Caroline A.E. Strömberg

The Origins of C4 Grasslands: Integrating Evolutionary and Ecosystem Science

Grassland Emergence The evolution of the C4 photosynthetic pathway from the ancestral C3 pathway in grasses led to the establishment of grasslands in warm climates during the Late Miocene (8 to 3 million years ago). This was a major event in plant evolutionary history, and their high rates of foliage production sustained high levels of herbivore consumption. The past decade has seen significant advances in understanding C4 grassland ecosystem ecology, and now a wealth of data on the geological history of these ecosystems has accumulated and the phylogeny of grasses is much better known. Edwards et al. (p. 587) review this multidisciplinary research area and attempt to synthesize emerging knowledge about the evolution of grass species within the context of plant and ecosystem ecology. The evolution of grasses using C4 photosynthesis and their sudden rise to ecological dominance 3 to 8 million years ago is among the most dramatic examples of biome assembly in the geological record. A growing body of work suggests that the patterns and drivers of C4 grassland expansion were considerably more complex than originally assumed. Previous research has benefited substantially from dialog between geologists and ecologists, but current research must now integrate fully with phylogenetics. A synthesis of grass evolutionary biology with grassland ecosystem science will further our knowledge of the evolution of traits that promote dominance in grassland systems and will provide a new context in which to evaluate the relative importance of C4 photosynthesis in transforming ecosystems across large regions of Earth.

Dinosaur Coprolites and the Early Evolution of Grasses and Grazers

Silicified plant tissues (phytoliths) preserved in Late Cretaceous coprolites from India show that at least five taxa from extant grass (Poaceae) subclades were present on the Indian subcontinent during the latest Cretaceous. This taxonomic diversity suggests that crown-group Poaceae had diversified and spread in Gondwana before India became geographically isolated. Other phytoliths extracted from the coprolites (from dicotyledons, conifers, and palms) suggest that the suspected dung producers (titanosaur sauropods) fed indiscriminately on a wide range of plants. These data also make plausible the hypothesis that gondwanatherian mammals with hypsodont cheek teeth were grazers.

Late Cretaceous origin of the rice tribe provides evidence for early diversification in Poaceae

Rice and its relatives are a focal point in agricultural and evolutionary science, but a paucity of fossils has obscured their deep-time history. Previously described cuticles with silica bodies (phytoliths) from the Late Cretaceous period (67-65 Ma) of India indicate that, by the latest Cretaceous, the grass family (Poaceae) consisted of members of the modern subclades PACMAD (Panicoideae-Aristidoideae-Chloridoideae-Micrairoideae-Arundinoideae-Danthonioideae) and BEP (Bambusoideae-Ehrhartoideae-Pooideae), including a taxon with proposed affinities to Ehrhartoideae. Here we describe additional fossils and show that, based on phylogenetic analyses that combine molecular genetic data and epidermal and phytolith features across Poaceae, these can be assigned to the rice tribe, Oryzeae, of grass subfamily Ehrhartoideae. The new Oryzeae fossils suggest substantial diversification within Ehrhartoideae by the Late Cretaceous, pushing back the time of origin of Poaceae as a whole. These results, therefore, necessitate a re-evaluation of current models for grass evolution and palaeobiogeography.

Evolution of hypsodonty in equids: testing a hypothesis of adaptation

Abstract The independent acquisition of high-crowned cheek teeth (hypsodonty) in several ungulate lineages (e.g., camels, equids, rhinoceroses) in the early to middle Miocene of North America has classically been used as an indication that savanna vegetation spread during this time. Implicit in this interpretation is the untested assumption that hypsodonty was an evolutionary response to feeding in open habitats, either due to a change in food source (from browse to graze) or to increased incorporation of airborne grit in the diet. I examined the adaptive explanation for hypsodonty in equids using criteria pertaining to process and pattern of adaptations set up in the comparative-methods literature. Specifically, I tested whether hypsodonty appeared coincident with or just after the spread of open, grass-dominated habitats in the Great Plains of North America. Phytolith (plant opal) analysis of 99 phytolith assemblages extracted from sediment samples from Montana/Idaho, Nebraska/Wyoming, and Colorado were used to establish the first continuous record of middle Eocene–late Miocene vegetation change in the northern to Central Great Plains. This record was compared with the fossil record of equids from the same area in a phylogenetic framework. The study showed that habitats dominated by C3 grasses were established in the Central Great Plains by early late Arikareean (≥21.9 Ma), at least 4 Myr prior to the emergence of hypsodont equids (Equinae). Nevertheless, the adaptive hypothesis for hypsodonty in equids could not be rejected, because the earliest savanna-woodlands roughly co-occurred with members of the grade constituting the closest outgroups to Equinae (“Parahippus”) showing mesodont dentition. Explanations for the slow evolution of full hypsodonty may include weak and changing selection pressures and/or phylogenetic inertia. These results suggest that care should be taken when using functional morphology alone to reconstruct habitat change.

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.

Decoupling the spread of grasslands from the evolution of grazer-type herbivores in South America

The evolution of high-crowned cheek teeth (hypsodonty) in herbivorous mammals during the late Cenozoic is classically regarded as an adaptive response to the near-global spread of grass-dominated habitats. Precocious hypsodonty in middle Eocene (∼38 million years (Myr) ago) faunas from Patagonia, South America, is therefore thought to signal Earths first grasslands, 20 million years earlier than elsewhere. Here, using a high-resolution, 43-18 million-year record of plant silica (phytoliths) from Patagonia, we show that although open-habitat grasses existed in southern South America since the middle Eocene (∼40 Myr ago), they were minor floral components in overall forested habitats between 40 and 18 Myr ago. Thus, distinctly different, continent-specific environmental conditions (arid grasslands versus ash-laden forests) triggered convergent cheek-tooth evolution in Cenozoic herbivores. Hypsodonty evolution is an important example where the present is an insufficient key to the past, and contextual information from fossils is vital for understanding processes of adaptation.

The Neogene transition from C3 to C4 grasslands in North America: assemblage analysis of fossil phytoliths

Abstract The rapid ecological expansion of grasses with C4 photosynthesis at the end of the Neogene (8-2 Ma) is well documented in the fossil record of stable carbon isotopes. As one of the most profound vegetation changes to occur in recent geologic time, it paved the way for modern tropical grassland ecosystems. Changes in CO2 levels, seasonality, aridity, herbivory, and fire regime have all been suggested as potential triggers for this broadly synchronous change, long after the evolutionary origin of the C4 pathway in grasses. To date, these hypotheses have suffered from a lack of direct evidence for floral composition and structure during this important transition. This study aimed to remedy the problem by providing the first direct, relatively continuous record of vegetation change for the Great Plains of North America for the critical interval (ca. 12-2 Ma) using plant silica (phytolith) assemblages. Phytoliths were extracted from late Miocene–Pliocene paleosols in Nebraska and Kansas. Quantitative phytolith analysis of the 14 best-preserved assemblages indicates that habitats varied substantially in openness during the middle to late Miocene but became more uniformly open, corresponding to relatively open grassland or savanna, during the late Miocene and early Pliocene. Phytolith data also point to a marked increase of grass short cells typical of chloridoid and other potentially C4 grasses of the PACMAD clade between 8 and 5 Ma; these data suggest that the proportion of these grasses reached up to ∼50–60% of grasses, resulting in mixed C3-C4 and highly heterogeneous grassland communities by 5.5 Ma. This scenario is consistent with interpretations of isotopic records from paleosol carbonates and ungulate tooth enamel. The rise in abundance of chloridoids, which were present in the central Great Plains since the early Miocene, demonstrates that the “globally” observed lag between C4 grass evolution/taxonomic diversification and ecological expansion occurred at the regional scale. These patterns of vegetation alteration imply that environmental change during the late Miocene–Pliocene played a major role in the C3-C4 shift in the Great Plains. Specifically, the importance of chloridoids as well as a decline in the relative abundance of forest indicator taxa, including palms, point to climatic drying as a key trigger for C4 dominance.

Linked canopy, climate, and faunal change in the Cenozoic of Patagonia

Fluctuations revealed in fossil forests The reconstruction of past vegetation unlocks the door to understanding ecological changes associated with climatic change. But it is also difficult. Dunn et al. developed a method for assessing changes in vegetation openness based on epidermal cell morphology from conserved plant tissue. Applying this method to fossil assemblages from Patagonia, they show how vegetation structure changed over the Cenozoic era (49 to 11 million years ago). These changes map onto the known climate changes over this period and can also be used to track how the evolution of herbivorous mammals responded to vegetation structure. Science, this issue p. 258 A reconstruction of leaf area index from plant microfossils reveals a 38-million-year record of habitat change. Vegetation structure is a key determinant of ecosystems and ecosystem function, but paleoecological techniques to quantify it are lacking. We present a method for reconstructing leaf area index (LAI) based on light-dependent morphology of leaf epidermal cells and phytoliths derived from them. Using this proxy, we reconstruct LAI for the Cenozoic (49 million to 11 million years ago) of middle-latitude Patagonia. Our record shows that dense forests opened up by the late Eocene; open forests and shrubland habitats then fluctuated, with a brief middle-Miocene regreening period. Furthermore, endemic herbivorous mammals show accelerated tooth crown height evolution during open, yet relatively grass-free, shrubland habitat intervals. Our Patagonian LAI record provides a high-resolution, sensitive tool with which to dissect terrestrial ecosystem response to changing Southern Ocean conditions during the Cenozoic.

The Neogene transition from C3 to C4 grasslands in North America: stable carbon isotope ratios of fossil phytoliths

Abstract C4 grasses form the foundation of warm-climate grasslands and savannas and provide important food crops such as corn, but their Neogene rise to dominance is still not fully understood. Carbon isotope ratios of tooth enamel, soil carbonate, carbonate cements, and plant lipids indicate a late Miocene–Pliocene (8-2 Ma) transition from C3 vegetation to dominantly C4 grasses at many sites around the world. However, these isotopic proxies cannot identify whether the C4 grasses replaced woody vegetation (trees and shrubs) or C3 grasses. Here we propose a method for reconstructing the carbon isotope ratio of Neogene grasses using the carbon isotope ratio of organic matter trapped in plant silica bodies (phytoliths). Although a wide range of plants produce phytoliths, we hypothesize that in grass-dominated ecosystems the majority of phytoliths will be derived from grasses, and will yield a grass carbon isotope signature. Phytolith extracts can be contaminated by non-phytolith silica (e.g., volcanic ash). To test the feasibility of the method given these potential problems, we examined sample purity (phytolith versus non-phytolith silica), abundance of grass versus non-grass phytoliths, and carbon isotope ratios of phytolith extracts from late Miocene–Pliocene paleosols of the central Great Plains. Isotope results from the purest samples are compared with phytolith assemblage analysis of these same extracts. The dual record spans the interval of focus (ca. 12-2 Ma), allowing us, for the first time, to investigate how isotopic shifts correlate with floral change. We found that many samples contained high abundances of non-biogenic silica; therefore, only a small subset of “pure” samples (>50% of phytoliths by volume) with good preservation were considered to provide reliable carbon isotope ratios. All phytolith assemblages contained high proportions (on average 85%) of grass phytoliths, supporting our hypothesis for grass-dominated communities. Therefore, the carbon isotope ratio of pure, well-preserved samples that are dominated by grass biosilica is considered a reliable measure of the proportion of C3 and C4 grasses in the Neogene. The carbon isotope ratios of the pure fossil phytolith samples indicate a transition from predominantly C3 grasses to mixed C3-C4 grasses by 5.5 Ma and then a shift to more than 80% C4 grasses by 3-2 Ma. With the exception of the Pliocene sample, these isotopic data are broadly concordant with phytolith assemblages that show a general increase in C4 grasses in the late Miocene. However, phytolith assemblage analysis indicates lower relative abundance of C4 grasses in overall vegetation than do the carbon isotopes from the same phytolith assemblages. The discrepancy may relate to either (1) incomplete identification of (C4) PACMAD phytoliths, (2) higher production of non-diagnostic phytoliths in C4 grasses compared to C3 grasses, or (3) biases in the isotope record toward grasses rather than overall vegetation. The impact of potential incomplete characterization of (C4) PACMAD phytoliths on assemblage estimates of proportion of C4, though important, cannot reconcile discrepancies between the methods. We explore hypothesis (2) by analyzing a previously published data set of silica content in grasses and a small data set of modern grass leaf assemblage composition using analysis of variance, independent contrasts, and sign tests. These tests suggest that C4 grasses do not have more silica than C3 grasses; there is also no difference with regard to production of non-diagnostic phytoliths. Thus, it is most likely that the discrepancy between phytolith assemblages and isotope ratios is a consequence of hypothesis (3), that the isotope signature is influenced by the contribution of non-diagnostic grass phytoliths, whereas the assemblage composition is not. Assemblage-based estimates of % C4 within grasses, rather than overall vegetation, are in considerably better agreement with the isotope-based estimates. These results support the idea that, in grass-dominated assemblages, the phytolith carbon isotope method predominantly records shifts in dominant photosynthetic pathways among grasses, whereas phytolith assemblage analysis detects changes in overall vegetation. Carbon isotope ratios of fossil phytoliths in conjunction with phytolith assemblage analysis suggest that the late Neogene expansion of C4 grasses was largely at the expense of C3 grasses rather than C3 shrubs/trees. Stable isotopic analysis of phytoliths can therefore provide unique information about grass community changes during the Neogene, as well as help test how grass phytolith morphology relates to photosynthetic pathway.

Functions of phytoliths in vascular plants: an evolutionary perspective

Summary Solid biosilica (phytoliths) deposited in plant tissues is thought to function as structural support, as a cost-effective alternative to lignin, and in herbivore defence, by limiting nutrient access/extraction and abrading herbivore mouthparts. It has been assumed that active phytolith accumulation evolved for these purposes, but these hypotheses remain untested. For example, an influential idea holds that grasses became so silica-rich through antagonistic co-evolution with mammalian grazers during the Cenozoic. We examine whether phytoliths fulfil criteria established for adaptations, focusing on three aspects. First, we evaluate the recent debate concerning whether plant silica wears herbivore mouthparts/teeth. Secondly, we test whether the evolutionary pattern of phytolith accumulation is consistent with adaptive hypotheses by mapping silica content onto time-calibrated land plant and grass phylogenies. Thirdly, we compare with palaeontological evidence for the timing of the ‘demand’ for the hypothesized function (structural support, herbivore defence). Our survey demonstrates that phytoliths meet several criteria for adaptations, but key aspects require further study. For example, phytoliths wear teeth but are likely less important than dietary grit, suggesting that silica deterrence is ineffective against large mammalian grazers. Mapping analysis indicates that active silica accumulation evolved numerous times, rather than being ancestral in land plants. However, a clear temporal link between these events and hypothesized functional ‘demands’ is still missing. For example, we find no convincing evidence for Cenozoic grass-grazer co-evolution. Synthesis. Phytoliths help support and defend plants today, but the adaptive origin of this trait requires further testing. Such tests should integrate the phylogenetic distributions of phytoliths with ecology and biomechanics and use fossil evidence to evaluate the correlation between functional ‘demand’ and plant evolution.