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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., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

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., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

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., (© 2014 The Authors New Phytologist © 2014 New Phytologist Trust.)

The processes by which the functions of interdependent tissues are coordinated as lineages diversify are poorly understood. Here, we examine evolutionary coordination of vascular, epidermal and cortical leaf tissues in the anatomically, ecologically and morphologically diverse woody plant family Proteaceae. We found that, across the phylogenetic range of Proteaceae, the sizes of guard, epidermal, palisade and xylem cells were positively correlated with each other but negatively associated with vein and stomatal densities. The link between venation and stomata resulted in a highly efficient match between potential maximum water loss (determined by stomatal conductance) and the leaf vascular system's capacity to replace that water. This important linkage is likely to be driven by stomatal size, because spatial limits in the packing of stomata onto the leaf surface apparently constrain the maximum size and density of stomata. We conclude that unified evolutionary changes in cell sizes of independent tissues, possibly mediated by changes in genome size, provide a means of substantially modifying leaf function while maintaining important functional links between leaf tissues. Our data also imply the presence of alternative evolutionary strategies involving cellular miniaturization during radiation into closed forest, and cell size increase in open habitats., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Tree species exceeding 70 m in height are rare globally. Giant gymnosperms are concentrated near the Pacific coast of the USA, while the tallest angiosperms are eucalypts (Eucalyptus spp.) in southern and eastern Australia. Giant eucalypts co-occur with rain-forest trees in eastern Australia, creating unique vegetation communities comprising fire-dependent trees above fire-intolerant rain-forest. However, giant eucalypts can also tower over shrubby understoreys (e.g. in Western Australia). The local abundance of giant eucalypts is controlled by interactions between fire activity and landscape setting. Giant eucalypts have features that increase flammability (e.g. oil-rich foliage and open crowns) relative to other rain-forest trees but it is debatable if these features are adaptations. Probable drivers of eucalypt gigantism are intense intra-specific competition following severe fires, and inter-specific competition among adult trees. However, we suggest that this was made possible by a general capacity of eucalypts for 'hyper-emergence'. We argue that, because giant eucalypts occur in rain-forest climates and share traits with rain-forest pioneers, they should be regarded as long-lived rain-forest pioneers, albeit with a particular dependence on fire for regeneration. These unique ecosystems are of high conservation value, following substantial clearing and logging over 150 yr. Contents Summary 1001 I. Introduction 1001 II. Giant eucalypts in a global context 1002 III. Giant eucalypts - taxonomy and distribution 1004 IV. Growth of giant eucalypts 1006 V. Fire and regeneration of giant eucalypts 1008 VI. Are giant eucalypts different from other rain-forest trees? 1009 VII. Conclusions 1010 Acknowledgements 1011 References 1011., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

This review uses proxies of past temperature and atmospheric CO(2) composition based on fossil leaves to illustrate the uncertainties in biologically based proxies of past environments. Most leaf-based proxies are geographically local or genetically restricted and therefore can be confounded by evolution, extinction, changes in local environment or immigration of species. Stomatal frequency proxies illustrate how genetically restricted proxies can be particularly vulnerable to evolutionary change. High predictive power in the modern world resulting from the use of a very narrow calibration cannot be confidently extrapolated into the past (the Ginkgo paradox). Many foliar physiognomic proxies of climate are geographically local and use traits that are more or less fixed for individual species. Such proxies can therefore be confounded by floristic turnover and biome shifts in the region of calibration. Uncertainty in proxies tends to be greater for more ancient fossils. I present a set of questions that should be considered before using a proxy. Good proxies should be relatively protected from environmental and genetic change, particularly through having high information content and being founded on biomechanical or biochemical principles. Some current and potential developments are discussed, including those that involve more mechanistically sound proxies and better use of multivariate approaches., (© 2011 The Author. New Phytologist © 2011 New Phytologist Trust.)

Higher leaf vein density (D(vein) ) enables higher rates of photosynthesis because enhanced water transport allows higher leaf conductances to CO(2) and water. If the total cost of leaf venation rises in proportion to the density of minor veins, the most efficient investment in leaf xylem relative to photosynthetic gain should occur when the water transport capacity of the leaf (determined by D(vein) ) matches potential transpirational demand (determined by stomatal size and density). We tested whether environmental plasticity in stomatal density (D(stomata) ) and D(vein) were linked in the evergreen tree Nothofagus cunninghamii to achieve a balance between liquid and gas phase water conductances. Two sources of variation were examined; within-tree light acclimation, and differences in sun leaves among plants from ecologically diverse populations. Strong, linear correlations between D(vein) and D(stomata) were found at all levels of comparison. The correlations between liquid- and vapour-phase conductances implied by these patterns of leaf anatomy were confirmed by direct measurement of leaf conductance in sun and shade foliage of an individual tree. • Our results provide strong evidence that the development of veins and stomata are coordinated so that photosynthetic yield is optimized relative to carbon investment in leaf venation., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

• Paleobotanists have long used models based on leaf size and shape to reconstruct paleoclimate. However, most models incorporate a single variable or use traits that are not physiologically or functionally linked to climate, limiting their predictive power. Further, they often underestimate paleotemperature relative to other proxies. • Here we quantify leaf-climate correlations from 92 globally distributed, climatically diverse sites, and explore potential confounding factors. Multiple linear regression models for mean annual temperature (MAT) and mean annual precipitation (MAP) are developed and applied to nine well-studied fossil floras. • We find that leaves in cold climates typically have larger, more numerous teeth, and are more highly dissected. Leaf habit (deciduous vs evergreen), local water availability, and phylogenetic history all affect these relationships. Leaves in wet climates are larger and have fewer, smaller teeth. Our multivariate MAT and MAP models offer moderate improvements in precision over univariate approaches (± 4.0 vs 4.8°C for MAT) and strong improvements in accuracy. For example, our provisional MAT estimates for most North American fossil floras are considerably warmer and in better agreement with independent paleoclimate evidence. • Our study demonstrates that the inclusion of additional leaf traits that are functionally linked to climate improves paleoclimate reconstructions. This work also illustrates the need for better understanding of the impact of phylogeny and leaf habit on leaf-climate relationships., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

Hydraulic dysfunction in leaves determines key aspects of whole-plant responses to water stress; however, our understanding of the physiology of hydraulic dysfunction and its relationships to leaf structure and ecological strategy remains incomplete. Here, we studied a morphologically and ecologically diverse sample of angiosperms to test whether the water potential inducing a 50% loss in leaf hydraulic conductance (P50(leaf)) is predicted by properties of leaf xylem relating to water tension-induced conduit collapse. We also assessed the relationships between P50(leaf) and other traits considered to reflect drought resistance and ecological strategy. Across species, P50(leaf) was strongly correlated with a theoretical predictor of vulnerability to cell collapse in minor veins (the cubed ratio of the conduit wall thickness to the conduit lumen breadth). P50(leaf) was also correlated with mesophyll traits known to be related to drought resistance, but unrelated to traits associated with carbon economy. Our data indicate a link between the structural mechanics of leaf xylem and hydraulic function under water stress. Although it is possible that collapse may contribute directly to dysfunction, this relationship may also be a secondary product of vascular economics, suggesting that leaf xylem is dimensioned to avoid wall collapse., (© The Authors (2010). Journal compilation © New Phytologist Trust (2010).)

The stomata of angiosperms respond to changes in ambient atmospheric concentrations of CO(2) (C(a)) in ways that appear to optimize water-use efficiency. It is unknown where in the history of land plants this important stomatal control mechanism evolved. Here, we test the hypothesis that major clades of plants have distinct stomatal sensitivities to C(a) reflecting a relatively recent evolution of water-use optimization in derived angiosperms. Responses of stomatal conductance (g(s)) to step changes between elevated, ambient and low C(a) (600, 380 and 100 micromol mol(-1), respectively) were compared in a phylogenetically and ecologically diverse range of higher angiosperms, conifers, ferns and lycopods. All species responded to low C(a) by increasing g(s) but only angiosperm stomata demonstrated a significant closing response when C(a) was elevated to 600 micromol mol(-1). As a result, angiosperms showed significantly greater increases in water-use efficiency under elevated C(a) than the other lineages. The data suggest that the angiosperms have mechanisms for detecting and responding to increases in C(a) that are absent from earlier diverging lineages, and these mechanisms impart a greater capacity to optimize water-use efficiency.

Glacial aridity of the Pleistocene was inhospitable for the cool temperate rainforest tree Nothofagus cunninghamii over most of its current range in southeastern Australia, particularly in eastern Tasmania. A chloroplast DNA phylogeographic study was undertaken to investigate whether this species was likely to have survived in situ or conforms to a dispersal model of postglacial recovery. Twenty-three chloroplast haplotypes were identified by PCR-RFLP and direct sequencing of 2164 base pairs from 213 N. cunninghamii individuals collected in a range-wide survey. Fine-scale haplotype distribution was investigated using PCR-RFLP in eastern Tasmania. Deep chloroplast divergence occurred in N. cunninghamii. The single haplotype of the sister species, N. moorei, was nested among N. cunninghamii haplotypes. The distribution of N. cunninghamii haplotypes supports: multiple glacial refugia in coastal and inland western Tasmania, the centre of haplotype diversity; glacial survival in the central highlands of Victoria, corroborating pollen data; and the long-term occupation of eastern Tasmania because of the presence of a unique deeply diverged chloroplast lineage. Nothofagus cunninghamii withstood glacial aridity within multiple regions in apparently nonequable climates. This finding contributes to a growing understanding of how the resilience of temperate species during glacial periods has shaped modern biota.

The lengths of fossil leaves of Nothofogus cunninghamii, and of closely related fossil species from sediments older than the Middle Pleistocene showed greater variation than fossil leaves from younger sediments and extant leaves from modern sediment samples and forest floor litter. Leaf sizes of samples of the other two extant evergreen species of Nothofagus subgenus Lophazonia, N. moorei and N.menziesii, were no more variable than modern N.cunninhamii. Leaf lengths of modern and Middle-Late Pleistocene N. cunninhamii and of modern N. menziesii were more or less unimodal in distribution, whereas the leaves of N. moorei follow bimodal distributions, and leaves from the Early Pleistocene Regatta Point and the Oligocene Link- Rapid River sediments tended to be bimodal and highly variable. Leaf lengths from the Oligo-Miocene Monpeelyata sediments were also highly variable, but the sample size was too small to determine if they follow bimodal distributions. The bimodality and high variability in the older sites are unlikely to have been due to taphonomic processes. Thus, within population variability appears to have declined during the Late Tertiary-Early Pleistocene, although the presence of cryptic species in the fossil record could also explain the results. Both a decline in variability within species, and the extinction of Nothofugus species, are consistent with a well documented Late Cainozoic decline in rainforest diversity in Tasmania. Log transformed leaf lengths of modern forest floor litter samples of N. cunninhamii were Strongly correlated with climatic parameters, particularly summer temperature, suggesting that this parameter is a strong determinant of leaf size in N. cunninhamii Changes in N. cunninhamii leaf lengths within Middle Pleistocene sediments were associated with floristic changes interpreted as transitions in glacial/interglacial cycles but these changes were small compared with changes predicted Iron climatic correlation of extant samples, probably because of limited Emetic variability in local populations at the time of deposition.