Elizabeth L. Spriggs

Contemporaneous and recent radiations of the world's major succulent plant lineages.

The cacti are one of the most celebrated radiations of succulent plants. There has been much speculation about their age, but progress in dating cactus origins has been hindered by the lack of fossil data for cacti or their close relatives. Using a hybrid phylogenomic approach, we estimated that the cactus lineage diverged from its closest relatives ≈35 million years ago (Ma). However, major diversification events in cacti were more recent, with most species-rich clades originating in the late Miocene, ≈10–5 Ma. Diversification rates of several cactus lineages rival other estimates of extremely rapid speciation in plants. Major cactus radiations were contemporaneous with those of South African ice plants and North American agaves, revealing a simultaneous diversification of several of the worlds major succulent plant lineages across multiple continents. This short geological time period also harbored the majority of origins of C4 photosynthesis and the global rise of C4 grasslands. A global expansion of arid environments during this time could have provided new ecological opportunity for both succulent and C4 plant syndromes. Alternatively, recent work has identified a substantial decline in atmospheric CO2 ≈15–8 Ma, which would have strongly favored C4 evolution and expansion of C4-dominated grasslands. Lowered atmospheric CO2 would also substantially exacerbate plant water stress in marginally arid environments, providing preadapted succulent plants with a sharp advantage in a broader set of ecological conditions and promoting their rapid diversification across the landscape.

Molecular Dating, Evolutionary Rates, and the Age of the Grasses

Many questions in evolutionary biology require an estimate of divergence times but, for groups with a sparse fossil record, such estimates rely heavily on molecular dating methods. The accuracy of these methods depends on both an adequate underlying model and the appropriate implementation of fossil evidence as calibration points. We explore the effect of these in Poaceae (grasses), a diverse plant lineage with a very limited fossil record, focusing particularly on dating the early divergences in the group. We show that molecular dating based on a data set of plastid markers is strongly dependent on the model assumptions. In particular, an acceleration of evolutionary rates at the base of Poaceae followed by a deceleration in the descendants strongly biases methods that assume an autocorrelation of rates. This problem can be circumvented by using markers that have lower rate variation, and we show that phylogenetic markers extracted from complete nuclear genomes can be a useful complement to the more commonly used plastid markers. However, estimates of divergence times remain strongly affected by different implementations of fossil calibration points. Analyses calibrated with only macrofossils lead to estimates for the age of core Poaceae ∼51-55 Ma, but the inclusion of microfossil evidence pushes this age to 74-82 Ma and leads to lower estimated evolutionary rates in grasses. These results emphasize the importance of considering markers from multiple genomes and alternative fossil placements when addressing evolutionary issues that depend on ages estimated for important groups.

Photosynthetic pathway and ecological adaptation explain stomatal trait diversity amongst grasses

• The evolution of C(4) photosynthesis in plants has allowed the maintenance of high CO(2) assimilation rates despite lower stomatal conductances. This underpins the greater water-use efficiency in C(4) species and their tendency to occupy drier, more seasonal environments than their C(3) relatives. • The basis of interspecific variation in maximum stomatal conductance to water (g(max) ), as defined by stomatal density and size, was investigated in a common-environment screening experiment. Stomatal traits were measured in 28 species from seven grass lineages, and comparative methods were used to test for predicted effects of C(3) and C(4) photosynthesis, annual precipitation and habitat wetness on g(max) . • Novel results were as follows: significant phylogenetic patterns exist in g(max) and its determinants, stomatal size and stomatal density; C(4) species consistently have lower g(max) than their C(3) relatives, associated with a shift towards smaller stomata at a given density. A direct relationship between g(max) and precipitation was not supported. However, we confirmed associations between C(4) photosynthesis and lower precipitation, and showed steeper stomatal size-density relationships and higher g(max) in wetter habitats. • The observed relationships between stomatal patterning, photosynthetic pathway and habitat provide a clear example of the interplay between anatomical traits, physiological innovation and ecological adaptation in plants.

C4 photosynthesis promoted species diversification during the Miocene grassland expansion.

Identifying how organismal attributes and environmental change affect lineage diversification is essential to our understanding of biodiversity. With the largest phylogeny yet compiled for grasses, we present an example of a key physiological innovation that promoted high diversification rates. C4 photosynthesis, a complex suite of traits that improves photosynthetic efficiency under conditions of drought, high temperatures, and low atmospheric CO2, has evolved repeatedly in one lineage of grasses and was consistently associated with elevated diversification rates. In most cases there was a significant lag time between the origin of the pathway and subsequent radiations, suggesting that the ‘C4 effect’ is complex and derives from the interplay of the C4 syndrome with other factors. We also identified comparable radiations occurring during the same time period in C3 Pooid grasses, a diverse, cold-adapted grassland lineage that has never evolved C4 photosynthesis. The mid to late Miocene was an especially important period of both C3 and C4 grass diversification, coincident with the global development of extensive, open biomes in both warm and cool climates. As is likely true for most “key innovations”, the C4 effect is context dependent and only relevant within a particular organismal background and when particular ecological opportunities became available.

Misconceptions on Missing Data in RAD-seq Phylogenetics with a Deep-scale Example from Flowering Plants

Abstract Restriction‐site associated DNA (RAD) sequencing and related methods rely on the conservation of enzyme recognition sites to isolate homologous DNA fragments for sequencing, with the consequence that mutations disrupting these sites lead to missing information. There is thus a clear expectation for how missing data should be distributed, with fewer loci recovered between more distantly related samples. This observation has led to a related expectation: that RAD‐seq data are insufficiently informative for resolving deeper scale phylogenetic relationships. Here we investigate the relationship between missing information among samples at the tips of a tree and information at edges within it. We re‐analyze and review the distribution of missing data across ten RAD‐seq data sets and carry out simulations to determine expected patterns of missing information. We also present new empirical results for the angiosperm clade Viburnum (Adoxaceae, with a crown age >50 Ma) for which we examine phylogenetic information at different depths in the tree and with varied sequencing effort. The total number of loci, the proportion that are shared, and phylogenetic informativeness varied dramatically across the examined RAD‐seq data sets. Insufficient or uneven sequencing coverage accounted for similar proportions of missing data as dropout from mutation‐disruption. Simulations reveal that mutation‐disruption, which results in phylogenetically distributed missing data, can be distinguished from the more stochastic patterns of missing data caused by low sequencing coverage. In Viburnum, doubling sequencing coverage nearly doubled the number of parsimony informative sites, and increased by >10X the number of loci with data shared across >40 taxa. Our analysis leads to a set of practical recommendations for maximizing phylogenetic information in RAD‐seq studies.

C4 photosynthesis boosts growth by altering physiology, allocation and size.

C4 photosynthesis is a complex set of leaf anatomical and biochemical adaptations that have evolved more than 60 times to boost carbon uptake compared with the ancestral C3 photosynthetic type1–3. Although C4 photosynthesis has the potential to drive faster growth rates4,5, experiments directly comparing C3 and C4 plants have not shown consistent effects1,6,7. This is problematic because differential growth is a crucial element of ecological theory8,9 explaining C4 savannah responses to global change10,11, and research to increase C3 crop productivity by introducing C4 photosynthesis12. Here, we resolve this long-standing issue by comparing growth across 382 grass species, accounting for ecological diversity and evolutionary history. C4 photosynthesis causes a 19–88% daily growth enhancement. Unexpectedly, during the critical seedling establishment stage, this enhancement is driven largely by a high ratio of leaf area to mass, rather than fast growth per unit leaf area. C4 leaves have less dense tissues, allowing more leaves to be produced for the same carbon cost. Consequently, C4 plants invest more in roots than C3 species. Our data demonstrate a general suite of functional trait divergences between C3 and C4 species, which simultaneously drive faster growth and greater investment in water and nutrient acquisition, with important ecological and agronomic implications.

Unpacking a century-old mystery: Winter buds and the latitudinal gradient in leaf form

Th is year marks the 100th anniversary of a seminal paper on plant form. In 1916, in the pages of the American Journal of Botany , Irving W. Bailey and Edmund W. Sinnott documented a remarkable observation: in wet tropical forests, the percentage of woody plant species with toothed or lobed leaves is close to zero, but it increases toward 100% moving north into cold-temperate regions ( Bailey and Sinnott, 1916 ). Th is latitudinal gradient has repeatedly been confi rmed (e.g., Little et al., 2010 ; Peppe et al., 2011 ) and is so robust that paleobotanists use the percentage of leaves with entire margins in paleofl oras as a proxy for mean annual temperature ( Wolfe, 1971 ). In the meantime, it has come to light that other aspects of leaf form may be correlated with climate, as temperate leaves also tend to be rounder, while tropical leaves are more elliptical ( Schmerler et al., 2012 ). But, why does leaf form vary so predictably? Th e short answer is that we still don’t know. Here we explore a new angle, focusing attention on changes in the rhythm of growth and leaf development that accompanied evolutionary shift s into strongly seasonal climates. First we must ask: Is this pattern due to many evolutionary shift s in leaf form as lineages moved from tropical into temperate forests (and vice versa)? Or, is it largely driven by just a few successful lineages in northern latitudes that happened to have teeth and lobes (e.g., maples, birches, oaks)? We still don’t have a clear idea of the number of tropical–temperate transitions in plants ( Donoghue and Edwards, 2014 ). Yet, the wide taxonomic distribution of lineages with both tropical and temperate ranges supports the assumption that there were multiple biome shift s accompanied by repeated evolutionary changes in leaf form (e.g., temperate Acer within Sapindaceae, Tilia within Malvaceae, Hamamelis within Hamamelidaceae, Fagus within Fagaceae). And, judging by our experience with Viburnum ( Schmerler et al., 2012 ; Spriggs et al., 2015 ), additional transitions are likely hidden within many of the clades that span these biomes ( Edwards and Donoghue, 2013 ; Donoghue and Edwards, 2014 ). Until now, adaptive explanations for the leaf-form gradient have focused on leaf function either later in development or in mature leaves. For instance, we know that leaf size and shape infl uence boundary layer dynamics; smaller and more dissected leaves facilitate gas exchange and transpirational cooling ( Gates, 1968 ). But, why then should leaves not instead be more dissected in tropical forests, where the air is oft en hot and still? A second explanation points to leaf teeth as sites of early-season gas exchange, arguing that rapid maturation of toothy margins provides a boost in photosynthate production when light and water are more available, before the formation of a full forest canopy ( Baker-Brosh and Peet, 1997 ; Royer and Wilf, 2006 ). Data vary in support of this hypothesis, and there has been no attempt to quantify the total contribution of photosynthesis in teeth of emerging leaves to a plant’s carbon budget, which we imagine is exceedingly small. Another hypothesis is that teeth serve as hydathodes that expel water that might otherwise fl ood developing leaf tissues early in the spring. Th is may be relevant for temperate species that use positive root pressure to remove freeze–thaw embolisms ( Lechowicz, 1984 ; Feild et al., 2005 ), but many species with leaf teeth do not generate positive xylem pressure. A fourth explanation is biomechanical: temperate leaves, it is said, are thinner and rely more heavily on structural support from their vein systems. In such leaves, the optimal tissue confi guration surrounding each major vein is wedge shaped, which in a pinnately veined leaf would result in a toothy margin ( Givnish, 1979 ). It has even been argued that teeth protect leaves against herbivores ( Brown and Lawton, 1991 ). Each of these hypotheses has some merit and might apply in particular cases. But, in our estimation, none of them is terribly well supported, and little attention has been paid to the alternative possibility that selection on other aspects of the organism might indirectly generate certain leaf characteristics, possibly affecting both teeth and shape simultaneously. Here we consider the idea that the repeated emergence of 1 Manuscript received 21 March 2016; revision accepted 22 April 2016. 2 Department of Ecology and Evolutionary Biology, Brown University, 80 Waterman St., Box G-W, Providence, Rhode Island 02912; and 3 Department of Ecology and Evolutionary Biology, Yale University, PO Box 208106, New Haven, Connecticut 06520-8106 4 Author for correspondence (e-mail: erika_edwards@brown.edu), phone: 401.863.2081 5 Present address: Biomedical Imaging Unit, University of Southampton, Southampton, SO16 6YD, UK doi:10.3732/ajb.1600129 O N T H E N AT U R E O F T H I N G S : E S S AY S New Ideas and Directions in Botany

Topological Data Analysis as a Morphometric Method: Using Persistent Homology to Demarcate a Leaf Morphospace

Current morphometric methods that comprehensively measure shape cannot compare the disparate leaf shapes found in seed plants and are sensitive to processing artifacts. We explore the use of persistent homology, a topological method applied as a filtration across simplicial complexes (or more simply, a method to measure topological features of spaces across different spatial resolutions), to overcome these limitations. The described method isolates subsets of shape features and measures the spatial relationship of neighboring pixel densities in a shape. We apply the method to the analysis of 182,707 leaves, both published and unpublished, representing 141 plant families collected from 75 sites throughout the world. By measuring leaves from throughout the seed plants using persistent homology, a defined morphospace comparing all leaves is demarcated. Clear differences in shape between major phylogenetic groups are detected and estimates of leaf shape diversity within plant families are made. The approach predicts plant family above chance. The application of a persistent homology method, using topological features, to measure leaf shape allows for a unified morphometric framework to measure plant form, including shapes, textures, patterns, and branching architectures.

Persistent homology demarcates a leaf morphospace

Current morphometric methods that comprehensively measure shape cannot compare the disparate leaf shapes found in seed plants and are sensitive to processing artifacts. We explore the use of persistent homology, a topological method applied across the scales of a function, to overcome these limitations. The described method isolates subsets of shape features and measures the spatial relationship of neighboring pixel densities in a shape. We apply the method to the analysis of 182,707 leaves, both published and unpublished, representing 141 plant families collected from 75 sites throughout the world. By measuring leaves from throughout the seed plants using persistent homology, a defined morphospace comparing all leaves is demarcated. Clear differences in shape between major phylogenetic groups are detected and estimates of leaf shape diversity within plant families are made. This approach does not only predict plant family, but also the collection site, confirming phylogenetically invariant morphological features that characterize leaves from specific locations. The application of a persistent homology method to measure leaf shape allows for a unified morphometric framework to measure plant form, including shape and branching architectures.

Correlation, causation, and the evolution of leaf teeth: A reply to Givnish and Kriebel

We are grateful to Givnish and Kriebel (2017) for providing a thorough review of the arguments that have been advanced to explain the latitudinal gradient in leaf form. Th eir paper was motivated by our recent “On Th e Nature of Th ings” essay presenting the “bud packing hypothesis” (BP) as an alternative explanation for why leaves in the temperate zone are so oft en toothed or lobed ( Edwards et al., 2016 ). Although Givnish and Kriebel (2017) added the BP hypothesis to their list of possible explanations and included it as one of many causal arrows in their synthetic model (see their fi g. 6 ), they were generally unconvinced by our arguments and instead strongly favored the “support and supply hypothesis” (SS) advanced by Givnish almost 40 years ago ( Givnish, 1979 ). Here we present new analyses that question the assumptions of the SS model and elaborate further on the possible connections between teeth and bud packing. Most importantly, we reiterate our plea for studies of bud development. But fi rst, let us note where we seem to agree. We all view leaf boundary layer dynamics, early season photosynthesis, and guttation through hydathodes as unlikely explanations for the latitudinal gradient in leaf margins. Although Givnish and Kriebel portray us as dismissing the idea that spinose teeth might sometimes defend against herbivores, we do not disagree at all. In fact, as we stressed ( Edwards et al., 2016 , p. 975), “Each of these hypotheses has some merit and might apply in particular cases.” As they rightly argued, large herbivores are especially likely to select for spinose teeth in short-statured plants in arid and semiarid environments. Our discussion focused instead on woody plants of mesic forests, where spinose leaves are rare (e.g., Ilex ). Finally, we strongly agree with Givnish and Kriebel that venation architecture deserves far more attention in relation to this problem, and we return to this topic below. However, we disagree with their other main points. Most of the Givnish and Kriebel commentary focused on demonstrating a correlation between leaf thickness and toothy margins, which they present as evidence in favor of the SS model. But, we have never doubted a relationship between leaf thickness and leaf teeth. In fact, Givnish and Kriebel used our data on Viburnum ( Schmerler et al., 2012 ; Chatelet et al., 2013 ) to show this relationship, which we were already well aware of. Because we disagree with their scoring of several species, we re-examined this relationship with a revised data set and recovered an even stronger relationship between thickness and margin type than they originally reported ( Fig. 1 ). However, it is very diffi cult to disentangle leaf thickness and leaf margins from leaf habit and longevity. In Viburnum —and we presume in many other clades—teeth and thickness are also strongly correlated with evolutionary shift s in leaf habit (evergreen vs. deciduous; Fig. 1 ), and, more importantly for our arguments, with changes in the rhythm of leaf production, leaf lifespan, and the extent of leaf development inside of resting buds. Our phylogenetic regression analyses recover a strong association between leaf thickness and margin type ( β = −3.361 ± 0.891 SE, p = 0.0002), but an equally strong relationship between leaf thickness and leaf habit ( β = 3.824 ± 0.944 SE, p = 0.0001), and, as we have shown previously ( Schmerler et al., 2012 ), another very strong relationship between leaf margin type and leaf habit ( p = 2e −12 ). Givnish and Kriebel performed two other analyses along these lines. First, they reduced a ~3500 species data set on leaf form, thickness, and habitat compiled by Royer et al. (2012) to ~600 species to repeat the original analyses in a phylogenetic context. As expected, they found a tight relationship between leaf thickness and margin type. Th ough we do not question this correlation, in general 1 Manuscript received 22 February 2017; revision accepted 30 March 2017. 2 Department of Ecology and Evolutionary Biology, Brown University, 80 Waterman Street, Box G-W, Providence, Rhode Island 02912 USA; 3 Present address: Biomedical Imaging Unit, University of Southampton, Southampton, SO16 6YD, UK; 4 Department of Ecology and Evolutionary Biology, Yale University, P.O. Box 208106, New Haven, Connecticut 06520-8106 USA 5 Author for correspondence (e-mail: erika_edwards@brown.edu), phone: 401.863.2081 doi:10.3732/ajb.1700075 C O M M E N TA R Y