Supporting Information Methods S1
Methods S1 Full methodology used to compile and index the dataset
Strategy
As a starting point, we followed Watson & Dallwitz (1992 onwards) and Sage et al.
(1999) in assuming that most genera are comprised entirely of either C3 or C4 species.
Forty years of research on photosynthetic pathway in grasses has proved that this
assumption is generally reasonable, although rare exceptions have been discovered in
recent years (e.g. Cerros-Tlatilpa & Columbus, 2009). CAM does not occur in grasses.
The current work proceeded in three stages.
The most recent census of photosynthetic pathway at the genus level was
compiled by Hattersley & Watson (1992), updated by Sage et al. (1999), and is archived
using the taxonomy of Grass Genera of the World (GGTW; Watson and Dallwitz, 1992
onwards). In large part this body of work was grounded in the original anatomical
observations of Metcalfe (1960). We first transposed genus-level information on
photosynthetic pathway from the GGTW taxonomy onto the generic level taxonomic
classification in Genera Graminum (Clayton & Renvoize, 1986) and GrassBase (Clayton et
al., 2002 onwards). The latter is the only nomenclatural scheme for grasses to contain a
complete treatment of synonymy at the genus and species levels. Secondly, we
identified genera that contained both C3 and C4 species, or C3–C4 intermediate species,
and carried out a species-level census of photosynthetic pathway based on published
evidence. Finally, we filled gaps in this species-level census with direct measurements
of stable carbon isotope ratio (13C) in herbarium specimens of 97 species. The result is
the most comprehensive survey of photosynthetic pathway yet completed for grasses.
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The delimitation of the genus Panicum in Genera Graminum is not strictly
phylogenetic (Clayton & Renvoize, 1986). Panicum s.l. is demonstrably polyphyletic
(Giussani et al., 2001; Aliscioni et al., 2003), and many previously recognized sections
have been excluded and recognized as independent genera (Zuloaga et al., 2006, 2007,
2010, 2012; Morrone et al., 2007, 2008; Sede et al., 2008, 2009; Soreng, 2010); Panicum
s.s. is uniformly C4, with the NAD-ME subtype.
Streptostachys s.l. is similarly
polyphyletic, with species recently segregated into new genera (Morrone & Zuloaga,
2009; Sede et al., 2009). However, these phylogenetic studies do not include all named
entities; therefore, to obtain a comprehensive database we use the names Panicum s.l.
and Streptostachys s.l. here in the broad sense in which they are applied in Genera
Graminum.
Genus-level census
Photosynthetic pathway is assigned to each genus (entirely C3, entirely C4, or both C3
and C4 species) in the GGTW database on the basis of anatomical and 13C evidence. We
attached these photosynthetic types as labels to both the accepted generic name and all
of its synonyms, as recognized in the Watson and Dallwitz (1992 onwards) taxonomy.
The labelled names and synonyms were then mapped from GGTW onto the taxonomy of
Genera Graminum (Clayton & Renvoize, 1986) and GrassBase (Clayton et al., 2002
onwards), automating the process with a Python script (ww.python.org).
We next checked through each of the genera accepted by Clayton & Renvoize
(1986) by hand, categorizing photosynthetic pathway using the labelled synonyms from
the GGTW dataset. In every case, there was unanimous agreement in photosynthetic
pathway between the synonyms for each genus, although the pathway was
uncategorized for some synonyms. Photosynthetic pathway remained unclassified for a
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number of genera. Those that belonged to the subfamilies Bambusoideae,
Ehrhartoideae or Pooideae (the “BEP” clade; GPWG, 2001) were assumed to be C3, since
C4 species have never been reported from these taxa. The majority of the remaining
genera were categorized using anatomical and 13C evidence from the literature. This
process left five monospecific genera with an unknown photosynthetic pathway
(Acostia, Lophopogon, Thedachloa, Taeniorachis and Veldkampia), and all but the last
two of these were categorized using 13C analysis of herbarium material.
Species-level census
Nine genera have previously been reported to contain both C3 and C4 species, and/or C3C4 intermediates: Alloteropsis, Aristida, Chaeotobromus, Eragrostis, Neurachne, Panicum
s.l., Steinchisma, Stipagrostis and Streptostachys s.l.. Using phylogenetic methods and
13C analysis, Ingram et al. (2011) showed that the only C3 member of Eragrostis is
misplaced in this C4 genus, and we did not consider it further here. A species-level
census of photosynthetic pathway in the remaining genera was carried out in two steps:
first using a literature search for anatomical, 13C and physiological evidence, and
secondly by carrying out a 13C analysis of herbarium material for the remaining
species.
The literature survey used several lines of evidence to ascribe photosynthetic
pathway. First, leaf anatomy of the Kranz type typically characterizes C 4 photosynthesis,
whereas the non-Kranz arrangement is found in C3 species (Downton, 1971). Second,
the 13C of plant tissues is diagnostic of photosynthetic pathway (Bender, 1971), with a
range of -10 to -15‰ for C4 species, and -22 to -31‰ for C3 species (95% confidence
intervals from the data compilation of Cerling et al., 1997). Third, we considered other
3
physiological characteristics of C4 photosynthesis, including a low CO2 compensation
point and O2-insensitivity (Björkman, 1971), and biochemical evidence of the C4
pathway, based largely on the previous data compilation of Brown (1977). For a small
number of Panicum s.l. species belonging to the sub-genus Dichanthelium, we assigned
photosynthetic pathway based on taxonomic inference, because all species of this subgenus studied to-date have been C3 (Brown & Smith, 1975). This process provided a
complete
species-level
dataset
for
Alloteropsis,
Neurachne,
Steinchisma
and
Streptostachys s.l..
Aristida longifolia (Cerros-Tlatilpa & Columbus, 2009) and Stipagrostis paradisea
(Sage et al., 1999) are the only representatives of their respective genera reported to
use the C3 photosynthetic pathway. We made the simplifying assumption that these
species are the only anomalous members of their genus, and primarily focused our
isotope survey on uncharacterized species from the genus Panicum s.l., and on following
up previous reports of both C3 and C4 photosynthetic pathways in Chaetobromus.
Stable carbon isotope analysis
Specimens of unclassified genera or species were sourced from the Herbarium at the
Royal Botanic Gardens, Kew, or by using Tropicos (www.tropicos.org) to locate other
herbaria that held specimens and obtain material by request. A small (2-5 cm long)
piece of leaf was cut from each specimen, and 13C determined on a 20 mg sub-sample
using a stable isotope mass spectrometer (ANCA GSL preparation module coupled to a
20–20 analyser, PDZ Europa, Cheshire, UK). Measurements on the same sample
typically had a reproducibility of ±0.5‰.
4
References
Aliscioni SS, Giussani LM, Zuloaga FO, Kellogg EA. 2003. A molecular phylogeny of
Panicum (Poaceae : Paniceae): tests of monophyly and phylogenetic placement
within the Panicoideae. American Journal of Botany 90: 796-821.
Bender MM. 1971. Variations in the 13C/12C ratios of plants in relation to the pathway
of photosynthetic carbon dioxide fixation. Phytochemistry 10: 1239-1244.
Björkman O. 1971. Comparative photosynthetic CO2 exchange in higher plants. In:
Hatch MD, Osmond CB, Slatyer RO, eds. Photosynthesis and photorespiration. New
York, USA: Wiley Interscience, 18-32.
Brown WV. 1977. The Kranz syndrome and its subtypes in grass systematics. Memoirs
of the Torrey Botanical Club 23: 1-97.
Brown WV, Smith BN. 1975. The genus Dicanthelium (Gramineae). Bulletin of the
Torrey Botanical Club 102: 10-13.
Cerling TE, Harris JM, MacFadden BJ, Leakey MG, Quade J, Eisenmann V,
Ehleringer JR. 1997. Global vegetation change through the Miocene / Pliocene
boundary. Nature 389: 153-158.
Cerros-Tlatipa R, Columbus JT. 2009. C3 photosynthesis in Aristida longifolia:
implication for photosynthetic diversification in Aristidoideae (Poaceae).
American Journal of Botany 96: 1379-1387.
Clayton WD, Renvoize SA. 1986. Genera Graminum. London, UK: HMSO.
Clayton WD, Vorontsova MS, Harman KT, Williamson H. 2002 onwards. GrassBase
– The Online World Grass Flora. Synonymy. http://www.kew.org/data/grassessyn.html. [accessed 15 March 2012; 11:30 GMT].
Downton WJS. 1971. Checklist of C4 species. In: Hatch MD, Osmond CB, Slatyer RO, eds.
Photosynthesis and photorespiration. New York, USA: Wiley Interscience.
5
Giussani LM, Cota-Sánchez JH, Zuloaga FO, Kellogg EA. 2001. A molecular phylogeny
of the grass subfamily Panicoideae (Paniceae) shows multiple origins of C4
photosynthesis. American Journal of Botany 88: 1993-2012.
Grass Phylogeny Working Group. 2001. Phylogeny and subfamilial classification of
the grasses (Poaceae). Annals of the Missouri Botanical Garden 88: 343-457.
Hattersley PW, Watson L. 1992. Diversification of photosynthesis. In: Chapman GP, ed.
Grass evolution and domestication. New York, USA: Cambridge University Press,
38-116.
Ingram AL, Christin P-A, Osborne CP. 2011. Molecular phylogenies disprove an
hypothesized C4 reversion in Eragrostis walteri (Poaceae: Chloridoideae). Annals
of Botany 107: 321-325.
Metcalfe CR. 1960. Anatomy of the monocotyledons. I. Gramineae. Oxford, UK:
Clarendon Press.
Morrone O, Denham SS, Aliscioni SS, Zuloaga FO. 2008. Parodiophyllochloa, a new
genus segregated from Panicum (Paniceae, Poaceae) based on morphological and
molecular data. Systematic Botany 33: 66-76.
Morrone O, Scataglini AM, Zuloaga FO. 2007. Cyphonanthus, a new genus segregated
from Panicum (Poaceae: Panicoideae: Paniceae) based on morphological,
anatomical and molecular data. Taxon 56: 521-532.
Morrone O, Zuloaga FO. 2009. Keratochlaena, el nombre correcto para Sclerochlamys
(Poaceae, Paniceae). Darwiniana 47: 231.
Sage RF, Li M, Monson RK. 1999. The taxonomic distribution of C4 photosynthesis. In:
Sage RF, Monson RK, eds. C4 plant biology. New York, USA: Academic Press, 551584.
6
Sede SM, Morrone O, Giussani LM, Zuloaga FO. 2008. Phylogenetic studies in the
Paniceae (Poaceae): a realignment of Section Lorea of Panicum. Systematic
Botany 33: 284-300.
Sede SM, Zuloaga FO, Morrone O. 2009. Phylogenetic studies in the Paniceae
(Poaceae-Panicoideae): Ocellochloa, a new genus from the New World.
Systematic Botany 34: 684-692.
Soreng RJ. 2010. Coleataenia Griseb. (1879): the correct name for Sorengia Zuloaga &
Morrone (2010) (Poaceae: Paniceae). Journal of the Botanical Research Institute
of Texas 4: 691–692.
Watson L, Dallwitz MJ. 1992 onwards. The grass genera of the world: descriptions,
illustrations, identification, and information retrieval; including synonyms,
morphology, anatomy, physiology, phytochemistry, cytology, classification,
pathogens, world and local distribution, and references. Version: 11th February
2012. http://delta-intkey.com
Zuloaga FO, Giussani LM, Morrone O. 2006. On the taxonomic position of Panicum
aristellum (Poaceae: Panicoideae: Paniceae). Systematic Botany 31: 497-505.
Zuloaga FO, Giussani L, Morrone O. 2007. Hopia, a new monotypic genus segregated
from Panicum (Poaceae: Panicoideae: Paniceae). Taxon 56: 145-156.
Zuloaga FO, Morrone O, Scataglini MA. 2012. Monograph of Trichanthecium, a new
genus segregated from Panicum (Poaceae, Paniceae) based on morphological and
molecular data. Systematic Botany Monographs 94: 1-101
Zuloaga FO, Scataglini AM, Morrone O. 2010. A phylogenetic evaluation of sects.
Agrostoideae, Megista, Prionitia and Tenera of Panicum (Panicoideae, Poaceae):
two new genera Stephostachys and Sorengia. Taxon 59: 1535-1546.
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