight the question of what initiated the sustained interval of Pliocene warmth.

Similar geometric rules govern the distribution of veins and stomata in petals, sepals and leaves.

Investment in leaf veins (supplying xylem water) is balanced by stomatal abundance, such that sufficient water transport is provided for stomata to remain open when soil water is abundant. This coordination is mediated by a common dependence of vein and stomatal densities on cell size. Flowers may not conform to this same developmental pattern if they depend on water supplied by the phloem or have high rates of nonstomatal transpiration. We examined the relationships between veins, stomata and epidermal cells in leaves, sepals and petals of 27 angiosperms to determine whether common spacing rules applied to all tissues. Regression analysis found no evidence for different relationships within organ types. Both vein and stomatal densities were strongly associated with epidermal cell size within organs, but, for a given epidermal cell size, petals had fewer veins and stomata than sepals, which had fewer than leaves. Although our data support the concept of common scaling between veins and stomata in leaves and flowers, the large diversity in petal vein density suggests that, in some species, petal veins may be engaged in additional functions, such as the supply of water for high cuticular transpiration or for phloem delivery of water or carbohydrates.

Wheat leaves embolized by water stress do not recover function upon rewatering.

New techniques now make it possible to precisely and accurately determine the failure threshold of the plant vascular system during water stress. This creates an opportunity to understand the vulnerability of species to drought, but first, it must be determined whether damage to leaf function associated with xylem cavitation is reparable or permanent. This question is particularly relevant in crop plants such as wheat, which may have the capacity to repair xylem embolism with positive root pressure. Using wheat (Triticum aestivum, Heron), we employed non-invasive imaging to find the water potential causing 50% xylem embolism (-2.87 ± 0.52 MPa) in leaves. Replicate plants were water-stressed to varying degrees to induce embolism ranging from minimal to substantial. Plants were then rewatered to determine the reversibility of xylem damage and photosynthetic inhibition in glasshouse conditions. Rewatering after drought-induced xylem cavitation did not induce visible refilling of embolized xylem, and embolized leaves showed photosynthetic impairment upon rewatering. This impairment was significant even after only 10-20% of leaf veins were embolized, and leaves accumulating >20% embolism died upon rewatering in 7/10 individuals. Photosynthetic damage and hydraulic decline occurred concurrently as wheat leaves dehydrated, and leaf shrinkage during drying was the best predictor of photosynthetic recovery.

Extended differentiation of veins and stomata is essential for the expansion of large leaves in Rheum rhabarbarum.

PREMISE OF THE STUDY: The densities of veins and stomata govern leaf water supply and gas exchange. They are coordinated to avoid overproduction of either veins or stomata. In many species, where leaf area is greater at low light, this coordination is primarily achieved through differential cell expansion, resulting in lower stomatal and vein density in larger leaves. This mechanism would, however, create highly inefficient leaves in species in which leaf area is greater at high light. Here we investigate the role of cell expansion and differentiation as regulators of vein and stomatal density in Rheum rhabarbarum, which produces large leaves under high light. METHODS: Rheum rhabarbarum plants were grown under full sunlight and 7% of full sunlight. Leaf area, stomatal density, and vein density were measured from leaves harvested at different intervals. KEY RESULTS: Leaves of R. rhabarbarum expanded at high light were six times larger than leaves expanded at low light, yet vein and stomatal densities were similar. In high light-expanded leaves, minor veins were continuously initiated as the leaves expanded, while an extended period of stomatal initiation, compared to leaves expanded at low light, occurred early in leaf development. CONCLUSIONS: We demonstrate that R. rhabarbarum adjusts the initiation of stomata and minor veins at high light, allowing for the production of larger leaves uncoupled from lower vein and stomatal densities. We also present evidence for an independent control of vein and stomatal initiation, suggesting that this adjustment must involve some unknown developmental mechanism.

Arbutin Derivatives Isolated from Ancient Proteaceae: Potential Phytochemical Markers Present in Bellendena, Cenarrhenes, and Persoonia Genera.

Extensive phytochemical studies of the paleoendemic Tasmanian Proteaceae species Bellendena montana, Cenarrhenes nitida, and Persoonia gunnii were conducted employing pressurized hot water extraction. As part of these studies, six novel glycosides were isolated, including rare examples of glycoside-containing natural products featuring tiglic acid esters. These polar molecules may represent potential phytochemical markers in ancient Proteaceae.

Insights into hominid evolution from the gorilla genome sequence.

Gorillas are humans' closest living relatives after chimpanzees, and are of comparable importance for the study of human origins and evolution. Here we present the assembly and analysis of a genome sequence for the western lowland gorilla, and compare the whole genomes of all extant great ape genera. We propose a synthesis of genetic and fossil evidence consistent with placing the human-chimpanzee and human-chimpanzee-gorilla speciation events at approximately 6 and 10 million years ago. In 30% of the genome, gorilla is closer to human or chimpanzee than the latter are to each other; this is rarer around coding genes, indicating pervasive selection throughout great ape evolution, and has functional consequences in gene expression. A comparison of protein coding genes reveals approximately 500 genes showing accelerated evolution on each of the gorilla, human and chimpanzee lineages, and evidence for parallel acceleration, particularly of genes involved in hearing. We also compare the western and eastern gorilla species, estimating an average sequence divergence time 1.75 million years ago, but with evidence for more recent genetic exchange and a population bottleneck in the eastern species. The use of the genome sequence in these and future analyses will promote a deeper understanding of great ape biology and evolution.

A high-resolution map of human evolutionary constraint using 29 mammals.

The comparison of related genomes has emerged as a powerful lens for genome interpretation. Here we report the sequencing and comparative analysis of 29 eutherian genomes. We confirm that at least 5.5% of the human genome has undergone purifying selection, and locate constrained elements covering ∼4.2% of the genome. We use evolutionary signatures and comparisons with experimental data sets to suggest candidate functions for ∼60% of constrained bases. These elements reveal a small number of new coding exons, candidate stop codon readthrough events and over 10,000 regions of overlapping synonymous constraint within protein-coding exons. We find 220 candidate RNA structural families, and nearly a million elements overlapping potential promoter, enhancer and insulator regions. We report specific amino acid residues that have undergone positive selection, 280,000 non-coding elements exapted from mobile elements and more than 1,000 primate- and human-accelerated elements. Overlap with disease-associated variants indicates that our findings will be relevant for studies of human biology, health and disease.

Conifer species adapt to low-rainfall climates by following one of two divergent pathways.

Water stress is one of the primary selective forces in plant evolution. There are characters often cited as adaptations to water stress, but links between the function of these traits and adaptation to drying climates are tenuous. Here we combine distributional, climatic, and physiological evidence from 42 species of conifers to show that the evolution of drought resistance follows two distinct pathways, both involving the coordinated evolution of tissues regulating water supply (xylem) and water loss (stomatal pores) in leaves. Only species with very efficient stomatal closure, and hence low minimum rates of water loss, inhabit dry habitats, but species diverged in their apparent mechanism for maintaining closed stomata during drought. An ancestral mechanism found in Pinaceae and Araucariaceae species relies on high levels of the hormone abscisic acid (ABA) to close stomata during water stress. A second mechanism, found in the majority of Cupressaceae species, uses leaf desiccation rather than high ABA levels to close stomata during sustained water stress. Species in the latter group were characterized by xylem tissues with extreme resistance to embolism but low levels of foliar ABA after 30 d without water. The combination of low levels of ABA under stress with cavitation-resistant xylem enables these species to prolong stomatal opening during drought, potentially extending their photosynthetic activity between rainfall events. Our data demonstrate a surprising simplicity in the way conifers evolved to cope with water shortage, indicating a critical interaction between xylem and stomatal tissues during the process of evolution to dry climates.

Cell expansion not cell differentiation predominantly co-ordinates veins and stomata within and among herbs and woody angiosperms grown under sun and shade.

BACKGROUND AND AIMS: It has been proposed that modification of leaf size, driven by epidermal cell size, balances leaf water supply (determined by veins) with transpirational demand (generated by stomata) during acclimation to local irradiance. We aimed to determine whether this is a general pattern among plant species with contrasting growth habits. METHODS: We compared observed relationships between leaf minor vein density, stomatal density, epidermal cell size and leaf size in four pairs of herbs and woody species from the same families grown under sun and shade conditions with modelled relationships assuming vein and stomatal densities respond passively to epidermal cell expansion. Leaf lignin content was also quantified to assess whether construction costs of herbaceous leaf veins differ from those of woody plants and the leaf mass fraction invested in veins. KEY RESULTS: Modelled relationships accurately described observed relationships, indicating that in all species, co-ordinated changes to the density of minor veins and stomata were mediated by a common relationship between epidermal cell size, vein density and stomatal density, with little or no impact from stomatal index. This co-ordination was independent of changes in leaf size and is likely to be an adaptive process driven by the significant proportion of biomass invested in veins (13·1 % of sun leaf dry weight and 21·7 % of shade leaf dry weight). Relative costs of venation increased in the shade, intensifying selective pressure towards economizing investment in vein density. CONCLUSIONS: Modulation of epidermal cell size appears to be a general mechanism among our experimental species to maintain a constant ratio between leaf anatomical traits that control leaf water fluxes independently of habit. We propose that this process may co-ordinate plasticity in hydraulic supply and demand in the majority of eudicot angiosperms.

Transient hybridization, not homoploid hybrid speciation, between ancient and deeply divergent conifers.

PREMISE OF THE STUDY: Homoploid hybrid speciation is receiving growing attention due the increasing recognition of its role in speciation. We investigate if individuals intermediate in morphology between the two species of the conifer genus Athrotaxis represent a homoploid hybrid species, A. laxifolia, or are spontaneous F1 hybrids. METHODS: A total of 1055 individuals of Athrotaxis cupressoides and A. selaginoides, morphologically intermediate individuals, and two putative hybrid swarms were sampled across the range of the genus and genotyped with 13 microsatellites. We used simulations to test the power of our data to identify the pure species, F1s, F2s, and backcross generations. KEY RESULTS: We found that Athrotaxis cupressoides and A. selaginoides are likely the most divergent congeneric conifers known, but the intermediates are F1 hybrids, sharing one allele each from A. cupressoides and A. selaginoides at six loci with completely species specific alleles. The hybrid swarms contain wide genetic variation with stronger affinities to the locally dominant species, A. selaginoides and A. selaginoides backcrosses outnumbering A. cupressoides backcrosses. In addition, we observed evidence for isolated advanced generation backcrosses within the range of the pure species. CONCLUSIONS: We conclude that, even though they can be large and long-lived, Athrotaxis hybrid swarms are on a trajectory of decline and will eventually be reabsorbed by the parental species. However, this process may take millennia and fossil evidence suggests that such events have occurred repeatedly since the early Quaternary. Given this timeline, our study highlights the many obstacles to homoploid hybrid speciation.

Fossil evidence for a hyperdiverse sclerophyll flora under a non-Mediterranean-type climate.

The spectacular diversity of sclerophyll plants in the Cape Floristic Region in South Africa and Australia's Southwest Floristic Region has been attributed to either explosive radiation on infertile soils under fire-prone, summer-dry climates or sustained accretion of species under inferred stable climate regimes. However, the very poor fossil record of these regions has made these ideas difficult to test. Here, we reconstruct ecological-scale plant species richness from an exceptionally well-preserved fossil flora. We show that a hyperdiverse sclerophyll flora existed under high-rainfall, summer-wet climates in the Early Pleistocene in southeastern Australia. The sclerophyll flora of this region must, therefore, have suffered subsequent extinctions to result in its current relatively low diversity. This regional loss of sclerophyll diversity occurred at the same time as a loss of rainforest diversity and cannot be explained by ecological substitution of species of one ecological type by another type. We show that sclerophyll hyperdiversity has developed in distinctly non-Mediterranean climates, and this diversity is, therefore, more likely a response to long-term climate stability. Climate stability may have both reduced the intensity of extinctions associated with the Pleistocene climate cycles and promoted the accumulation of species richness by encouraging genetic divergence between populations and discouraging plant dispersal.

Environmental adaptation in stomatal size independent of the effects of genome size.

Cell sizes are linked across multiple tissues, including stomata, and this variation is closely correlated with genome size. These associations raise the question of whether generic changes in cell size cause suboptimal changes in stomata, requiring subsequent evolution under selection for stomatal size. We tested the relationships among guard cell length, genome size and vegetation type using phylogenetically independent analyses on 67 species of the ecologically and structurally diverse family, Proteaceae. We also compared how genome and stomatal sizes varied at ancient (among genera) and more recent (within genus) levels. The observed 60-fold range in genome size in Proteaceae largely reflected the mean chromosome size. Compared with variation among genera, genome size varied much less within genera (< 6% of total variance) than stomatal size, implying evolution in stomatal size subsequent to changes in genome size. Open vegetation and closed forest had significantly different relationships between stomatal and genome sizes. Ancient changes in genome size clearly influenced stomatal size in Proteaceae, but adaptation to habitat strongly modified the genome-stomatal size relationship. Direct adaptation to the environment in stomatal size argues that new proxies for past concentrations of atmospheric CO2 that incorporate stomatal size are superior to older models based solely on stomatal frequency.

Linking changes in community composition and function under climate change.

Climate change is expected to directly alter the composition of communities and the functioning of ecosystems across the globe. Improving our understanding of links between biodiversity and ecosystem functioning across large spatial scales and rapid global change is a major priority to help identify management responses that will retain diverse, functioning systems. Here we address this challenge by linking projected changes in plant community composition and functional attributes (height, leaf area, seed mass) under climate change across Tasmania, Australia. Using correlative community-level modeling, we found that projected changes in plant community composition were not consistently related to projected changes in community mean trait values. In contrast, we identified specific mechanisms through which alternative combinations of projected functional and compositional change across Tasmania could be realized, including loss/replacement of functionally similar species (lowland grasslands/grassy woodlands) and loss of a small number of functionally unique species (lowland forests). Importantly, we demonstrate how these linked projections of change in community composition and functional attributes can be utilized to inform specific management actions that may assist in maintaining diverse, functioning ecosystems under climate change.