CurrBiol18- suppl data

Please cite this article in press as: Christin et al., Oligocene CO2 Decline Promoted C4 Photosynthesis in Grasses, Current Biology (2008), doi:10.1016/j.cub.2007.11.058 Supplemental Data Oligocene CO2 Decline Promoted C4 Photosynthesis in Grasses S1 Pascal-Antoine Christin, Guillaume Besnard, Emanuela Samaritani, Melvin R. Duvall, Trevor R. Hodkinson, Vincent Savolainen, and Nicolas Salamin Supplemental Experimental Procedures Taxon Sampling The GenBank database was screened for sequences of rbcL and ndhF, both plastid markers, which were downloaded when available for the same grass species. This data set was completed by sequencing 109 and 97 additional sequences from rbcL and ndhF, respectively (Table S1). A set of primers (Table S2) were used to amplify rbcL and ndhF in one and two overlapping fragments, respectively. The PCR reaction mixture contained w100 ng of genomic DNA template, 10 ml of 53 GoTaq Reaction Buffer, 0.15 mM dNTPs, 0.2 mM of each primer, 2 mM of MgCl2, and 1 unit of Taq polymerase (GoTaq DNA Polymerase, Promega, Madison, WI) in a total volume of 50 ml. The samples were incubated for 3 min at 94 C, followed by 35 cycles consisting of 1 min at 94 C, 30 s at the annealing temperature (Table 2), and 2 min at 72 C. The last cycle was followed by a 7 min extension at 72 C. PCR products were purified with QIAquick PCR Purification Kit (QIAGEN, GmbH, Germany) and sequenced with internal primers with the Big Dye 3.1 Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA), according to the provider instructions, and were separated on an ABI Prism 3100 genetic analyzer (Applied Biosystems). All sequences have been deposited in the EMBL database (accession numbers indicated in Table S1). Our sampling incorporated all described and supposed C4 lineages and, as far as possible, aimed to reduce the branch between the stem group node (divergence of the clade from its sister group) and crown group node (first divergence of two species from the same clade) of the different C4 lineages to give a more accurate representation of the branching events. Our final data set contained 187 grass species (Table S1). Finally, outside the grass family, 46 other commelinids species and four Asparagales species, for which both rbcL and ndhF were available in GenBank, were added (Table S1). Extending the sampling outside the grass family allowed the use of supplementary fossil calibration points and increased the time span considered in our study. Phylogenetic Analyses and Dating Sequences were translated into amino acids and aligned with Clustal W [S1]. Nucleotide alignment was then visually checked and manually edited. A phylogenetic tree was inferred from both markers simultaneously by Bayesian inference with MrBayes 3.1 [S2], under a GTR substitution model with base frequencies, gamma shape parameter, and proportion of invariant sites estimated from the data (GTR+I+G). Best-fit substitution models were determined with hierarchical likelihood ratio tests. Base frequencies, shape parameter, proportion of invariant sites, and substitution rates were optimized separately for each gene. Two different analyses, each consisting of four chains, were run for 10,000,000 generations. Trees were sampled at every 1,000 generations, and the burn-in period was set to 3,000,000 generations. A maximum-likelihood analysis was also performed with the RaxML software [S3] under the GTRMIX model. The topology obtained was then used for Bayesian molecular dating, as per the recommendations of [S4]. This procedure takes into account changes in rates of evolution with time. Model parameters were estimated with baseml [S5] for the two genes separately. The software estbranches [S6] was then used to optimize branch lengths and the variance-covariance matrix. Finally, the posterior distributions of substitution rates and divergence times were approximated with a Bayesian MCMC procedure implemented in the software multidivtime [S7, S8]. The MCMC was run for 1,000,000 generations after a burn-in period of 100,000. The sampling frequency was set to 100 generations. During the Bayesian molecular dating, the outgroup (in this case, the Asparagales) is removed because it is not possible to date the divergence between the ingroup and the outgroup of a phylogenetic tree. An accurate tree calibration requires several time boundaries to be set on several nodes. These constraints were chosen to be as conservative as possible. First, an upper bound for the root of the tree was set arbitrarily to 140 My, a date higher than a previous estimate (120 Mya; [S9]). Several fossil descriptions were used to constrain node ages. Unfortunately, fossils only give a lower bound (i.e., minimal age) because the first fossil could potentially have been found long after the emergence of the lineage. A fossil attributed to Distichlis and dated from 14 Mya was used to set the lower bound of the Distichlis-Tragus divergence node [S10]. The earliest well-described remains of grasses are the grass spikelets described by [S11]. These fossils from the Palaeocene-Eocene boundary have characteristics from the BEP (Bambusoideae, Ehrhartoideae, and Pooideae subfamilies) and PACMAD (Panicoideae, Arundinoideae, Chloridoideae, Micrairoideae, Aristidoideae, and Danthonioideae subfamilies) clades that were absent from the more outlying grass genera [S12]. This description was used to set a lower and an upper bound of 55 My and 60 My, respectively, to the stem node (divergence of the clade from its sister group) of the BEP-PACMAD clade. Based on the earliest report of Poaceae pollen [S13], a lower bound of 60 My was also set to the crown node (first divergence of two species from the same clade) of the Poaceae. Outside the grass family, a listing of stem group and crown group fossils for many plant families [S14] was used to set the lower bound of Bromeliaceae to 37.5 Mya, of the Arecaceae to 77.4 Mya, and the upper bound of Arecaceae, based on a stem group fossil, to 84 Mya. Supplemental References S1. Thompson, J.D., Higgins, D.J., and Gibson, T.J. (1994). Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and matrix choice. Nucleic Acids Res. 22, 4673–4680. S2. Ronquist, F., and Huelsenbeck, J.P. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. S3. Stamastakis, A. (2006). RaxML-VI-HPC: maximum likelihoodbased phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688–2690. S4. Rutschmann, F. (2006). Molecular dating of phylogenetic trees: a brief review of current methods that estimate divergence times. Divers. Distrib. 12, 35–48. S5. Yang, Z.H. (1997). PAML: a program package for phylogenetic analysis by maximum likelihood. Comput. Appl. Biosci. 13, 555–556. S6. Thorne, J.L., Kishino, H., and Painter, I.S. (1998). Estimating the rate of evolution of the rate of molecular evolution. Mol. Biol. Evol. 15, 1647–1657. S7. Kishino, H., Thorne, J.L., and Bruno, W.J. (2001). Performance of a divergence time estimation method under a probabilistic model of rate evolution. Mol. Biol. Evol. 18, 352–361. S8. Thorne, J.L., and Kishino, H. (2002). Divergence time and evolutionary rate estimation with multilocus data. Syst. Biol. 51, 689–702. S9. Jansen, T., and Bremer, K. (2004). The age of major monocot groups inferred from 800+ rbcL sequences. Bot. J. Linn. Soc. 146, 385–398. S10. Dugas, D.P., and Retallack, G.J. (1993). Middle Miocene fossil grasses from Fort Ternan, Kenya. J. Paleontol. 67, 113–128. S11. Crepet, W.L., and Feldman, G.D. (1991). The earliest remains of grasses in the fossil record. Am. J. Bot. 78, 1010–1014. Please cite this article in press as: Christin et al., Oligocene CO2 Decline Promoted C4 Photosynthesis in Grasses, Current Biology (2008), doi:10.1016/j.cub.2007.11.058 S2 Figure S1. Phylogenetic Tree Inferred from rbcL and ndhF Sequences Bayesian analysis was performed with MrBayes 3.1 [S2], under a GTR+I+G substitution model. Posterior support values of branches are indicated above the branches. Outside the grasses (Poaceae), families are compressed. Inside the grass family, subfamilies are compressed. Braces delimit orders. The tree was rooted with Asparagales species. S12. Bremer, K. (2002). Gondwana evolution of the grass alliance of families (Poales). Evolution Int. J. Org. Evolution 56, 1374– 1387. S13. Linder, H.P. (1987). The evolutionary history of the Poales/Restionales: a hypothesis. Kew Bull. 42, 297–318. S14. Magallon, S., and Sanderson, M.J. (2001). Absolute diversification rates in angiosperm clades. Evolution Int. J. Org. Evolution 55, 1762–1780. S15. GPWG-Grass Phylogeny Working Group (2001). Phylogeny and subfamilial classification of the grasses (Poaceae). Ann. Mo. Bot. Gard. 88, 373–457. S16. Duvall, M.R., Davis, J.I., Clark, L.G., Noll, J.D., Goldman, D.H., and Sánchez-Ken, J.G. (2007). Phylogeny of the grasses (Poaceae) revisited. Aliso 23, 237–247. S17. De Luna, E., Newton, A.E., Withey, A., Gonzalez, D., and Mishler, B.D. (1999). The transition to pleurocarpy: a phylogenetic Please cite this article in press as: Christin et al., Oligocene CO2 Decline Promoted C4 Photosynthesis in Grasses, Current Biology (2008), doi:10.1016/j.cub.2007.11.058 S3 (Continued) Please cite this article in press as: Christin et al., Oligocene CO2 Decline Promoted C4 Photosynthesis in Grasses, Current Biology (2008), doi:10.1016/j.cub.2007.11.058 S4 Please cite this article in press as: Christin et al., Oligocene CO2 Decline Promoted C4 Photosynthesis in Grasses, Current Biology (2008), doi:10.1016/j.cub.2007.11.058 S5 Figure S2. Phylogenetic Tree Inferred from rbcL and ndhF Sequences Bayesian analysis was performed with MrBayes 3.1 [S2], under a GTR+I+G substitution model. When greater than 50%, Bayesian support values are indicated above branches and support values obtained from 100 bootstrap replicates with RAxML [S3] are indicated below branches. (A) Species relationships inside the Panicoideae subfamily. (B) Species relationships inside the grass family. (C) Phylogenic relationships among commelinid species and four Asparagales species that were used to root the tree. analysis of the main diplolepidous lineages based on rbcL sequences and morphology. Bryologist 102, 634–650. S18. Olmstead, R.G., and Sweere, J.A. (1994). Combining data in phylogenetic systematics: an empirical approach using three molecular data sets in the Solanaceae. Syst. Biol. 43, 467–481. Table S1. Species List Taxon Voucher ndhF rbcL Photosynthetic Type AY147774 AY149360 C3 AJ535761 AF074229 C3 AF508368 Z69211 C3 AF508369 AJ311066 C3 AY044555 AY044625 C3 ASPARAGALES Asphodelaceae Asphodelus albus Miller Orchidaceae Spiranthes cernua (L.) L.C. Rich. Themidaceae Dichelostemma multiflorum (Benth.) Heller Dichelostemma volubile (Kellogg) Heller ARECALES Arecaceae Acrocomia aculeata (Jacq.) Lodd. ex Mart. (Continued) Please cite this article in press as: Christin et al., Oligocene CO2 Decline Promoted C4 Photosynthesis in Grasses, Current Biology (2008), doi:10.1016/j.cub.2007.11.058 S6 Table S1. Continued Taxon Aphandra natalia (Balslev & A.J. Hend.) Barfod Bentinckia nicobarica (Kurz) Becc. Howea belmoreana (C. Moore & F. Muell.) Becc. Livistona speciosa Kurz Mauritia flexuosa L. f. Phytelephas aequatorialis Spruce Prestoea acuminata (Willd.) H.E. Moore Ravenea hildebrandti H. Wendl. ex Bouche Roystonea oleracea (Jacq.) O.F. Cook Scheelea butyracea (Mutis ex L. f.) H. Karst. ex H. Wendl. Washingtonia filifera (L. Linden) H. Wendl. Voucher ndhF rbcL Photosynthetic Type AY044532 AY044621 C3 AF453468 AY012499 C3 AF453472 AY012492 C3 AY044528 AY044524 AY044533 AY012463 AY012473 AY012512 C3 C3 C3 AY044553 AY012487 C3 AY044544 AY012475 C3 AY044554 AJ404805 C3 AY044571 AY044636 C3 AY044527 AY012465 C3 AY624109 AY624127 AF312247 AF312250 C3 C3 AY624120 AF312240 C3 AY624124 AF312238 C3 AY624126 AF312245 C3 AY656083 AY656112 C3 AY656093 AY656121 C3 AY656098 AF378781 C3 AY656097 AY656124 C3 AY124995 AF243845 C3 AY465646 AM235157 C3 AY147766 L19977 C3 L75905 AY614422 C3 L75898 AY614500 C3 AY191213 AY465696 C3 U79230 AM110250 C3 AF191810 Y12997 C3 AF163472 AY725943 C3 COMMELINALES Commelinaceae Callisia repens (Jacq.) L. Gibasis geniculata (Jacq.) Rohw. Palisota ambigua (P. Beauv.) C.B.Clarke Tradescantia soconuscana Matuda Weldenia candida Schultes f. ZINGIBERALES Marantaceae Calathea crotalifera S. Wats. Ischnosiphon helenae Anderss. Pleiostachya pruinosa (Regel) K. Schum. Stachyphrynium jagorianum (K. Koch) K. Schum. Strelitziaceae Phenakospermum guyannense (Rich.) Endl. Strelitzia reginae Aiton POALES Bromeliaceae Ananas comosus (L.) Merr. Guzmania wittmackii (Andre) Andre ex Mez Tillandsia bergeri Mez Sparganiaceae Sparganium eurycarpum Engelm. Typhaceae Typha angustifolia L. Cyperaceae Carex echinochloe (C.B. Clarke) Kuk. Dulichium arundinaceum (L.) Britt. Please cite this article in press as: Christin et al., Oligocene CO2 Decline Promoted C4 Photosynthesis in Grasses, Current Biology (2008), doi:10.1016/j.cub.2007.11.058 S7 Table S1. Continued Taxon Gahnia deusta (R. Br.) Benth. Kobresia simpliciuscula (Wahlenb.) Mackenzie Centrolepidaceae Centrolepis monogyna (Hook. f.) Benth. Juncaceae Juncus effusus L. Thurniaceae Prionium serratum (Thunb.) Drege Mayacaceae Mayaca fluviatilis Aubl. Eriocaulaceae Eriocaulon aquaticum (Hill) Druce Syngonanthus flavidulus (Michx.) Ruhland Tonina fluviatilis Aubl. Xyridaceae Orectanthe sceptrum (Oliver) Maguire Xyris jupicai L.C. Rich. Restionaceae Baloskion tetraphyllum (Labill.) B.G. Briggs & L.A.S. Johnson Elegia fenestrata Pillans Flagellariaceae Flagellaria indica L. Ecdeiocoleaceae Ecdeiocolea monostachya F. Muell. Joinvilleaceae Joinvillea ascendens Gaud. ex Brongn. & Gris Joinvillea plicata (Hook. f.) Newell & B.C. Stone Poaceae Early Diverging Taxa Anomochloa marantoidea Brongn. Guaduella marantifolia Franch. Streptochaeta sodiroana Hack. Pharus latifolius L. Bambusoideae Arundinaria sp. Arundinaria gigantea (Walter) Muhl. Buergersiochloa bambusoides Pilger Chimonobambusa marmorea (Mitford) Makino Chusquea circinata Soderstr. & C.E. Calderon Guadua sp. Lithachne humilis Soderstr. Olyra sp. Otatea acuminata (Munro) C.E. Calderon & Soderstr. Voucher ndhF rbcL Photosynthetic Type AY129253 U49231 C3 AF163462 U49232 C3 EF153941 EF153951 C3 AF547015 AY216612 C3 AF547019 U49223 C3 DQ058414 AJ419948 C3 DQ188990 AF036877 C3 DQ188989 AY298850 C3 DQ188993 AY123237 C3 AY438616 AY123241 C3 AF547021 AY465698 C3 AF251444 AF148761 C3 AF547016 AY123238 C3 U22008 L12678 C3 AY622313 AJ286559 C3 U21973 NA C3 NA L01471 C3 U21992 AF021875 C3 AF164777 AJ746291 C3 AY622318 AM887878 C3 U21993 AY357724 C3 AM849130 U21846 AM849366 AY618660 C3 C3 AF182341 AY632362 C3 U21969 AJ746176 C3 U21991 U13227 C3 T.R. Hodkinson, s.n. (TCD) AM849156 U21977 AM849367 U13231 C3 C3 T.R. Hodkinson, s.n. (TCD) AM849172 AF182350 AM849369 AJ746271 C3 C3 T.R. Hodkinson 575 (TCD) T.R. Hodkinson, s.n. (TCD) (Continued) Please cite this article in press as: Christin et al., Oligocene CO2 Decline Promoted C4 Photosynthesis in Grasses, Current Biology (2008), doi:10.1016/j.cub.2007.11.058 S8 Table S1. Continued Taxon Pariana radiciflora Sagot ex Doell. Phyllostachys sp. Streptogyna americana C.E. Hubb. Ehrhartoideae Ehrharta erecta Lam. Microlaena stipoides (Labill.) R. Br. Oryza nivara S.D.Sharma et Shastry Oryza sativa L. Zizania latifolia (Griseb.) Turcz. ex Stapf Pooideae Agrostis stolonifera L. Aira caryophyllea L. Ampelodesmos mauritanica (Poir.) T. Durand & Schinz Arrhenatherum elatius (L.) P. Beauv. Avena sativa L. Brachypodium cf. pinnatum (L.) P. Beauv. Brachypodium pinnatum (L.) P. Beauv. Bromus hordeaceus L. Dactylis glomerata L. Elymus farctus (Viv.) Runemark ex Melderis Glyceria grandis S.Watson Holcus lanatus L. Hordeum vulgare L. Lygeum cf. spartum L. Melica cupanii Guss. Melica uniflora Retz. Nardus sp. Phaenosperma globosa Murno ex Oliver Stipa sp. Triticum aestivum L. Aristidoideae Aristida adscensionis L. Aristida purpurea Nutt. var. longiseta (Steud.) Vasey Aristida rhiniochloa Hochst. Sartidia jucunda (Schweick.) De Winter Stipagrostis acutiflora (Trin. & Rupr.) De Winter Stipagrostis pennata (Trin.) De Winter Stipagrostis plumosa (L.) Munro ex T. Anders. Stipagrostis zeyheri (Nees) De Winter Arundinoideae Amphipogon strictus R. Br. Arundo donax L. Cyperochloa hirsuta Lazarides & L.Watson Gynerium sagittatum (Aubl.) Beauv. Molinia caerulea (L.) Moench Voucher ndhF rbcL Photosynthetic Type AF182354 AY632369 C3 T.R. Hodkinson, s.n. (TCD) AM849182 U21965 AM849380 AY618663 C3 C3 G. Besnard 04-2007 (G) G. Besnard 03-2006 (G) AM887887 AM849152 AM887883 AM849377 C3 C3 AP006728 AP006728 C3 AY522329 AM887888 AY522329 AM887884 C3 C3 G. Besnard 69-2006 (G) NC_008591 AM849198 AF251448 NC_008591 AM849361 AJ746172 C3 C3 C3 P.A. Christin 07-2005 (G) AM849127 AY395529 C3 G. Besnard 24-2004 (G) U22000 AM849133 L15300 AM849347 C3 C3 AY622312 AY632361 C3 AM849134 AM849145 AM849122 AY395531 AY395535 AM849350 C3 C3 C3 AY622314 AY632364 C3 AM849157 U22003 AM849164 AY622315 AM849165 AM849168 U22006 AJ746279 AY137456 AM849395 AY632365 AJ746294 AM849379 AY632370 C3 C3 C3 C3 C3 C3 C3 T.R. Hodkinson, s.n. (TCD) AM849190 AB042240 AM849372 AB042240 C3 C3 T. Renaud 01-2005 (G) AM849125 U21966 AM849349 NA C4 C4 T. Renaud et al., 12-2003 (G) G.L. Rossouw 216 (PRE) AM849126 AM849346 C4 AM849216 AM849419 C3 F. Anthelme 02-2006 AM849191 AM849357 C4 - AM887886 AM887885 C4 F. Anthelme 03-2006 AM849210 AM849416 C4 AF251455 U31378 C4 AF251456 U88403 C3 U21998 AY847139 U13226 U88404 C3 C3 AF251466 U31105 C3 U21995 AY632367 C3 - G. Besnard 68-2006 (G) P.A. Christin 08-2005 (G) P.A. Christin 06-2005 (G) G. Besnard 05-2002 (G) T.R. Hodkinson, s.n. (TCD) G. Besnard 03-2003 (G) T.R. Hodkinson, s.n. (TCD) Please cite this article in press as: Christin et al., Oligocene CO2 Decline Promoted C4 Photosynthesis in Grasses, Current Biology (2008), doi:10.1016/j.cub.2007.11.058 S9 Table S1. Continued Taxon Phragmites australis (Cav.) Trin. ex Steud. Chloridoideae Calamovilfa sp. Centropodia forskalii (Vahl) Cope Centropodia glauca (Nees) Cope Centropodia mossamedensis (Rendle) Cope Chloris gayana Kunth Crypsis schoenoides (L.) Lam. Cynodon dactylon (L.) Pers. Dactyloctenium aegyptium (L.) P. Beauv. Distichlis spicata (L.) Greene Eleusine indica (L.) Gaertn. Enteropogon prieurii (Kunth) Clayton Eragrostis capensis (Thunb.) Trin. Eragrostis minor Host Lepturus repens (G. Forst.) R. Br. Spartina anglica C.E. Hubb. Spartina pectinata Bosc ex Link Sporobolus festivus Hochst. ex A. Rich. Tetrapogon cenchriformis (A. Rich.) Clayton Tragus racemosus (L.) All. Tripogon minimus (A. Rich.) Hochst. ex Steud. Uniola paniculata L. Zoysia japonica Steud. Danthonioideae Austrodanthonia laevis (Vickery) H.P. Linder Cortaderia richardii (Endl.) Zotov Karroochloa purpurea (L. f.) Conert & Turpe Merxmuellera disticha (Nees) Conert Merxmuellera macowanii (Stapf) Conert Merxmuellera rangei (Pilg.) Conert Merxmuellera stricta (Schrad.) Conert Micrairoideae Eriachne festucacea F. Muell. Eriachne triseta Nees ex Steud. Isachne arundinacea (Sw.) Griseb. Isachne mauritiana Kunth Voucher ndhF rbcL Photosynthetic Type U21997 U29900 C3 AM849135 AM849137 AM849352 AM849365 C4 C4 AF251462 U31100 C4 NA DQ887125 C4 G. Besnard 04-2006 (G) Chase 9264 (K) AM849205 AM849140 AM849409 AM887879 C4 C4 G. Besnard 70-2006 (G) AM849142 AM849393 C4 T. Renaud 05-2005 (G) AM849144 AM887877 C4 AF251464 AY632363 C4 - AM849151 AM887880 C4 T. Renaud 11-2005 (G) AM849153 AM849397 C4 G. Besnard 59-2006 (G) AM849209 U31104 C4 G. Besnard 01-2003 (G) T.R. Hodkinson 272 (TCD) AM849154 AM849163 AM849338 AM887876 C4 C4 T.R. Hodkinson 153 (TCD) AM849186 AM849382 C4 AF251465 AJ784821 C4 T. Renaud et al., 192-2003 (G) T. Renaud et al., 224-2003 (G) AM849188 AM849383 C4 AM849192 AM849396 C4 Chase 19533 (K) AM849193 AM887881 C4 T. Renaud 08-2005 (G) AM849194 AM849342 C4 Chase 9278 (K) AF251463 AM849197 AY632373 AM849385 C4 C4 AF251460 DQ887097 C3 AM849139 AJ784830 C3 AF251458 U31437 C3 V.R. Clark et al., 355 (G) AM849206 AM849413 C3 Chase 9256 (K) AM849184 U31438 C3 AF251461 AY640153 C3 AM849207 AM849414 C3 DQ897931 NA C4 S. Jacobs 8795 (NSW) AM849155 AY626351 C4 G. Sanchez-Ken 680 (ISC) AY847119 AM849337 C3 G. Besnard 09-2006 (G) AM849162 AM849388 C3 T.R. Hodkinson, s.n. (TCD) F. Anthelme 01-2006 Chase 22022 (K) V.R. Clark & G. Coombs 270 (G) (Continued) Please cite this article in press as: Christin et al., Oligocene CO2 Decline Promoted C4 Photosynthesis in Grasses, Current Biology (2008), doi:10.1016/j.cub.2007.11.058 S10 Table S1. Continued Taxon Voucher ndhF rbcL Photosynthetic Type Micraira spiciforma M. Lazarides Micraira subulifolia F. Muell. Panicoideae Centotheceae Centotheca lappacea (L.) Desv. Chasmanthium latifolium (Michx.) Yates Danthoniopsis petiolata (J. Phipps) W. Clayton Loudetia simplex (Nees) C.E. Hubb. Orthoclada laxa (Rich.) P. Beauv. Thysanolaena latifolia (Roxb. ex Hornem.) Honda Thysanolaena maxima (Roxb.) Kuntze Tristachya leucothrix Nees Zeugites pittieri Hack. Andropogoneae Andropogon gerardii Vitman Arthraxon cf. lanceolatus (Roxb.) Hochst. Arundinella deppeana Nees ex Steud. Arundinella hirta (Thunb.) C. Tanaka Bothriochloa ischaemum (L.) Keng Bothriochloa saccharoides (Sw.) Rydb. Capillipedium parviflorum (R. Br.) Stapf Coix lacryma-jobi L. Cymbopogon citratus (D.C. ex Nees) Stapf Eulalia aurea (Bory) Kunth Hyparrhenia hirta (L.) Stapf Imperata cylindrica (L.) Raeusch. Ischaemum koleosatchys (Steud.) Hack. Microstegium sp. Pogonatherum paniceum (Lam.) Hack. Saccharum officinarum L. Sorghum bicolor (L.) Moench Vetiveria zizanioides (L.) Nash ex Small Zea mays L. Paniceae Acroceras tonkinense (Balansa) C.E. Hubb. ex Bor Alloteropsis semialata (R. Br.) A. Hitchc. S. Jacobs 8850 (NSW) AM849166 AM849378 C3 AY622316 AY632366 C3 T.R. Hodkinson 235 (TCD) AY847122 AM887875 C3 Chase 19162 (K) AY029694+AM849138 U31101 C3 U21988 AY618661 C4 AY847117 NA C4 T.R. Hodkinson 522 (TCD) AY847128 AM887874 C3 G. Besnard 58-2006 (G) AM849202 AM849406 C3 U21984 U31380 C3 AM849195 AM849384 C4 U21987 AY632374 C3 AF117391 AJ784818 C4 G. Besnard 67-2006 (G) AM849128 AJ784819 C4 G. Sanchez-Ken 676 (ISC) AM849214 AM849417 C4 AF117393 NA C4 G. Besnard 01-2006 (G) AM849131 AM887873 C4 G. Besnard 66-2006 (G) AM849146 AM849353 C4 G. Besnard 03-2004 (G) AM849136 AM849398 C4 G. Besnard 72-2006 (G) T.R. Hodkinson 129 (TCD) AF117403 AM849141 AM887882 AM887872 C4 C4 G. Besnard 16-2006 (G) AM849213 AM849410 C4 P.A. Christin 03-2005 (G) AM849124 AM849351 C4 Marsden 3 AM849161 AJ784826 C4 G. Besnard 05-2006 (G) AM849211 AM849411 C4 G. Besnard 20-2004 (G) P. Rondeau 03-2005 (G) AM849167 AM849183 AM849387 AM849381 C4 C4 AP006714 AP006714 C4 G. Besnard 10-2007 (G) U21981 AM849341 C4 P. Rondeau 02-2005 (G) AM849196 AM849394 C4 X86563 X86563 C4 A.T. & S.S. 630 (TCD) AM849121 AM849376 C3 A.T. & S.S. 837 (TCD) AM849123 AM849345 C4 H. Beck 5018 Please cite this article in press as: Christin et al., Oligocene CO2 Decline Promoted C4 Photosynthesis in Grasses, Current Biology (2008), doi:10.1016/j.cub.2007.11.058 S11 Table S1. Continued Taxon Altoparadisium chapadense Filg., Davidse, Zuloaga & Morrone Anthaenantiopsis rojasiana L. Parodi Arthropogon lanceolatus Filg. Arthropogon villosus Nees Axonopus anceps (Mez) A. Hitchc. Brachiaria deflexa (Schumach.) C.E. Hubb. ex Robyns Brachiaria villosa (Lam.) A. Camus Cenchrus echinatus L. Cenchrus setiger Vahl Cyrtococcum patens (L.) A. Camus Digitaria cf. ciliaris (Retz.) Koeler Digitaria didactyla Willd. Digitaria sanguinalis (L.) Scop. Echinochloa colona (L.) Link Echinochloa crus-galli (L.) P. Beauv. Echinolaena inflexa (Poir.) Chase Homolepis glutinosa (Sw.) Zuloaga & Soderstr. Homolepis isocalycia (G. Meyer) Chase Hymenachne amplexicaulis (Rudge) Nees Ichnanthus panicoides P. Beauv. Ichnanthus sp. Ichnanthus vicinus (Bailey) Merr. Leptocoryphium lanatum (Kunth) Nees Melinis minutiflora P. Beauv. Mesosetum chaseae Luces Neurachne alopecuroides R. Br. Neurachne munroi (F. Muell.) F. Muell. Neurachne tenuifolia S.T .Blake Ophiochloa hydrolithica Filg., Davidse & Zuloaga Oplismenopsis najada (Hackel & Arechav.) L. Parodi Oplismenus compositus (L.) P. Beauv. Oplismenus undulatifolius (Ard.) Roem. & Schult. Otachyrium versicolor (Döll) Henrard Voucher ndhF rbcL Photosynthetic Type Filgueiras & Fonseca 2987 (SI) AY029619 AM849402 C4 AY029620 NA C4 Filgueiras 3588 (SI) AY029621+AM849129 AM849348 C3 Filgueiras, s.n. (SI) AY029622 AM849401 C4 AY029623 NA C4 G. Besnard 47-2006 (G) AM849200 AM849407 C4 T. Renaud 09-2005 (G) AM849132 AM849375 C4 G. Besnard 15-2004 (G) G. Besnard 10-2004 (G) AF499151 AF499153 AM849143 AM849359 L14622 AM849331 C4 C4 C3 T. Renaud 10-2005 (G) AM849148 AM849336 C4 G. Besnard 28-2006 (G) G. Besnard 01-2005 (G) AM849203 AM849147 AM849404 AJ746264 C4 C4 AY029631 NA C4 G. Besnard 04-2001 (G) AM849149 AM887871 C4 Zuloaga 6943 (SI) AY029633 AM849335 C3 AY029637 NA C3 Zuloaga 6938 (SI) AY029636 AM849403 C3 A.T. & S.S. 635 (TCD) AM849158 AM849392 C3 M. Gonzalez 03-2005 (G) AM849159 AM849362 C3 T.R. Hodkinson, s.n. (TCD) A.T. & S.S. 657 (TCD) AM849199 AM849160 AM849358 AM849354 C3 C3 AY029640 NA C4 AM849212 AM849412 C4 AY029641 NA C3 AM849215 AM849418 C3 NA X55828 C4 NA X55827 C3 AY029642 AM849400 C4 AY188453 NA C3 G. Besnard 15-2006 (G) AM849169 AM849368 C3 P.A. Christin 09-2005 (G) AM849171 AM849360 C3 AY029643 NA C3 G. Besnard 18-2006 (G) A. Strid 20373 (G) Filgueiras, s.n. (SI) (Continued) Please cite this article in press as: Christin et al., Oligocene CO2 Decline Promoted C4 Photosynthesis in Grasses, Current Biology (2008), doi:10.1016/j.cub.2007.11.058 S12 Table S1. Continued Taxon Ottochloa nodosa (Kunth) Dandy Panicum anceps Michx. Panicum capillare L. Panicum coloratum L. Panicum laetum Kunth Panicum laxum Sw. Panicum millegrana Poir. Panicum miliaceum L. Panicum ovuliferum Trin. Panicum pilosum Sw. Panicum prionitis Nees Panicum stoloniferum Poir. Panicum validum Mez. Panicum virgatum L. Paspalum cf. conjugatum Berg. Paspalum dilatatum Poir. Paspalum cf. nutans Lam. Paspalum quadrifarium Lam. Pennisetum glaucum (L.) R. Br. Pennisetum longissimum S.L. Chen & Y.X. Jin Poecilostachys bromoides Stapf Pseudechinolaena polystachya (Kunth) Stapf Sacciolepis indica (L.) Chase Setaria italica (L.) P. Beauv. Setaria palmifolia (J. König) Stapf Setaria plicata (Lam.) T. Cooke Spinifex littoreus (Burm. f.) Merrill Steinchisma hians (Elliott) Nash Stenotaphrum dimidiatum (L.) Brongen. Streptostachys asperifolia Desv. Streptostachys ramosa Zuloaga & Soderstr. Tatianyx arnacites (Trin.) Zuloaga & Soderstrom Urochloa maxima (Jacq.) Webster Zuloagaea bulbosa (Kunth) Bess. Voucher ndhF rbcL Photosynthetic Type A.T. & S.S. 646 (TCD) AM849173 AM849370 C3 P.A. Christin 10-2005 (G) G. Besnard 63-2006 (G) T. Renaud 06-2005 (G) Zuloaga 6756 (SI) Zuloaga 6766 (SI) AY188455 AM849174 AM849208 AM849175 AY029655 AM849176 NA AM849355 AM849415 AM849391 AM849373 AM849363 C4 C4 C4 C4 C3 C3 G. Besnard 65-2006 (G) Zuloaga 6803 (SI) AY188472 AM849177 AM849333 AM849364 C4 C3 M. Gonzalez 04-2005 (G) Zuloaga 6794 (SI) Zuloaga, s.n. (SI) AY188480 AY029652 AY188488 AM849330 AM849374 AM849421 C3 C4 C3 G. Besnard 25-2004 (G) AY188495 U21986 AM849180 NA AY632368 AM849399 C4 C4 C4 T.R. Hodkinson 128 (TCD) AM849178 AM849371 C4 G. Besnard 06-2006 (G) AM849204 AM849405 C4 Chase 22020 (K) AM849179 AM849332 C4 AF499149 L14623 C4 G. Besnard 23-2004 (G) AM849181 AM849356 C4 G. Besnard 43-2006 (G) AM849201 AM849408 C3 Zuloaga 6798 (SI) AY029676 AM849344 C3 A.T. & S.S. 654 (TCD) AY029677 AM887870 C3 AF499140 X79900 C4 G. Besnard 73-2006 (G) AF499144 AM849389 C4 G. Besnard 22-2004 (G) AM849185 AM849386 C4 A.T. & S.S. 657 (TCD) AM849187 AM849340 C4 Zuloaga 6773 (SI) AY029685 AM849339 C3-C4 intermediate P. Rondeau 04-2005 (G) AM849189 AM849343 C4 AY029687 NA C3 Zuloaga 6960 (SI) AY029686 AM849420 C4 Zuloaga 6965 (SI) AY029688 AM849422 C4 P. Rondeau 05-2005 (G) AY029649 AM849390 C4 T.R. Hodkinson, s.n. (TCD) AY864833 AM849334 C4 A.T. & S.S = A. Teerawatananon and S. Sungkaew; NA, not available. List of species sampled and accession numbers of ndhF and rbcL sequences. The photosynthetic type was deduced from the literature. Taxonomy is adapted from [S15] according to [S16]. Voucher location is indicated, when available, in parentheses. Accession numbers of sequences obtained in this study are in bold. Please cite this article in press as: Christin et al., Oligocene CO2 Decline Promoted C4 Photosynthesis in Grasses, Current Biology (2008), doi:10.1016/j.cub.2007.11.058 S13 Table S2. Primers Used for the Amplification of rbcL and ndhF Sequences Primer Sequence Reference Ta ( C) rbcL_1_for rbcL_1388_rev ndhf_1_for ndhf_1318_rev ndhf_972_for ndhf_2110_rev ATGTCACCACAAACAGAAACTAAAGC TTCCATAYTTCACAAGCTGC ATGGAACATACATATCAATA CGAAACATATAAAATGCAGT GTCTCAATTGGGTTATATGATG CCCCCTACATATTTGATACCTTCTC [S17] This study This study This study [S18] This study 48 48 47 47 50 50 Ta, annealing temperature.