Although temporal calibration is widely recognized as critical for obtaining accurate divergence-time estimates using molecular dating methods, few studies have evaluated the variation resulting from different calibration strategies. Depending on the information available, researchers have often used primary calibrations from the fossil record or secondary calibrations from previous molecular dating studies. In analyses of flowering plants, primary calibration data can be obtained from macro- and mesofossils (e.g., leaves, flowers, and fruits) or microfossils (e.g., pollen). Fossil data can vary substantially in accuracy and precision, presenting a difficult choice when selecting appropriate calibrations. Here, we test the impact of eight plausible calibration scenarios for Nothofagus (Nothofagaceae, Fagales), a plant genus with a particularly rich and well-studied fossil record. To do so, we reviewed the phylogenetic placement and geochronology of 38 fossil taxa of Nothofagus and other Fagales, and we identified minimum age constraints for up to 18 nodes of the phylogeny of Fagales. Molecular dating analyses were conducted for each scenario using maximum likelihood (RAxML + r8s) and Bayesian (BEAST) approaches on sequence data from six regions of the chloroplast and nuclear genomes. Using either ingroup or outgroup constraints, or both, led to similar age estimates, except near strongly influential calibration nodes. Using "early but risky" fossil constraints in addition to "safe but late" constraints, or using assumptions of vicariance instead of fossil constraints, led to older age estimates. In contrast, using secondary calibration points yielded drastically younger age estimates. This empirical study highlights the critical influence of calibration on molecular dating analyses. Even in a best-case situation, with many thoroughly vetted fossils available, substantial uncertainties can remain in the estimates of divergence times. For example, our estimates for the crown group age of Nothofagus varied from 13 to 113 Ma across our full range of calibration scenarios. We suggest that increased background research should be made at all stages of the calibration process to reduce errors wherever possible, from verifying the geochronological data on the fossils to critical reassessment of their phylogenetic position.

SummaryThe 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.

Gondwanan rainforest lineages contribute significantly to modern rainforest community assembly and often co-occur in widely separated assemblages far from their early fossil records. Understanding how and where lineages from ancient Gondwanan assemblages co-occur today has implications for the conservation of global rainforest vegetation, including in the Old World tropics.

Sunken, covered, and encrypted stomata have been anecdotally linked with dry climates and reduced transpiration and therefore have been used to infer dry palaeoclimates from fossils. This study assesses the evolutionary and ecological associations of such stomatal protection in a model system-the diverse southern hemisphere family Proteaceae. Analyses were based on the morphology of over 1400 Australian, South African, New Caledonian, New Zealand, and South American species, anatomy of over 300 of these species, and bioclimatic data from all 1109 Australian species. Ancestral state reconstruction revealed that five or six evolutionary transitions explain over 98% of the dry climate species in the family, with a few other, minor invasions of dry climates. Deep encryption, i.e., stomata in deep pits, in grooves, enclosed by tightly revolute margins or strongly overarched by cuticle, evolved at least 11 times in very dry environments. Other forms of stomatal protection (sunken but not closely encrypted stomata, papillae, and layers of hairs covering the stomata) also evolved repeatedly, but had no systematic association with dry climates. These data are evidence for a strong distinction in function, with deep encryption being an adaptation to aridity, whereas broad pits and covered stomata have more complex relations to climate.

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.