Improving our understanding of environmental controls on the distribution of C3 and C4 grasses

A number of studies have demonstrated the ecological sorting of C₃ and C₄ grasses along temperature and moisture gradients. However, previous studies of C₃ and C₄ grass biogeography have often inadvertently compared species in different and relatively unrelated lineages, which are associated with different environmental settings and distinct adaptive traits. Such confounded comparisons of C₃ and C₄ grasses may bias our understanding of ecological sorting imposed strictly by photosynthetic pathway. Here, we used MaxEnt species distribution modeling in combination with satellite data to understand the functional diversity of C₃ and C₄ grasses by comparing both large clades and closely related sister taxa. Similar to previous work, we found that C₄ grasses showed a preference for regions with higher temperatures and lower precipitation compared with grasses using the C₃ pathway. However, air temperature differences were smaller (2 °C vs. 4 °C) and precipitation and % tree cover differences were larger (1783 mm vs. 755 mm, 21.3% vs. 7.7%, respectively) when comparing C₃ and C₄ grasses within the same clade vs. comparing all C₄ and all C₃ grasses (i.e., ignoring phylogenetic structure). These results were due to important differences in the environmental preferences of C₃ BEP and PACMAD clades (the two main grass clades). Winter precipitation was found to be more important for understanding the distribution and environmental niche of C₃ PACMADs in comparison with both C₃ BEPs and C₄ taxa, for which temperature was much more important. Results comparing closely related C₃–C₄ sister taxa supported the patterns derived from our modeling of the larger clade groupings. Our findings, which are novel in comparing the distribution and niches of clades, demonstrate that the evolutionary history of taxa is important for understanding the functional diversity of C₃ and C₄ grasses, and should have implications for how grasslands will respond to global change.     

Implications of quantum yield differences on the distributions of C3 and C4 grasses

Summary The implications of a reduced quantum yield (initial slope of the photosynthetic light response curve) in C₄ plants and temperature dependence of quantum yield in C₃ plants on total canopy primary production were investigated using computer simulations. Since reduced quantum yield represents the only known disadvantage of the C₄ photosynthetic pathway, simulations were conducted with grass canopies (high LAI and hence photosynthesis in most leaves will be light-limited) to see if quantum yield is a significant factor in limiting the primary production and thus distributions of C₄ grasses. Simulations were performed for three biogeographical or environmental conditions: the Great Plains region of North America, the Sonoran Desert of North America, and shade habitats. For all three cases, the simulations predicted either spatial or temporal gradients in the abundances of C₄ grasses identical to the abundance patterns of C₄ grasses observed in the field. It is thus concluded that while the C₄ photosynthetic mechanism may be highly advantageous in specific environments, it may be disadvantageous in others.C.I.W.-D.P.B. Publication No. 598     

Phylogenetic niche conservatism in C4 grasses

Photosynthetic pathway is used widely to discriminate plant functional types in studies of global change. However, independent evolutionary lineages of C₄ grasses with different variants of C₄ photosynthesis show different biogeographical relationships with mean annual precipitation, suggesting phylogenetic niche conservatism (PNC). To investigate how phylogeny and photosynthetic type differentiate C₄ grasses, we compiled a dataset of morphological and habitat information of 185 genera belonging to two monophyletic subfamilies, Chloridoideae and Panicoideae, which together account for 90 % of the world’s C₄ grass species. We evaluated evolutionary variance and covariance of morphological and habitat traits. Strong phylogenetic signals were found in both morphological and habitat traits, arising mainly from the divergence of the two subfamilies. Genera in Chloridoideae had significantly smaller culm heights, leaf widths, 1,000-seed weights and stomata; they also appeared more in dry, open or saline habitats than those of Panicoideae. Controlling for phylogenetic structure showed significant covariation among morphological traits, supporting the hypothesis of phylogenetically independent scaling effects. However, associations between morphological and habitat traits showed limited phylogenetic covariance. Subfamily was a better explanation than photosynthetic type for the variance in most morphological traits. Morphology, habitat water availability, shading, and productivity are therefore all involved in the PNC of C₄ grass lineages. This study emphasized the importance of phylogenetic history in the ecology and biogeography of C₄ grasses, suggesting that divergent lineages need to be considered to fully understand the impacts of global change on plant distributions.     

Drought limitation of photosynthesis differs between C3 and C4 grass species in a comparative experiment

Phylogenetic analyses show that C₄ grasses typically occupy drier habitats than their C₃ relatives, but recent experiments comparing the physiology of closely related C₃ and C₄ species have shown that advantages of C₄ photosynthesis can be lost under drought. We tested the generality of these paradoxical findings in grass species representing the known evolutionary diversity of C₄ NADP‐me and C₃ photosynthetic types. Our experiment investigated the effects of drought on leaf photosynthesis, water potential, nitrogen, chlorophyll content and mortality. C₄ grasses in control treatments were characterized by higher CO₂ assimilation rates and water potential, but lower stomatal conductance and nitrogen content. Under drought, stomatal conductance declined more dramatically in C₃ than C₄ species, and photosynthetic water‐use and nitrogen‐use efficiency advantages held by C₄ species under control conditions were each diminished by 40%. Leaf mortality was slightly higher in C₄ than C₃ grasses, but leaf condition under drought otherwise showed no dependence on photosynthetic‐type. This phylogenetically controlled experiment suggested that a drought‐induced reduction in the photosynthetic performance advantages of C₄ NADP‐me relative to C₃ grasses is a general phenomenon.     

Phylogenetic signals in the realized climate niches of Chinese grasses (Poaceae)

Explaining relationships between species richness and biogeographical patterns over a broad geographic scale is a central issue of biogeography and macroecology. We document the realized climate niches for grasses in China’s nature reserves and discuss its formation mechanism using grass richness data combined with climatic, physiological, and phylogenetic data. Our results suggest that climate niche structure of grasses is phylogenetically conservative for BEP (Bambusoideae, Ehrhartoideae, and Pooideae) and PACMAD (Panicoideae, Arundinoideae, Chloridoideae, Micrairoideae, Aristidoideae, and Danthonioideae) clades along temperature gradients and for Chloridoideae and Panicoideae along precipitation gradients. At the national scale, the divergence patterns of climate niches between two major clades are more distinguishable than between C₃ and C₄ grasses. High rates of climate niche evolution are found in C₄ clades in the subtropical forest region. There appears to be a strong association between elevation gradients and grass diversity: the specific environmental conditions (e.g. energy) and the rapid shifts of climate conditions drive high grass diversification. Evolutionary conservatism of climate niches may be influenced by the specific adaptive ability to changing environmental conditions within NAD-ME/NADP-ME clades. Our results indicate that adaptations to major climate changes may be accomplished by C₄ grass nodes of high climate niche evolutionary rates in China’s nature reserves.     

Trait differences between grass species along a climatic gradient in South and North America

Question: Are trait differences between grasses along a gradient related to climatic variables and/or photosynthetic pathway? Location: Temperate grassland areas of South and North America. Methods: In a common garden experiment, we cultivated C₃ and C₄ grasses from grasslands under different climatic conditions, and we measured a set of 12 plant traits related to size and resource capture and utilization. We described (1) interspecific plant trait differences along a climatic gradient defined by the precipitation and temperature at the location where each species is dominant and (2) the association between those plant trait differences and the photosynthetic pathway of the species. Results: Trait differences between grasses were related to the precipitation at the area where each species is dominant, and to the photosynthetic pathway of the species. Leaf length, leaf width, plant height, leaf area per tiller, specific leaf area, leaf δ¹³C ratio, and nitrogen resorption efficiency increased while leaf dry matter content and nitrogen concentration in senesced leaves decreased as precipitation increased. A proportion of these changes along the gradient was related to the photosynthetic pathway because dominant grass species in cold areas with low precipitation are mainly C₃ and those from warm and wet areas are C₄. Conclusions: A previous worldwide analysis showed that traits of graminoid species measured in situ changed slightly along climatic gradients (< 10% variance explained). In contrast, under a common environment we observed that (1) grass traits changed strongly along a climatic gradient (30‐85% variance explained) and, (2) a proportion of those changes were related to the association between photosynthetic pathway of the species and precipitation.     

Preference for C4 shade grasses increases hatchling performance in the butterfly,Bicyclus safitza

The Miocene radiation of C₄ grasses under high‐temperature and low ambient CO₂ levels occurred alongside the transformation of a largely forested landscape into savanna. This inevitably changed the host plant regime of herbivores, and the simultaneous diversification of many consumer lineages, including Bicyclus butterflies in Africa, suggests that the radiations of grasses and grazers may be evolutionary linked. We examined mechanisms for this plant–herbivore interaction with the grass‐feeding Bicyclus safitza in South Africa. In a controlled environment, we tested oviposition preference and hatchling performance on local grasses with C₃ or C₄ photosynthetic pathways that grow either in open or shaded habitats. We predicted preference for C₃ plants due to a hypothesized lower processing cost and higher palatability to herbivores. In contrast, we found that females preferred C₄ shade grasses rather than either C₄ grasses from open habitats or C₃ grasses. The oviposition preference broadly followed hatchling performance, although hatchling survival was equally good on C₄ or C₃ shade grasses. This finding was explained by leaf toughness; shade grasses were softer than grasses from open habitats. Field monitoring revealed a preference of adults for shaded habitats, and stable isotope analysis of field‐sampled individuals confirmed their preference for C₄ grasses as host plants. Our findings suggest that plant–herbivore interactions can influence the direction of selection in a grass‐feeding butterfly. Based on this work, we postulate future research to test whether these interactions more generally contribute to radiations in herbivorous insects via expansions into new, unexploited ecological niches.     

The photosynthetic response of C3 and C4 bioenergy grass species to fluctuating light

Bioenergy grass species are a renewable energy source, but their productivity has not been fully realized. Improving photosynthetic efficiency has been proposed as a mechanism to increase the productivity of bioenergy grass species. Fluctuating light, experienced by all field grown crops, is known to reduce photosynthetic efficiency. This experiment aimed to evaluate the photosynthetic performance of both C₃ and C₄ bioenergy grass species under steady state and fluctuating light conditions by examining leaf gas exchange. The fluctuating light regime used here decreased carbon assimilation across all species when compared to expected steady state values. Overall, C₄ species assimilated more carbon than C₃ species during the fluctuating light regime, with both photosynthetic types assimilating about 16% less carbon than expected based on steady state measurements. Little diversity was observed in response to fluctuating light among C₃ species, and photorespiration partially contributed to the rapid decreases in net photosynthetic rates during high to low light transitions. In C₄ species, differences among the four NADP-ME species were apparent. Diversity observed among C₄ species in this experiment provides evidence that photosynthetic efficiency in response to fluctuating light may be targeted to increase C₄ bioenergy grass productivity.     

The small scale spatial pattern of C3 and C4 grasses depends on shrub distribution

At micro‐site scale, the spatial pattern of a plant species depends on several factors including interactions with neighbours. It has been seen that unfavourable effects generate a negative association between plants, while beneficial effects generate a positive association. In grasslands, the presence of shrubby species promotes a particular microenvironment beneath their canopy that could affect differently the spatial distribution of plants with different tolerance to abiotic conditions. We measured photosynthetic active radiation, air temperature and wind speed under shrub canopies and in adjacent open sites and analysed the spatial distribution of four grass species (two C₃ and two C₄) in relation to shrub canopy in a grazed sub‐humid natural grassland in southern Uruguay. Radiation, air temperature and wind speed were lower under shrubs than in adjacent open sites. The spatial distribution of grasses relative to the shrub canopy varied depending on the photosynthetic metabolism of grasses. C₄ grasses showed a negative association or no correlation with the shrubs, whereas C₃ grasses showed a positive association. Our results highlight the importance of the photosynthetic metabolism of the grasses in the final outcome of interactions between grasses and shrubs. Micro‐environmental conditions generated underneath shrubs create a more suitable site for the establishment of C₃ than for C₄ grasses. These results show that facilitation could be more important than previously thought in sub‐humid grasslands.     

Ecological responses of dominant grasses along two climatic gradients in the Great Plains of the United States

Abstract. Few empirical data exist to examine the influence of regional scale environmental gradients on productivity patterns of plant species. In this paper we analyzed the productivity of several dominant grass species along two climatic gradients, mean annual precipitation (MAP) and mean annual temperature (MAT), in the Great Plains of the United States. We used climatic data from 296 weather stations, species production data from Natural Resource Conservation Service rangeland surveys and a geographic information system to spatially integrate the data. Both MAP and MAT were significantly related to annual above-ground net primary production (ANPP). MAP explained 54% to 89% of the variation in ANPP of two C₄ short-grasses, Bouteloua gracilis and Buchloë dactyloides, and two C₄ tall-grasses, Andropogon gerardii and Schizachyrium scoparium (= Andropogon scoparius). MAT explained 19% to 41% of the variation in ANPP of two C₄ grasses, B. gracilis and B. dactyloides, and 41% to 66% of the variation in ANPP of two C₃ grasses, Agropyron smithii and Stipa comata. ANPP patterns for species along both gradients were described by either linear, negative exponential, logistic, normal or skewed curves. Patterns of absolute ANPP (g/m²) for species differed from those of relative ANPP (%) along the MAP gradient. Responses were similar for species with common functional characteristics (e.g. short-grasses, tall-grasses, C₃, C₄). Our empirical results support asymmetric responses of species to environmental gradients. Results demonstrate the importance of species attributes, type of environmental gradient and measure of species importance (relative or absolute productivity) in evaluating ecological response patterns.     

Traits explain sorting of C4 grasses along a global precipitation gradient

1. Species distributions are closely associated with moisture availability, but the underlying mechanisms remain unresolved. Drought relations are especially important for plants such as C₄ grasses that dominate seasonally dry ecosystems. Here, we test the hypothesis that C₄ grass species sampled across global precipitation gradients show variation in survival under drought that can be explained by their traits.
2. Our experiment subjected 18 C₄ grass species to a lethal drought under controlled environmental conditions. The number of days until death was measured, along with root traits, senescence, and aspects of hydraulic function.
3. We identified two strategies: Drought‐avoiding species that stayed green as the water potential declined and drought‐tolerating species that senesced more quickly but could extend survival via drought‐tolerant meristems.
4. Plants that stay‐green for longer occupied drier habitats and had the longest survival under drought, facilitated by narrow root diameter and isohydric stomatal behavior. Plants that senesced quickly had thicker roots, an anisohydric strategy, and occupied wetter habitats.
5. Global distributions of C₄ grasses can be predicted by variation in rates of senescence, meristem survival, root traits, and stomatal strategy, showing the value of these traits for understanding plant distributions in relation to climate.

     

Salt tolerance evolves more frequently in C4 grass lineages

Salt tolerance has evolved many times in the grass family, and yet few cereal crops are salt tolerant. Why has it been so difficult to develop crops tolerant of saline soils when salt tolerance has evolved so frequently in nature? One possible explanation is that some grass lineages have traits that predispose them to developing salt tolerance and that without these background traits, salt tolerance is harder to achieve. One candidate background trait is photosynthetic pathway, which has also been remarkably labile in grasses. At least 22 independent origins of the C₄ photosynthetic pathway have been suggested to occur within the grass family. It is possible that the evolution of C₄ photosynthesis aids exploitation of saline environments, because it reduces transpiration, increases water‐use efficiency and limits the uptake of toxic ions. But the observed link between the evolution of C₄ photosynthesis and salt tolerance could simply be due to biases in phylogenetic distribution of halophytes or C₄ species. Here, we use a phylogenetic analysis to investigate the association between photosynthetic pathway and salt tolerance in the grass family Poaceae. We find that salt tolerance is significantly more likely to occur in lineages with C₄ photosynthesis than in C₃ lineages. We discuss the possible links between C₄ photosynthesis and salt tolerance and consider the limitations of inferring the direction of causality of this relationship.     

Insect herbivory on C3 and C4 grasses

Summary This study tested the hypothesis that grasses with the C₄ photosynthetic pathway are avoided as a food source by insect herbivores in natural communities. Insects were sampled from ten pairs of C₃–C₄ grasses and their distributions analyzed by paired comparisons tests. Results showed no statistically significant differences in herbivore utilization of C₃–C₄ species. However, there was a trend towards heavier utilization of C₃ species when means for both plant groups were compared. In particular, Homoptera and Diptera showed heavier usage of C₃ plants. Significant correlations between insect abundances and plant protein levels suggest that herbivores respond to the higher protein content of C₃ grasses. ¹³C values for six of the most common grasshopper species in the study area indicated that three species fed on C₃ plants, two species fed on C₄ plants, and one species consumed a mixture of C₃ and C₄ tissue.Welder Wildlife Refuge Contribution Number 213     

Geographical and environmental distribution of C3 and C4 grasses in the Sinai,Negev, and Judean deserts

Summary The relation between photosynthetic pathway and habitat of the grass species recorded in the desert regions of Sinai, Negev, and Judea was investigated. The climatic conditions and micro-environments in the study area vary considerably, and the distribution of the various species is found to conform to specific patterns which reveal the adaptive advantages of the different photosynthetic pathways. There is also a distinct correlation between the phytogeographic origin of the grass species and the photosynthetic pathways that they utilize.The survey shows that the majority of the grass species in the region are of the C₃ type and all except one of these species belong to the Holarctic domain. This is in accordance with the fact that the region forms part of the Mediterranean winter rainfall regime and that C₃ species have an adaptive advantage where minimum temperatures are low during the winter growing season.The occurence of C₄ species increases with decreasing rainfall and they dominate in those districts where temperatures are high throughout the year. These C₄ grasses are of both Holarctic and Palaeotropic origin according to the classification adopted here, but they are essentially all elements of the Saharo-Arabian, Irano-Turanian, Sudanian, or Tropical phytogeographic regions and are not typical of the Mediterranean or Euro-Siberian floras. The plants with multi-regional distributions that occur in Mediterranean communities may well be intrusive.Analysis of the three subtypes of the C₄ species suggests that the malate-forming NADP-me grasses grow where water stress is not a dominating factor, while the aspartateforming NAD-me grasses are more successful under xeric conditions. The PEP-ck species are not abundant and form an intermediate group between the NADP-me and NAD-me subtypes.     

Germination sensitivities to water potential among co‐existing C3 and C4 grasses of cool semi‐arid prairie grasslands

An untested theory states that C₄ grass seeds could germinate under lower water potentials (Ψ) than C₃ grass seeds. We used hydrotime modelling to study seed water relations of C₄ and C₃ Canadian prairie grasses to address Ψ divergent sensitivities and germination strategies along a risk‐spreading continuum of responses to limited water. C₄ grasses were Bouteloua gracilis, Calamovilfa longifolia and Schizachyrium scoparium; C₃ grasses were Bromus carinatus, Elymus trachycaulus, Festuca hallii and Koeleria macrantha. Hydrotime parameters were obtained after incubation of non‐dormant seeds under different Ψ PEG 6000 solutions. A t‐test between C₃ and C₄ grasses did not find statistical differences in population mean base Ψ (Ψ_b(50)). We found idiosyncratic responses of C₄ grasses along the risk‐spreading continuum. B. gracilis showed a risk‐taker strategy of a species able to quickly germinate in a dry soil due to its low Ψ_b(50) and hydrotime (θ_H). The high Ψ_b(50) of S. scoparium indicates it follows the risk‐averse strategy so it can only germinate in wet soils. C. longifolia showed an intermediate strategy: the lowest Ψ_b(50) yet the highest θ_H. K. macrantha, a C₃ grass which thrives in dry habitats, had the highest Ψ_b(50), suggesting a risk‐averse strategy for a C₃ species. Other C₃ species showed intermediate germination patterns in response to Ψ relative to C₄ species. Our results indicate that grasses display germination sensitivities to Ψ across the risk‐spreading continuum of responses. Thus seed water relations may be poor predictors to explain differential recruitment and distribution of C₃ and C₄ grasses in the Canadian prairies.     

Neotropical C3/C4 grass distributions – present,past and future

Changes in C₄ grass distribution and abundance are frequently observed in Quaternary, Holocene and future environmental‐change scenarios. However, the factors driving these dynamics are not fully understood, and conflicting theories have been reported. In this paper, we present a very large dataset of modern altitudinal distribution profiles of C₃ and C₄ grasses covering the entire Neotropical Andes, which was compared with actual climate data. The results of multivariate analysis demonstrate that, in the Neotropical Andes, mean annual temperature is the main factor governing the modern altitudinal distribution of C₃ and C₄ grass species. The C₃ and C₄ grass distributions were compared with simulations based on the Lund‐Potsdam‐Jena dynamic global vegetation model (LPJ‐DGVM), which allowed the present grass distribution to be estimated. Finally, the DGVM was employed to simulate past and future scenarios, using the IPCC's climate projections for 2100 and PMIP2 models for the Holocene Optimum (HO, 6000 years bp ) and the Last Glacial Maximum (LGM, 21 000 years bp ). The results were found to be significantly different from those obtained using a simple photosynthetic model. According to LPJ forced with the PMIP2 models for the LGM, during the LGM, the C₄ grasses would not have reached higher altitudes than found in the present day.     

Ecological selection pressures for C4 photosynthesis in the grasses

Grasses using the C₄ photosynthetic pathway dominate grasslands and savannahs of warm regions, and account for half of the species in this ecologically and economically important plant family. The C₄ pathway increases the potential for high rates of photosynthesis, particularly at high irradiance, and raises water-use efficiency compared with the C₃ type. It is therefore classically viewed as an adaptation to open, arid conditions. Here, we test this adaptive hypothesis using the comparative method, analysing habitat data for 117 genera of grasses, representing 15 C₄ lineages. The evidence from our three complementary analyses is consistent with the hypothesis that evolutionary selection for C₄ photosynthesis requires open environments, but we find an equal likelihood of C₄ evolutionary origins in mesic, arid and saline habitats. However, once the pathway has arisen, evolutionary transitions into arid habitats occur at higher rates in C₄ than C₃ clades. Extant C₄ genera therefore occupy a wider range of drier habitats than their C₃ counterparts because the C₄ pathway represents a pre-adaptation to arid conditions. Our analyses warn against evolutionary inferences based solely upon the high occurrence of extant C₄ species in dry habitats, and provide a novel interpretation of this classic ecological association.     

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.     

Macro-Climatic Distribution Limits Show Both Niche Expansion and Niche Specialization among C4 Panicoids

Grasses are ancestrally tropical understory species whose current dominance in warm open habitats is linked to the evolution of C₄ photosynthesis. C₄ grasses maintain high rates of photosynthesis in warm and water stressed environments, and the syndrome is considered to induce niche shifts into these habitats while adaptation to cold ones may be compromised. Global biogeographic analyses of C₄ grasses have, however, concentrated on diversity patterns, while paying little attention to distributional limits. Using phylogenetic contrast analyses, we compared macro-climatic distribution limits among ~1300 grasses from the subfamily Panicoideae, which includes 4/5 of the known photosynthetic transitions in grasses. We explored whether evolution of C₄ photosynthesis correlates with niche expansions, niche changes, or stasis at subfamily level and within the two tribes Paniceae and Paspaleae. We compared the climatic extremes of growing season temperatures, aridity, and mean temperatures of the coldest months. We found support for all the known biogeographic distribution patterns of C₄ species, these patterns were, however, formed both by niche expansion and niche changes. The only ubiquitous response to a change in the photosynthetic pathway within Panicoideae was a niche expansion of the C₄ species into regions with higher growing season temperatures, but without a withdrawal from the inherited climate niche. Other patterns varied among the tribes, as macro-climatic niche evolution in the American tribe Paspaleae differed from the pattern supported in the globally distributed tribe Paniceae and at family level.     

The age of the grasses and clusters of origins of C4 photosynthesis

At high temperatures and relatively low CO₂ concentrations, plants can most efficiently fix carbon to form carbohydrates through C₄ photosynthesis rather than through the ancestral and more widespread C₃ pathway. Because most C₄ plants are grasses, studies of the origin of C₄ are intimately tied to studies of the origin of the grasses. We present here a phylogeny of the grass family, based on nuclear and chloroplast genes, and calibrated with six fossils. We find that the earliest origins of C₄ likely occurred about 32 million years ago (Ma) in the Oligocene, coinciding with a reduction in global CO₂ levels. After the initial appearance of C₄ species, photosynthetic pathway changed at least 15 more times; we estimate nine total origins of C₄ from C₃ ancestors, at least two changes of C₄ subtype, and five reversals to C₃. We find a cluster of C₄ to C₃ reversals in the Early Miocene correlating with a drop in global temperatures, and a subsequent cluster of C₄ origins in the Mid‐Miocene, correlating with the rise in temperature at the Mid‐Miocene climatic optimum. In the process of dating the origins of C₄, we were also able to provide estimated times for other major events in grass evolution. We find that the common ancestor of the grasses (the crown node) originated in the upper Cretaceous. The common ancestor of maize and rice lived at 52 ± 8 Ma.