The Age of the Grasses and Clusters of Origins of C4 Photosynthesis

Global Change Biology (2008) 14, 2963–2977, doi: 10.1111/j.1365-2486.2008.01688.x

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

ALBERTO VICENTINI*1, JANET C. BARBERw ,SANDRAS.ALISCIONIz, LILIANA M. GIUSSANI§ andELIZABETH A. KELLOGG* *Department of Biology, University of Missouri, St Louis, One University Boulevard, St Louis, MO 63121, USA, wDepartment of Biology, Saint Louis University, 3507 Laclede Avenue, St Louis, MO 63103, USA, zCa´tedra de Botańica Agrıćola, Facultad de Agronomıá, Universidad de Buenos Aires, Av. San Martin 4453, C1417DSE, Buenos Aires, Argentina, §Instituto de Botańica Darwinion, Labardeń 200, Casilla de Correo 22, San Isidro, Buenos Aires 1642, Argentina

Abstract

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. Keywords: evolution, Miocene climate, photosynthesis, Poaceae

Received 23 April 2008 and accepted 27 May 2008

calories. Finally, species currently used or being studied Introduction as sources of biofuels, such as maize, sugar cane,

About a quarter of the world’s terrestrial primary switchgrass and Miscanthus,areC4. productivity comes from C4 plants (Lloyd & Farquhar, The C4 pathway is derived from the simpler and more 1994), most of which, whether computed in terms of ancient C3 carbon ﬁxation mechanism found in the number of species or in terms of biomass, are grasses majority of plants, and is presumed to be an adaptation

(Sage et al., 1999a, b). Grasses that use the C4 photosyn- to the low atmospheric CO2 levels and high O2 levels that thetic pathway dominate the Great Plains of North have prevailed since the Oligocene (Sage, 2004), in which

America, the grasslands of Africa and Australia, and the C3 pathway is inherently inefficient (Ehleringer & are components of most grassland and savanna biomes Monson, 1993). Rubisco (ribulose 1,5,-bisphosphate car- in the warmer parts of the world. In addition, C4 grasses boxylase oxygenase), the enzyme that fixes CO2 in plants, such as maize, sorghum, sugar cane and the various binds O2,aswellasCO2, a process known as photo- species of millets provide a significant source of human respiration, which increasingly reduces the efficiency of photosynthesis as temperatures increase (Ehleringer & Correspondence: Elizabeth A. Kellogg, tel. 1 1 314 516 6217, Monson, 1993; Tipple & Pagani, 2007). In contrast, fax 1 1 314 516 6233, e-mail: [email protected] C4 plants minimize photorespiration by sequestering 1Present address: Center for Tropical Forest Science, Smithsonian Rubisco in cells around the veins, and use a different Tropical Research Institute, Apartado Postal 0843-03092, Panama´, primary carbon acceptor (phosphoenol pyruvate) in the Panama. mesophyll. C4 photosynthesis is thus more efficient than r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd 2963 2964 A. VICENTINI et al.

C3, particularly at high temperatures, but it requires A test of Sage’s hypothesis requires (a) a well- more energy and only becomes advantageous when supported phylogeny of the grasses, including repre- atmospheric pCO2 (partial pressure of CO2)isatits sentatives of the various C4 lineages; (b) inference of the current relatively low level (below 500 ppm) and ancestral photosynthetic pathway at the nodes (specia- when temperatures are relatively high (above 25 1C) tion events) in the phylogeny; and (c) assigning dates to (Ehleringer et al., 1997). the nodes. Some years ago, we produced a phylogeny of

The grass family (Poaceae) includes more C4 species the subfamily Panicoideae based on the chloroplast than all other plant families combined (Clayton & gene ndhF (Giussani et al., 2001) and identiﬁed nodes

Renvoize, 1986; Sage et al., 1999a). The C4 pathway in the phylogeny where C4 might have originated. has originated independently in four of the 13 grass That study formed the basis of recent work by Christin subfamilies (Sinha & Kellogg, 1996; Kellogg, 1999; et al. (2008) and the present paper. Because the Giussani et al., 2001; Christin et al., 2008), including chloroplast is maternally inherited, its history reﬂects multiple changes in subfamily Panicoideae (Giussani only one line of evidence for the history of the plants. et al., 2001; Duvall et al., 2003). C4 also appears in Accordingly, we chose to verify the phylogeny of Gius- subfamilies Chloridoideae, Aristidoideae, and Micrair- sani et al. (2001) by assembling data for a nuclear gene, oideae. The various C4 lineages differ in leaf anatomy phytochrome B (phyB), and present those results here. (Prendergast et al., 1987), the tissue-speciﬁc expression In contrast, Christin et al. (2008) continued to use of photosynthetic enzymes (Sinha & Kellogg, 1996), chloroplast markers, including ndhF, but expanded the and the primary enzymes used in metabolizing the sampling of C4 species to include representatives in four-carbon molecules. The differences support the other grass subfamilies. hypothesis of independent gains of the pathway. A phylogeny estimates the numbers of mutations

The great C4 grasslands of today expanded in the Late separating extant species from their ancestors, but con- Miocene, 6–8 million years ago (Ma) (Wang et al., 1994; verting the number of mutations to an estimate of time Fox & Koch, 2003), but the earliest fossil grass leaf with requires a calibration point, generally a fossil. An in-

C4 anatomy is dated at 12.5 Ma (Nambudiri et al., 1978; dividual fossil provides a minimum age for a taxonomic Jacobs et al., 1999), and silica bodies (phytoliths) group. For example, Thomasson (1978) described a diagnostic of the C4 subfamily Chloridoideae have been fossil that can be assigned to the modern genus identiﬁed at about 19 Ma (Stro¨mberg, 2005). C4 origins Dichanthelium; the formation in which the fossil was thus precede C4 grassland expansions. found is dated at 8 Ma. Thus, the genus Dichanthelium Global CO2 levels in most of the Tertiary were much must be at least 8 Ma old, although it could clearly be higher than at present, well over 1000 ppm and ﬂuctu- older. Other reliable fossil dates are provided by a fossil ating to even higher levels during the Eocene (Cerling, of Setaria at 7 Ma (Elias, 1942), and the chloridoid 1991; Ekart et al., 1999; Pearson & Palmer, 2000; Royer phytoliths at 19 Ma (Stro¨mberg, 2005). et al., 2004). The Oligocene saw a drop in atmospheric Multiple fossils have been suggested to provide a

CO2, with modern (i.e. preindustrial) levels reached by date for the earliest grasses, and these need to be the Early Miocene (Pagani et al., 1999, 2005). Global assessed in any effort to date the grass phylogeny. temperature also fluctuated during this time, falling Unequivocal grass pollen is known from 55 Ma from a high during the Eocene climatic optimum to (Jacobs et al., 1999), and pollen that may be from a grass recent relatively low global temperatures, with much or from a close relative is dated to 70 Ma (Linder, 1986; variation along the way (Zachos et al., 2001; Lunt et al., Herendeen & Crane, 1995). A two-flowered grass 2007). The connection between global temperature and spikelet fossil from the Early Eocene (Crepet & other climatic parameters (seasonality, aridity) is not Feldman, 1991; 55 Ma) does not match any extant simple, and remains a subject of active research. taxon, but indicates that multiflowered spikelets had Ehleringer et al. (1991) proposed low atmospheric arisen by the Early Tertiary. A set of morphologically

CO2 levels to explain broad ecological expansion of C4 diverse phytoliths from the North American Great grasslands in the Late Miocene, an idea that was later Plains suggests that all the major subfamilies had pursued by Ehleringer et al. (1997) and Cerling et al. diverged by 35 Ma (Stro¨mberg, 2005). However, a recent (1997). More recently, it has become clear that the study of fossilized dinosaur dung (coprolites) in preindustrial CO2 levels were achieved much earlier, India (Prasad et al., 2005) places grass diversiﬁcation between ca. 35 and 25 Ma during the Oligocene (Pagani much earlier, at 65–67 Ma. Because India was not et al., 2005), prompting (Sage, 2001, 2004) to suggest a connected to any landmass at that date, Prasad et al. possible correlation between the Oligocene drop in CO2 (2005) interpreted this to mean that the radiation of the and the origins (rather than expanded distribution) of family occurred even earlier, before the breakup of

C4 species. Gondwana (80 Ma).

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Here, we present a calibrated phylogeny of the grasses, (Sambrook et al., 1989). Approximately 1.2 kb of exon 1 focusing particularly on subfamily Panicoideae, and of phyB was amplified following Mathews et al. (2000). incorporating both chloroplast and nuclear markers. We Except in maize (which has two copies Sheehan et al., investigate the effects of various fossil calibrations and 2004), phyB is apparently single copy in grasses find that (1) all but the coprolite calibration provide (Mathews & Sharrock, 1996); thus, strong PCR products similar estimates for C4 origins; (2) the earliest C4 origins were purified and sequenced directly. PCR products correlate with the drop in global CO2 levels in the were cloned before sequencing for 36 taxa that Oligocene, confirming the results of Christin et al. (2008) amplified weakly. Fragments were sequenced on both and the earlier hypothesis of Sage (2004); and (3) C4 strands with the external amplification primers and two origins are not correlated in any simple way with global internal primers (Mathews et al., 2000). Sequence temperatures (i.e. warm global temperatures may not divergence among multiple clones was o1% and all lead to C4 origins). Our most striking result, however, is the cloned sequences of a taxon clustered together in (4) that changes in photosynthetic pathway are clustered preliminary analyses, so consensus sequences were in time, suggesting that global climatic forces in addition generated for each species, coding ambiguities as to temperature may have facilitated transitions between polymorphic. All the sequences of ndhF and additional

C3 and C4. Our data also provide estimates for dates of phyB sequences were downloaded from GenBank. critical nodes of the grass phylogeny, including the Genbank accession numbers are listed in Table S1. divergence of maize from rice. Multiple alignments of both ndhF and phyB were produced in MacClade (Maddison & Maddison, 2000); alignment was easily done by eye. Sequences were Materials and methods translated to verify the presence of an open reading frame. Following the procedures used by the Grass Taxon sampling Phylogeny Working Group (2001), 13 taxa were We assembled sequence data for 97 grasses plus three represented by an ndhF sequence of one species and a more distantly related monocots (Muscari in Hycintha- phyB sequence of another; this assumes that the relevant ceae, Flagellaria in Flagellariaceae, and Joinvillea in taxa are monophyletic, an assumption corroborated by Joinvilleaceae). The software used for dating the tree data in the literature. also requires a still more distant relative that is pruned from the tree during the analysis, so we included the Phylogenetic analysis eudicot Arabidopsis. All the subfamilies of grasses are represented except for the recently emended Micrair- We first tested whether the new data from the nuclear oideae for which phyB sequences were unavailable. We gene indicated the same phylogeny as the published focused on subfamily Panicoideae, which comprises ndhF data. We analyzed the phyB and ndhF datasets three large clades (Giussani et al., 2001; Aliscioni et al., individually with parsimony and Bayesian approaches, 2003): Andropogoneae, Paniceae taxa with chromosome and retrieved trees in which major clades were strongly numbers in multiples of 10 (the x 5 10 Paniceae), and supported and identical in composition (deposited in Paniceae taxa with chromosome numbers in multiples TreeBase). Nonetheless, there were several points of of 9 (the x 5 9 Paniceae). Multiple representatives of conflict between the trees. Conflict was highly localized each clade are included here (Table S1). and involved relationships of only four regions of the tree, within, rather than between, major clades. Accordingly, we combined the two datasets. DNA sequencing For the combined dataset, MrModeltest 2.2 (Nylan- Sequences of the nuclear gene phyB were generated der, 2004) chose the GTR 1 I 1 G as the best model for to maximize comparability with the available ndhF each gene. Two Bayesian analyses were performed with (chloroplast) data. Many of the ndhF data in GenBank MrBayes (Huelsenbeck & Ronquist, 2001) running on (and used by Christin et al., 2008) were produced by the Beowulf cluster at University of Missouri, St Louis, several of us (Giussani et al., 2001; Aliscioni et al., 2003), with the following options: ngen 5 10 000 000, print- so we were able to produce phyB sequences from either freq 5 1000, samplefreq 5 1000, and burnin 5 1000. A the same DNA used in our previous study, or new DNA majority rule consensus tree was obtained with the extracted from the silica-dried ndhF voucher material. 18 000 trees saved for two independent runs and the In a few cases, we extracted new DNA from living plant frequency of each node was recorded as the Bayesian material. Total genomic DNA was extracted with the posterior probability (Fig. S1). The 50% majority rule cetyl-trimethylammonium bromide method (Doyle & consensus tree was fully dichotomous and identical to Doyle, 1987) and purified via cesium chloride gradients the maximum likelihood tree. Because assignment of r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, 14, 2963–2977 2966 A. VICENTINI et al. dates requires an outgroup that is then pruned from the analysis, we rooted the trees using two unambiguous outgroups, Arabidopsis and Muscari. These outgroups are omitted from all the figures for clarity. ry for the node. Poll. Spike. Phyt. Molecular dating Dating used a Bayesian relaxed-clock method (Thorne & Kishino, 2002) as implemented in Multidivtime L67 (Rutschmann, 2005). Model parameters, and branch-lengths and their variance–covariance matrices were estimated separately for the ndhF and phyB partitions (Estbranches step). Estimation of rates and chronogram sampling was performed with 10 000 Markov chain samples (numsamps) taken at every 1000 cycles (sampfreq) starting after 1 000 000 initial cycles (burnin). Priors for parameters rtrate and rtratesd in the command file multicntrl.dat were selected by taking the median amount of evolution from tips to the ingroup root and dividing this figure by the prior age for the ingroup node (rttm parameter), as suggested in the Multidivtime manual (Rutschmann, 2005). The rttm parameter was set to 1.5 [i.e. 150 Ma with a standard deviation of 0.01 (rttmsd)], and bigtime was set to 10 (i.e. 1000 Ma). Other parameters were the default values: brownmean 5 1, brownsd 5 1, minab 5 1, newk 5 0.1, othk 5 0.5, thek 5 0.5. Multiple calibrations were tested, using fossils, as described in the text (Table 1).

Ancestral state reconstruction

Species were coded as C3 or C4; among the C4 species, we distinguished among the NADP malic enzyme, NAD malic enzyme, and phosphoenolpyruvate . (2005) L80 carboxykinase (PCK) subtypes (Table S1). The photo- . (2005) synthesis type of Aristida was set to NADP, although et al et al Aristida differs from other NADP plants in having a mberg (2005)mberg (2005) L19 L19 L19 L19 L35 L19 L19 L35 double bundle sheath (Prendergast & Hattersley, 1987), ¨ ¨ Thomasson (1978) L8 L8 L8 L8 L8 L8 carbon reduction in both sheaths, lack of a suberized Elias (1942) L7 L7 L7 L7 L7 L7 lamella in any bundle sheath cells, and a distinctive pattern of expression of photosynthetic genes (Sinha & Kellogg, 1996). Likewise, Sporobolus was coded as NAD, although different species of Sporobolus exhibit either the NAD or PCK subtypes. Steinchisma, the only true

C3/C4 intermediate in this sample of taxa, was coded as Dichanthelium NADP. Outside the genus Steinchisma, the only known Setaria

C3/C4 intermediate in Poaceae is Neurachne minor (Hat- tersley & Stone, 1986), which was not available for this study.

To infer the photosynthetic pathway of ancestral species, Fossil dates used for the different calibration schemes Pagel’s (1994) maximum likelihood method for discrete characters was used for both the unconstrained ML tree GEA 19C 35E 55–70 55 Chloridoid 80 Pollen NA phytoliths Multiﬂower spikelet Continental drift Stro Crepet Stro & Feldman (1991) Prasad Linder (1986) L55/U70 L70 L55 L55 L70 Time is indicated in millionsNodes of are years. indicated L in or Figs U 1 before and the S2. number of millions of years indicates whether the number served as a lower (L) or upper (U) bounda W8 E 67 Coprolites Prasad Table 1 Node Age (Ma) Fossil Reference Pollen55-70 Pol-lenL70 Spikelet Phytolith Gondwana Coprolite and the chronograms resulting from the dating analyses. b7

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For each tree, we tested different models of transition rates (Fig. S1) was well supported and consistent with pre- (equal, symmetrical and all-different models) and a range vious phylogenetic results (GPWG, 2001; Sańchez-Ken of initial likelihood values (ip 5 0–1) using function ace of et al., 2007; Bouchenak-Khelladi et al., 2008; Christin package Ape in R (Paradis et al., 2004). The best combina- et al., 2008). We retrieved a large clade including Bam- tion of transition rate model and initial likelihood setting busoideae, Ehrhartoideae, and Pooideae (the BEP clade) was then obtained given the lowest absolute Akaike sister to the clade including Panicoideae, Arundindoi- Information Criterion (AIC) score for the tested recon- deae, Centothecoideae, Chloridoideae, Micrairoideae, structions. In all cases, the best model was the ‘equal rates’ Aristidoideae, and Danthonioideae (the PACCMAD model regardless of the initial ip parameter (the default clade). All the C4 origins are in the latter clade, as found 0.01 was then used). Thus, these data indicate that gains in all the previous studies (e.g. GPWG, 2001). and losses of C4 photosynthesis are equally likely. We estimated dates for the internal nodes, and con- We also explored the panicoid portion of the tree using verted the tree to a chronogram (Fig. 1), using multiple parsimony optimization on the individual gene trees fossil calibrations. We assigned the minimum age for under ACCTRAN and DELTRAN optimizations. As the stem nodes of the Chloridoideae, Dichanthelium, and expected, the precise number of gains and losses varied Setaria (nodes G, W, and b) at 19, 8, and 7 Ma, respec- depending on the weighting scheme and also somewhat tively, based on fossil phytoliths and anthecia (Elias, depending on the sample of taxa. For example, for the 1942; Thomasson, 1978; Stro¨mberg, 2005) (Table 1). (The phyB trees, there were two to five gains and six to nine stem node for a group is the earliest date at which a losses of C4 under ACCTRAN optimization, whereas distinctive feature might have arisen. For example, the DELTRAN optimization showed three to six gains and phytoliths for Chloridoideae must have originated by five to seven losses. Similarly, optimization on the ndhF node H, but could have originated much earlier im- trees found 2–12 gains and zero to nine losses under mediately after the divergence of the chloridoid ances- ACCTRAN, and 5–13 gains and zero to six losses under tor from its sister group at node G. G is the stem node, DELTRAN. Nevertheless, changes (of whatever direction) whereas H is the crown node.) were optimized to the same nodes as identified by the We tested the effects of using different dates to likelihood weighting employed here (see Pollen.Spike.Phyt. calibrate deep nodes in the phylogeny (Table 1). The calibration in Fig. S2). two grass pollen fossils [one at 55 Ma unequivocally a grass, the other at 70 Ma possibly a grass (Linder, 1986; Herendeen & Crane, 1995)], let us date the stem node of Simulation of transitions of photosynthetic pathways the family (A in Fig. 1) as 55 or 70 Ma (calibrations With LASER (Rabosky, 2006), we created 1000 simulated PollenL55 and PollenL70). Spikelets arose between nodes datasets, each with as many transitions in photosynthetic B and C, and multiflowered spikelets between nodes C pathway as observed, generated under a pure birth con- and D (GPWG, 2001). Accordingly, we used the date of stant rate speciation model (Rabosky, 2006). These simu- the Eocene grass spikelet fossil (Crepet & Feldman, lated datasets were then scaled to vary within the age of 1991) to constrain the stem node (C) of the multiflow- the grasses (from node B in Fig. 1 to the present), and we ered spikelet clade to 55 Ma (Spikelet calibration). Phy- defined a set of time windows of different sizes (varying tolith data from the North American Great Plains from 1 to 10 Ma with increments of 0.5). Then, for each suggest that the BEP clade and the PACCMAD clade time –window, we tested at different points in time (from had diverged by 35 Ma (Stro¨mberg, 2005), providing a 50 Ma to the present with increments of 0.5 Ma) whether minimum date for node E (Phytolith calibration). The the number of observed transition events was greater than dinosaur coprolite (Prasad et al., 2005), however, places expected under a constant rate of transitions (number of the same node much earlier, at 65–67 Ma (Coprolite transition events in the 1000 simulated datasets). As the calibration), although Prasad et al. (2005) preferred a observed date for transitions, we used the mean age of Gondwanan age for the node (80 Ma; Gondwana cali- each event (Fig. 3a–d). We did this separately for C3–C4 bration). All the calibrations thus used four fossils – and C4–C3 events for each of the four calibration schemes chloridoid phytoliths, Dichanthelium, Setaria, and one of discussed in the text. six possible dates for the deep nodes (Table 1). Dates estimated from the PollenL55, PollenL70, Spike- let, and Phytolith calibrations were similar for all nodes, Results so the latter three were combined with those of the three more recent fossils to provide an estimate of dates based Phylogenies and dating on six fossils (Poll.Spike.Phyt calibration; Figs 1 and 2; The phylogenetic tree based on combined DNA Tables 1 and 2). Dates estimated from Poll.Spike.Phyt sequences from the chloroplast and the nucleus were similar to dates estimated by other authors (Bre- r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, 14, 2963–2977 2968 A. VICENTINI et al.

F H

V J W

X K Paniceae.X=9 c b Y

C Z

U N Photosynthesis type E

T C4 NAD S M Paniceae.X=10 C4 NADP

C4 PCK R O C3 Q L P

D Andropogoneae

A Pooideae

Bambusoideae

r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, 14, 2963–2977 CLUSTERED ORIGINS OF C4 PHOTOSYNTHESIS 2969 mer, 2002; Gaut, 2002; Christin et al., 2008) who used conditions favorable for C4 grasses in the late Oligo- fewer points of calibration. Values for the Coprolite and cene, even at CO2 levels around 800 ppm (Lunt et al., Gondwana calibrations were consistently older than the 2007). The Gondwana and Coprolite calibrations, in con- others (Table 2; Fig. 2). trast, place node J at 51.8 and 44.3 Ma, when global

Independent gains of C4 occurred in Aristidoideae, pCO2 was over 1000 ppm (Berner, 2004). Chloridoideae, Panicoideae, and multiple other Our data also suggest that C4 could have originated lineages (Fig. 1 and Fig. S2). The earliest possible change even earlier, possibly soon after the stem node of to C4 occurred some time after the divergence of the Aristidoideae (node F). The Poll.Spike.Phyt. and Pol- stem node of the Aristidoideae (node F), making node F lenL55 calibrations date node F at 42–44 Ma, but this is the maximum age of C4 grasses. Likewise, the dates of a maximum date, so the calibration does not necessarily the stem node of the chloridoids (G) and of Danthoniop- contradict a correlation between pCO2 and C4 origin sis (J) are maxima. Other switches between photosyn- (Figs 3a and 4a). The Coprolite and Gondwana calibra- thetic pathways could be assigned to intervals between tions, in contrast, place both node F and J much earlier, dated nodes (Fig. 3). near or at the Eocene climatic optimum (Figs 3 and 4),

indicating no correlation between pCO2 and C4. Finally, we correlated C4 origins with the global deep- 18 Clustered changes of photosynthetic pathway and sea oxygen isotopic (@ O) signal (Zachos et al., 2001), a correlation with climate proxy for temperature. Poll.Spike.Phyt. dates the first unequivocal C4 origins (node J) coincident with the We found that evolutionary changes in photosynthetic glacial aberration Oi-1 (Zachos et al., 2001) (Figs 3a pathway were clustered in geological time. We simu- and 4a). Clusters of C3–C4 changes occur during the lated the pattern of C4 origins and reversals assuming a Mid-Miocene climatic optimum (MMCO), 17–15 Ma, constant rate of change throughout grass evolution and also during a much cooler period 12–11 Ma. A 18 (Rabosky, 2006), and tested whether significantly more cluster of reversals to C3 correlated with @ O values changes occurred in any window of time than predicted suggesting a cool climate somewhat before the MMCO, for constant rates. Transition times were significantly at 21–18 Ma. The PollenL55 calibration indicates a clustered regardless of calibration and window size second cluster of reversals to C3 at 13–10 Ma. Thus, (Fig. 3). Four to five C3–C4 transitions and two to four the origin of C4 clades is not a simple response to reversals occurred within 10 Ma (the maximum win- elevated global temperature, and likely reflects more dow-size tested), depending on calibration scheme. complex environmental changes. Small window sizes (o5 Ma) resolved two separate time periods containing significantly more C3–C4 transitions (42) than random, as well as one period of Discussion clustered reversals. Notably, for all the analyses with all calibrations, C3–C4 clusters occurred during different Dates of C4 origins periods of geological time than did the C4–C3 clusters (Figs 3 and 4). Our best estimate of the earliest unequivocal C4 origin We correlated the estimated dates of C4 transitions (after node I and before J) is between 34.3 ( 6.2) and with published values for global pCO2 (Pagani et al., 31.7 ( 5.9) Ma, using the six-fossil (Poll.Spike.Phyt.) 2005). The earliest unequivocal origin of C4 occurred at calibration. This is comparable with the results of node J (second red dot from the left in Fig. 4), Christin et al. (2008), who place the oldest C4 between 31.7 5.9 Ma by the Poll.Spike.Phyt. calibration, or 32.0 ( 4.4) and 25.0 ( 4.0) Ma. The standard devia- slightly later with the PollenL55 calibration, suggesting tions of their estimate and ours overlap. Our data place that C4 originated as global pCO2 fell to the range in the earliest unambiguous C4 at the common ancestor of which C4 is energetically favorable (Table 2; Figs 3 and Danthoniopsis and other PACCMAD taxa, whereas 4). This is consistent with climatic models that indicate Christin et al. (2008) place the earliest C4 as the ancestor

Fig. 1 Chronogram for the Poll.Spike.Phyt. (six-fossil) calibration (Table 2) with the maximum likelihood reconstruction of photosynth- 18 esis pathway evolution. Graph of pCO2 is from Pagani et al. (2005) and @ O from Zachos et al. (2001). Nodes labeled A and B refer to stem and crown nodes of grasses, respectively; other labeled nodes mark changes in the photosynthetic pathway (see also Fig. S2a–d). Except for Joinvillea and Flagellaria, all taxa are members of the Poaceae (grasses). Grass subfamilies are indicated by vertical bars. Danthoniopsis and Gynerium are assigned to different subfamilies by different authors, so are left unassigned here. Dashed red line indicates time at which pCO2 ﬁrst drops below 500 ppm. r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, 14, 2963–2977 2970 A. VICENTINI et al.

130 Calibrations

120 PollenL55 Coprolite PollenL70 Gondwana 110 Spikelet Poll.Spike.Phyt 100 Phytolith

Age estimates (Ma) 50

A BCDEFGHIJKLMNOPQRSTUVWXYZabc Nodes of interest

Fig. 2 Age estimates of seven calibration schemes (Table S2). Nodes listed are indicated in Figs 1 and S2.

of the core Chloridoideae. The difference reflects differ- imposing both a lower limit (55 Ma) and an upper limit ences in taxonomic sampling in this portion of the tree (60 Ma) on the date, based on the Spikelet calibration. By (broader in Christin et al., 2008) and in the amount of imposing an upper limit, all dates higher in the tree are DNA sequence per taxon (broader in the present study). forced to be somewhat younger. (We used a similar The phylogeny of the PACCMAD clade has always constraint in early analyses, but felt that the results were been problematical (GPWG, 2001), and neither the tree artifactually precise, so reverted to using only the lower of Christin et al. (2008) nor the one presented here limits presented here.) provides strong support for relationships among the With their broader taxonomic sample, Christin et al. major lineages of the clade. (2008) were able to estimate dates for additional C4 Table 2 shows the dates of all C4 origins estimated by origins, beyond those presented here. These results Christin et al. (2008) and the present study. Comparing are summarized at the end of Table 2. They place the our estimates based on the six-fossil (Poll.Spike.Phyt.), origin of C4 in Micrairoideae at ca. 11 Ma, and date the calibration shows that none of the estimates are signifi- stem nodes of the C4 species of Neurachne and Alloter- cantly different, as indicated by broad overlap of con- opsis at 4 and 15 Ma, respectively. fidence intervals. Our Spikelet calibration is the same as An important difference between the taxonomic that used by Christin et al. (2008), as is the PollenL55 sample of Christin et al. (2008) and that of the present calibration. Because these two calibrations plus study is in the Aristidoideae. Because we had only PollenL70 and Phytolith all gave comparable dates for a single representative of this subfamily, we could

C4 origins, it is not surprising that our results agree with provide only a maximum possible date for the C4 origin those of Christin et al. In addition, one of our two (44.4 7.5 Ma). Christin et al. (2008) included all the datasets (ndhF) was the same as that used by Christin three genera of the subfamily, the C4 Aristida, Stipagros- et al., again explaining the general congruence of our tis, and the C3 Sartidia. The latter two are native to results. southern Africa and are not easy to collect, so their Table 2 also shows that the mean dates estimated by inclusion is a valuable addition to the literature. Christin et al. (2008) are generally slightly younger than Importantly, Sartidia is strongly supported as sister ours. Christin et al. (2008) constrained node D by to Stipagrostis, which leads to an estimate of two

r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, 14, 2963–2977 ora compilation Journal r Table 2 Dates of origins of C4 under different calibration schemes (this paper) compared with origins hypothesized by Christin et al. (2008) 08TeAuthors The 2008

Node Label Clade Pollen L55 Pollen L70 Spikelet Phytolith Coprolite Gondwana Poll. Spike. Phyt.* Christin et al. (2008)

w A [2] Grass stem node – Pollen fossil 89.5 12.7 90.6 11.7 93.8 11 90 12.2 111.8 11.3 121.2 10.9 93.8 10.9 B [3] Grass crown node 66.1 11.3 67 11 70.9 9.3 66.7 11 91.9 9 104.4 8.8 70.9 9.2 C [5] Multiﬂowered spikelet fossil 61.6 10.7 62.5 10.5 66.3 8.7 62.2 10.3 86.5 8.4 98.8 8.2 66.2 8.6

r D [6] Puelioideae-REST 52 9.7 52.6 9.6 55.7 8.4 52.6 9.4 77.1 6.4 89.8 6 55.6 8.3

08BakelPbihn Ltd, Publishing Blackwell 2008 E [7] BEP–PACCMAD divergence – NA phytoliths 48.4 9.1 48.9 9 51.8 849 8.8 72.7 5.5 85.2 5 51.6 7.9 and Coprolite fossil F [22]z Aristida – PACCMAD crown node 41.6 8.3 42.1 8.3 44.5 7.6 42 862.4 6.6 73.1 6.9 44.4 7.5 G [25]z Sporobolus 1 Eragrostis – Chloridoid fossil 35.3 7.3 35.6 7.2 37.7 6.7 35.6 752.6 6.4 61.6 6.9 37.6 6.6 H [97] Sporobolus 1 Eragrostis 21 5.8 21.2 5.9 22.4 5.8 21.3 5.7 31.4 6.8 36.8 8 22.5 5.7 25.0 4.0 I [26]z Danthoniopsis-Zea clade 32.2 6.7 32.5 6.7 34.4 6.3 32.5 6.6 47.9 6.2 56.1 6.8 34.3 6.2 z J [27] Danthoniopsis-Zea clade – Earliest unambiguous C4 29.8 6.3 30.1 6.3 31.8 5.9 30.1 6.2 44.3 6.1 51.8 6.7 31.7 5.9 K [28] Gynerium 27.6 5.9 27.8 5.9 29.4 5.5 27.9 5.8 40.9 5.7 47.9 6.4 29.3 5.5 L [30] Paniceae. X 5 10 22.9 5 23.1 5.1 24.4 4.8 23.1 533.9 5.3 39.7 5.9 24.3 4.9 21.9 3.9 LSEE RGN FC OF ORIGINS CLUSTERED M [47] Paniceae. X 5 10 17.4 4.2 17.6 4.2 18.6 4.1 17.6 4.1 25.9 4.9 30.3 5.4 18.5 4.1 O [48] Tatianyx-Altoparadisium clade 15.9 3.9 16.1 3.9 17 3.8 16.1 3.9 23.6 4.5 27.7 5.1 16.9 3.8 12.3 3.2

lblCag Biology Change Global P[49] Tatianyx-Altoparadisium clade 14 3.6 14.1 3.6 14.9 3.5 14.1 3.5 20.8 4.2 24.3 4.8 14.8 3.5 11.3 3.0 Q [53] Homolepis 6.8 2.3 6.9 2.4 7.3 2.4 6.9 2.3 10 3.1 11.7 3.6 7.2 2.4 R[55] Panicum prionitis 10.6 2.9 10.7 3 11.3 310.6 2.9 15.7 3.7 18.3 4.2 11.2 2.9 10.4 2.9 S [58]z Paspalum 11 3.1 11.1 3.1 11.7 3.2 11.1 3.1 16.3 3.9 19 4.5 11.7 3.1 14.1 3.4 N [59]z Anthaenantia§ 1 Axonopus clade 16.5 4 16.6 4 17.5 416.6 424.4 4.7 28.6 5.3 17.4 4 14.3 3.5 T [61] Echinolaena 13 3.6 13.1 3.6 13.8 3.6 13 3.5 19.3 4.4 22.5 5.1 13.7 3.5 U [66] Steinchisma hians 3.6 1.8 3.6 1.8 3.8 1.9 3.6 1.8 5.3 2.5 6.2 2.9 3.8 1.8

, V [67] C3-grade within Paniceae. X 5 9 19.4 4.5 19.6 4.6 20.6 4.4 19.6 4.5 28.7 5.1 33.7 5.8 20.6 4.5 13.8 3.5 14 W [68] C -grade within the Paniceae. X 5 9–Dichanthelium fossil 16.7 4 16.8 4 17.7 416.8 424.6 4.7 28.8 5.4 17.8 4 18.5 3.7

2963–2977 , 3 X [69] Multiphoto-pathways clade in Paniceae. X 5 916 3.8 16.1 3.9 17 3.8 16.1 3.8 23.5 4.5 27.5 5.2 17 3.9 16.4 3.6 Y [70] Multiphoto-pathways clade in Paniceae. X 5 9 14.9 3.6 15 3.7 15.8 3.6 15.1 3.6 21.9 4.3 25.7 5 15.9 3.7 21.2 3.9

Z[71] Panicum s.s. (Panicum C4NAD) 12.3 3.1 12.5 3.2 13.1 3.1 12.5 3.1 18.1 3.9 21.2 4.5 13.1 3.2

a[72] Panicum s.s. (Panicum C4NAD) 6.6 2.1 6.7 2.1 7 2.1 6.7 2.1 9.7 2.7 11.4 3.2 7.1 2.2 b [75] Urochloa (PCK clade) – Setaria fossil 11.1 2.9 11.3 2.9 11.8 2.9 11.3 2.9 16.3 3.6 19.1 4.2 11.8 3 4 PHOTOSYNTHESIS c [82] Urochloa (PCK clade) 7.1 2 7.2 2.1 7.6 2.1 7.2 2.1 10.4 2.7 12.3 3.2 7.6 2.2 Echinochloa 2.5 1.2 2.5 1.3 2.6 1.3 2.5 1.3 3.6 1.8 4.3 2.1 2.6 1.3 4.4 2.8 Eriachne stem 11.5 3.6 Eriachne crown 6.6 2.8 Stipagrostis stem 15.1 4.6 Stipagrostis crown 7.5 3.1 Centropodia stem 22.0 4.6 Neurachne munroi 4.4 3.3 Alloteropsis semialata 15.3 3.5

*Boldface column indicates our best estimate of dates based on data presented here. w

Numbers are in millions of years, plus or minus standard deviations. 2971 zIndicates nodes in which trees in the present paper differ from the tree in Christin et al. (2008). §Anthaenantia lanata is the currently accepted name for the taxon called Leptocoryphium lanatum by Christin et al. (2008). 2972 A. VICENTINI et al.

(a) PollenL55 (e) PollenL55

(b) Poll.Spike.Phyt (f) Poll.Spike.Phyt

(d) Gondwana (h) Gondwana

r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, 14, 2963–2977 CLUSTERED ORIGINS OF C4 PHOTOSYNTHESIS 2973 independent origins of C4 in the subfamily, at or after clusters, in that the origins of C4 in Eriachne, Neurachne, the crown node. Because of the position of Aristidoi- and Alloteropsis – not included here because of lack of deae in the PACCMAD clade, this also affects the phyB sequences – all fall within the time intervals estimate of the earliest origin of C4. The only caveat is identified for changes in other lineages. that the analytical method (parsimony) that Christin We found significant clustering with all the et al. (2008) used for inferring independent origins can calibrations, indicating that the result was insensitive make the timing of changes appear more precise than is to absolute dates. We undertook an additional set of warranted by the data (Pagel, 1999), and thus it would simulations to test whether the result was sensitive to be of interest to re-analyze their dataset in a maximum our use of the mean estimated date for each change, likelihood framework. Interestingly, the recent phylo- rather than a sample within the range. For these genetic analysis of Bouchenak-Khelladi et al. (2008) simulations, rather than using the mean estimated date, suggests that Aristidoideae might have originated we sampled a random date within the range of possible somewhat later than estimated by either Christin et al. dates for that transition event (Fig. 3a–d). We then (2008) or the present study. tested whether we still observed clusters of events. As The coprolite fossil has not been considered pre- we expected, the signal of clustering was not strong, viously as a possible calibration for the grass phylogeny, given the broad range of possible dates sampled, but we beyond its initial description in 2005 (Prasad et al., still found an excess of changes in the Mid- to Late- 2005), and was not included by Christin et al. (2008). Miocene in a number of the simulations. However, by This fossil, which underlies both the Gondwana and the choosing dates from within the confidence intervals for

Coprolite calibrations presented here, places C4 origins each node independently, we effectively destroyed the in the Eocene or possibly as early as the Paleocene, correlations between dates imposed by the tree. For when global CO2 levels were far higher than those in example, if we randomly sampled 13.4 Ma for node N which C4 is predicted to be favored. These dates contra- (the mean minus one standard deviation) and 17.2 Ma dict the expectation, based on the well-characterized for node T (the mean plus one standard deviation), it energetics of the pathway, that C4 is only favored at CO2 would violate the relative positions of those nodes in levels closer to 500 ppm. the phylogeny, which requires that node N must be We did not attempt to incorporate the putative bam- earlier than node T; in other words, the dates cannot busoid fossil Programinis burmitis, from the Early Cre- vary independently of each other. We found taceous of Burma (Poinar, 2004). The identity of the it remarkable that any signiﬁcant clustering appeared fossil is not certain, nor is the date. If the fossil is indeed at all, given this extensive disruption of the signal in a multiﬂowered grass spikelet, then node C would be the data.

Early Cretaceous, with resulting dates older than those The distinction between clusters of C4–C3 transitions from the Coprolite calibration. We also tested 82 Ma as and those of C3–C4 suggests an effect of cycles of the upper bound for node B, as suggested in Bremer climate change, because global pCO2 has remained (2002), but observed little effect. relatively low since the Early Miocene or earlier (Zachos et al., 2001). Both regional temperature and seasonal

distribution of precipitation affect the distribution of C4 Clustered C4 origins grasses (Hattersley, 1992; Sage et al., 1999b; Huang et al., The clustering of changes in photosynthetic pathway is 2001; Osborne, 2008), and regional climates are con- one of the striking results of this study, and points to nected in a complex way to global temperatures, ocean periods of earth history in which the selective regime circulation patterns, and polar glaciation (Pagani et al., changed to favor one pathway over the other. The work 1999; Vlastelic et al., 2005; Meehl et al., 2007). C4 origins of Christin et al. (2008) supports the identiﬁcation of in times of global cooling, such as that in the Early

Fig. 3 (a–d) Time of change events in photosynthetic pathway for four calibration schemes, Pollen55–70, Pollen.Spike.Phyt., Coprolite, and Gondwana. The maximum age of autapomorphic events is indicated by an arrow with length equal mean SD. Non autapomorphic events are indicated by a box with length equal to the range between maxima and minima age estimates; the line indicating the standard deviations. (e–h) Results of a simulation testing whether the rate of transitions from C3–C4 and from C4–C3 was signiﬁcantly higher for the observed dataset than for a model in which the rate of transition was constant, at different points in geological time (x) and for a range of time window sizes (y). Signiﬁcant departures (at Po0.05) from constant rate of transitions are indicated in red for C3–C4 transitions and in blue for C4–C3 transitions. The color gradient indicates the number of events observed in each time window (light gray area indicates searched space but in which the null model was not rejected). pCO2 mean levels for the Miocene indicated are from Pagani et al. (2005), and the @18O data are from Zachos et al. (2001). Note that the axes of the two graphs for each calibration scheme run in opposite directions. r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, 14, 2963–2977 2974 A. VICENTINI et al.

Oligocene, are consistent with the hypothesis that The C4 clades originating in the Mid-Miocene are the glacial conditions correlate with increased seasonality Tatianyx-Altoparadisium and the Anthaenantia-Axonopus at low latitudes (Pagani et al., 1999). However, we clades of the x 5 10 Paniceae, Alloteropsis semialata, and also find C4 origins in relatively warm periods such the large speciose (ca. 900 species) clade of the as the MMCO (17–15 Ma; Zachos et al., 2001). This x 5 9 Paniceae that includes Digitaria, Panicum sensu observation fits other climatic models (Lunt et al., stricto, the clade exhibiting the PCK subtype of C4 2007) correlating high global temperature with conti- photosynthesis and the ‘bristle clade’ that includes nental aridity. foxtail and pearl millet. The origin and subsequent diversification of these clades in the Mid-Miocene is consistent with the recent observation by Osborne (2008) that current abundance of Paniceae species is (a) PollenL55 correlated with moist, aseasonal conditions. The relationship among global temperature, regional hydrology, and seasonality are poorly understood for the Oligocene and Early to Middle Miocene. Ku¨ rschner et al. (2008), using stomatal indices of fossil trees,

estimate broad ﬂuctuations of pCO2 levels and global mean temperature within ranges that could affect the selective value of different photosynthetic pathways, although the range of variation they ﬁnd is more extreme than in other studies. Additional data on (b) Poll.Spike.Phyt paleoclimate will undoubtedly shed light on the origin

of C4 clades.

Age of major grass lineages Estimated dates for all the nodes under the various calibration schemes are listed in Table S2 and Fig. S3. For many of the calibration schemes, the standard deviations overlap, indicating that the various calibrations converge on similar estimates for the age of particular events. (c) Coprolite Our analysis provides the most robust estimate to date for the origin of the grass family, and for the common ancestor of the BEP and PACCMAD clades (Table 2). We estimate that the stem node of the grasses occurred at 93.8 10.9 Ma, somewhat older than the estimates provided by Gaut (2002) (77 Ma) and by Bremer (2002) (82 Ma). This most likely reﬂects the broader sampling in the present study as well as the use of more fossil calibration points. We place the

(d) Gondwana

Fig. 4 (a–d) Timing of changes in photosynthetic pathway and 18 variation in levels of pCO2 (Pagani et al., 2005) and @ O (Zachos et al., 2001). Each photosynthetic change is represented by a dot at the mean age for the event and at the mean @18O value for the time period in which the change has occurred (see Fig. S3A–C for error estimates on dates). Vertical bars indicate time periods

showing signiﬁcantly higher transition rates, with C3–C4 in red

and C4–C3 in blue; the width of the bar indicates the minimum time window that includes those events and departs signiﬁ- cantly from a constant rate of transitions (see also Fig. 3e–h). Climatic events indicated are from Zachos et al. (2001).

r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, 14, 2963–2977 CLUSTERED ORIGINS OF C4 PHOTOSYNTHESIS 2975 common ancestor of the BEP and PACCMAD clades at References 51.6 7.9 Ma, approximately the same as the dates Aliscioni SS, Giussani LM, Zuloaga FO, Kellogg EA (2003) A suggested by Gaut (2002) (50 Ma) and by Bremer molecular phylogeny of Panicum (Poaceae: Paniceae): test (2002) (55 Ma). This ancestor is also the common of monophyly and phylogenetic placement within the ancestor of maize and rice, so serves to calibrate Panicoideae. American Journal of Botany, 90, 796–821. divergence of those two genomes. Berner RA (2004) Atmospheric carbon dioxide over Phanerozoic In contrast to all the other fossil calibrations, the time. In: The Phanerozoic Carbon Cycle: CO2 and O2 (ed. Berner Gondwana and Coprolite calibrations suggest that the RA), pp. 72–99. Oxford University Press, Oxford, UK. stem node of the grass family occurred around Bouchenak-Khelladi Y, Salamin N, Savolainen V, Forest F, 111–120 Ma, appreciably earlier than the oldest putative vanderBank M, Chase MW, Hodkinson TR (2008) Large multi-gene phylogenetic trees of the grasses (Poaceae): grass pollen fossil (70 Ma). These dates are compar- progress towards complete tribal and generic taxon sampling. able with those postulated for the base of the monocots Molecular Phylogenetics and Evolution, 47, 488–505. (Janssen & Bremer, 2004). Thus, if either of these two Bremer K (2002) Gondwanan evolution of the grass alliance of calibrations is correct, then most other molecular clock families (Poales). Evolution, 56, 1374–1387. dates for monocots need to be revised. Alternatively, the Cerling TE (1991) Carbon dioxide in the atmosphere: evidence coprolite fossil itself may need to be re-evaluated. from Cenozoic and Mesozoic paleosols. American Journal of Science, 291, 377–400. Cerling TE, Harris JM, MacFadden BJ, Leakey MG, Quade J, Wisenmann V, Ehleringer JR (1997) Global vegetation change Conclusions through the Miocene/Pliocene boundary. Nature, 389, 153–158. Christin P-A, Besnard G, Samaritani E, Duvall MR, Hodkinson

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Sa´nchez-Ken JG, Clark LG, Kellogg EA, Kay EE (2007) Reinstate- ment and emendation of subfamily Micrairoideae. Systematic Supporting Information Botany, 32, 71–80. Additional Supporting Information may be found in the on- Sheehan MJ, Farmer PR, Brutnell TP (2004) Structure and line version of this article: expression of maize phytochrome family homeologs. Genetics, 167, 1395–1405. Fig. S1: Consensus tree from independent Bayesian analyses Sinha NR, Kellogg EA (1996) Parallelism and diversity in multi- of the combined ndhf and phyB datasets with reconstruction ple origins of C photosynthesis in the grass family. American 4 of the pattern of photosynthesis type evolution. Numbers at Journal of Botany, 83, 1458–1470. nodes are posterior probabilities. This tree contains 15 more Stro¨mberg CAE (2005) Decoupled taxonomic radiation and species than shown in Fig. 1; four are C species of the BEP ecological expansion of open-habitat grasses in the Cenozoic 3 clade, and the remainder are C species in Andropogoneae of North America. Proceedings of the National Academy of 4 or the multi-photosynthetic pathways clade. Their inclu- Sciences USA, 102, 11980–11984. sion/exclusion has no effect on optimization of C origins Thomasson JR (1978) Observations on the characteristics of the 4 on the tree. lemma and palea of the late Cenozoic grass Panicum elegans. Fig. S2: Chronograms for the four calibration schemes, Pollen, American Journal of Botany, 65, 34–39. Spikelet, Phytolith and Coprolite, with the respective re- Thorne JL, Kishino H (2002) Divergence time estimation and rate construction of photosynthetic pathway evolution. A max- evolution within multilocus data sets. Systematic Biology, 51, imum parsimony (MP) reconstruction is shown for the 689–702. Pollen chronogram, and the maximum likelihood (ML) Tipple BJ, Pagani M (2007) The early origins of terrestrial C 4 reconstructions are shown for the other three chronograms photosynthesis. Annual Review of Earth and Planetary Science, ( see Fig.1 for the ML reconstruction for Pollen. Spike. 35, 435–461. Phyt.). Note that the pattern of photosynthesis type evolu- Vlastelic I, Carpentier M, Lewin E (2005) Miocene climate change tion is basically the same in all schemes and the MP and ML recorded in the chemical and isotopic (Pb, Nd, Hf) signature of reconstructions indicate transitions at the same nodes. Southern Ocean sediments. Geochemistry, Geophysics, and Geo- Fig. S3: The same tree as Fig. 1, but with nodes numbered to systems, 6, Q03003, doi: 10.1029/2004GC000819. correspond to estimated dates presented in Table S2. Wang Y, Cerling TE, MacFadden BJ (1994) Fossil horses and Table S1: Taxa and GenBank numbers. carbon isotopes: new evidence for Cenozoic dietary, habitat, Table S2: Dates for all nodes in the grass phylogeny. and ecosystem changes in North America. Palaeogeography, Palaeoclimatology, Palaeoecology, 107, 269–279. Please note: Wiley-Blackwell are not responsible for the con- Zachos J, Pagani M, Sloan L, Thomas E, Billups K (2001) Trends, tent or functionality of any supporting materials supplied rhythms, and aberrations in global climate 65 Ma to present. by the authors. Any queries (other than missing material) Science, 292, 686–693. should be directed to the corresponding author for the article.

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