Systematic Botany (2009), 34(2): pp. 312–323 © Copyright 2009 by the American Society of Plant Taxonomists A Morphology-Based Cladistic Analysis of Digitaria (Poaceae, Panicoideae, Paniceae) Andrea S. Vega,1 Gabriel H. Rua,1 Liliana T. Fabbri,1 and Zulma E. Rúgolo de Agrasar2 1 Cátedra de Botánica Agrícola, Facultad de Agronomía, Universidad de Buenos Aires, Avenida San Martín 4453, C1417DSE Buenos Aires, Argentina 2 Instituto de Botánica Darwinion, Labardén 200, Casilla de correo 22, B1642HYD San Isidro, Argentina 1 Author for correspondence (avega@agro.uba.ar) Communicating Editor: Kenneth M. Cameron Abstract—A phylogenetic analysis was performed on 67 species of Digitaria belonging to four subgenera and 26 of the 32 sections recognized in Henrard’s monograph. The analysis was based on 113 discrete and six continuous morphological characters. In the resulting topologies the genus Digitaria was monophyletic. In spite of the low support for most groupings, several clades were recovered. The subdivision of Digitaria in the four subgenera proposed by Henrard was not supported since the large subgenus Digitaria appears as a paraphyletic assemblage within which the other three subgenera are nested. Nevertheless, the monophyly of some of Henrard’s sections was supported. This is the first approach to the phylogeny of Digitaria. Resumen—Se llevó a cabo un análisis cladístico. Se incluyeron 67 especies de Digitaria pertenecientes a los 4 subgéneros y a 26 de las 32 secciones reconocidas en la monografía de Henrard. El análisis fue basado en 113 caracteres morfológicos discretos y 6 continuos En las topologías resultantes el género Digitaria resultó monofilético. A pesar del bajo soporte de la mayoría de los grupos, fueron recuperados varios clados. Los resultados no fueron consistentes con la subdivisión de Digitaria en 4 subgéneros propuesta por Henrard, ya que el extenso subgénero Digitaria aparece como un agregado parafilético dentro del cual se hallan anidados los otros tres subgéneros. Sin embargo, la monofilia de algunas secciones fue confirmada. El presente estudio constituye el primer análisis filogenético de Digitaria. Keywords—Cladistic, Digitaria, Paniceae, phylogeny, Poaceae, systematics. The genus Digitaria Haller emend. A. S. Vega & Rúgolo (Vega and Rúgolo de Agrasar 2001) comprises ca. 220 species distributed in tropical, subtropical, and temperate areas worldwide (Watson and Dallwitz 1992 onwards). It includes foraging species, minor cereals, turf plants, and soil binders, as well as some weeds (Henrard 1950; Veldkamp 1973; Rúgolo de Agrasar 1974; Clayton and Renvoize 1986; Nicora and Rúgolo de Agrasar 1987; Guzmán et al. 1989; Molina Sánchez 1990; Watson and Dallwitz 1992 onwards). Species of Digitaria can be recognized by their cartilaginous upper florets, with fertile lemmas having noninrolled, membranous margins, scarcely exposing the fertile palea. This character is used to distinguish Digitaria from allied genera: Axonopus P. Beauv., Panicum L. and Paspalum L. Another taxonomic character of Digitaria is the position of fertile lemmas toward the axis of the inflorescences; lower glumes and lemmas are abaxial, and upper glumes and lemmas are adaxial. This character distinguishes Digitaria from Axonopus, the latter also without a lower glume (Henrard 1950). An historical survey of Digitaria shows that several authors have considered it as an infrageneric entity (section, series, or subgenus) within Panicum and Paspalum (Haller 1768; Trinius 1826; Nees von Esenbeck 1829; Steudel 1853; Gray 1856; Bentham 1878 pp. 463–464; Hackel 1901; Camus 1912). Walter (1788) described Syntherisma as a new genus, and Nees (1829) described Trichachne, including Acicarpa Raddi in its synonymy. Hackel (1901) first divided Panicum subg. Digitaria into three well-marked, but not quite natural, series: Solitaria, Binata, and Ternata. Digitaria ser. Solitaria comprises species with single, sessile spikelets along the inflorescence branches; D. ser. Binata includes species with one spikelet subsessile and the other pedicellate, and in D. ser. Ternata each short paraclade comprises three or more spikelets, one subsessile and the others pedicellate. In her treatment of Paniceae, Chase (1906) recognized three genera, Valota Adans. (= Trichachne Nees), Syntherisma Walter, and Leptoloma Chase. According to Chase (1906), Valota includes species with lanceolate-acuminate upper florets and upper glumes and lower lemmas clothed with long hairs exceeding the length of the spikelets; Syntherisma, as well as Leptoloma, has elliptic upper florets, and upper glumes and lower florets clothed with short hairs or glabrous. Syntherisma differed from Leptoloma by having “spikelets disposed in 1-sided racemes which are digitate or racemose” rather than “panicles divergent at maturity” (Chase 1906). According to Stapf (1919) the varied and distinguishable types of hairs in the spikelets, when correlated with other characters, constitute a guide to the main groups of species in Digitaria. This author defined two sections of Digitaria in Tropical Africa, sect. Setariopsis and sect. Eu-Digitaria, the latter composed of nine subsections. Hitchcock (1927, 1950) recognized Digitaria, Leptoloma, and Trichachne as valid genera, and considered Valota as a synonym of Trichachne. Parodi (1928) mentioned the necessity of transferring the species of Valota (Trichachne) to Digitaria on the basis of morphological characteristics, and Henrard (1950) formally transferred species in Valota, Syntherisma, and Leptoloma to Digitaria, while making some nomenclatural changes. Even though taxonomic studies on Digitaria have been carried out by several authors (Hackel 1901; Chase 1906; Stapf 1919; Rúgolo de Agrasar 1968, 1969, 1970, 1974, 1976, 1990, 1992, 1993, 1994; Veldkamp 1973; Webster 1983; Rúgolo de Agrasar and Sánchez 1989; Wipff and Hatch 1994; CantoDorow 2001; Canto-Dorow and Longhi-Wagner 2001; Vega and Rúgolo de Agrasar 2001, 2002a, 2002b, 2003, 2005, 2006a, 2006b, 2007; Renvoize et al. 2006, among others), the only available comprehensive treatment is Henrard’s (1950) monograph. He subdivided Digitaria into four subgenera: Leptoloma (Chase) Henrard, Setariopsis (Stapf) Henrard, Solitaria (Hack.) Henrard, and Digitaria, the latter with 32 sections (Table 1). Nevertheless, Henrard’s classification has been partially questioned (Veldkamp 1973; Rúgolo de Agrasar 1974) and the relationships between species and groups of species remain unclear. Some recent molecular studies on Panicoideae throw some light on the systematic position of Digitaria within the sub312 2009] VEGA ET AL.: PHYLOGENY OF DIGITARIA 313 Table 1. Classification of Digitaria with subgenera and sections recognized by Henrard (1950). The total number of species included in each subgenus or section is mentioned between brackets, including additions (*) from Rúgolo de Agrasar (1976, 1992) and Wipff and Hatch (1994). Taxa considered in this study are listed below. Digitaria Subg. Solitaria (Hack.) Henrard (8 spp.) Subg. Setariopsis (Stapf) Henrard (9 spp.) Subg. Leptoloma (Chase) Henrard (4 spp.*) Subg. Digitaria Sect. Aequiglumae Henrard (16 spp.) Sect. Atrofuscae Henrard (10 spp.) Sect. Biformes Henrard (2 spp.) Sect. Calvulae (Stapf) Henrard (18 spp.) Sect. Capitipilae Henrard (4 spp.) Sect. Cirripilae (Stapf) Henrard (12 spp.) Sect. Clavipilae (Stapf) Henrard (22 spp.) Sect. Corynotrichae Henrard (10 spp.) Sect. Debiles Henrard (1 spp.) Sect. Erianthae Henrard (33 spp.) Sect. Flaccidulae (Stapf) Henrard (4 spp.) Sect. Gibbosae Henrard (1 spp.) Sect. Glabratae Henrard (9 spp.) Sect. Heteranthae Henrard (2 spp.) Sect. Horizontales Henrard (18 spp.) Sect. Laniflorae Henrard (2 spp.) Sect. Leianthae Henrard (8 spp.) Sect. Leucostachyae Henrard (1 spp.) Sect. Monodactylae (Stapf) Henrard (1 spp.) Sect. Orthotrichae Henrard (14 spp.) Sect. Parviflorae Henrard (11 spp.) Sect. Parviglumae Henrard (7 spp.) Sect. Pennatae (Stapf) Henrard (8 spp.) Sect. Remotae Henrard (2 spp.) Sect. Sanguinales (Stapf) Henrard (26 spp.) Sect. Subeffusae Henrard (4 spp.) Sect. Transversales Henrard (1 spp.) Sect. Trichachne (Nees) Henrard (10 spp.*) D. mariannensis Merr. D. diagonalis (Nees) Stapf D. minutiflora Stapf D. arenicola (Swallen) Beetle D. cognata (Schult.) Pilg. D. pubiflora (Vasey) Wipff D. eriostachya Mez D. aequiglumis (Hack. & Arechav.) Parodi D. fuscescens (J. Presl) Henrard D. curvinervis (Hack.) Fernald D. bicornis (Lam.) Roem. & Schult. D. badia (Scribn. & Merr.) Fernald D. phaeotrix (Trin.) Parodi var. hackelii (Arechav.) Henrard D. maitlandii Stapf & C. E. Hubb. D. gazensis Rendle D. botryostachya Stapf D. balansae Henrard D. ternata (A. Rich.) Stapf D. thouaresiana (Flüggé) Camus D. atra Luces emend A. S. Vega & Rúgolo D. argyrostachya (Steud.) Fernald D. melanochila Stapf D. bonplandii Henrard D. gerdesii (Hack.) Parodi D. chaseae Henrard D. debilis (Desf.) Willd. D. eriantha Steud. D. nodosa Parl. D. nitens Rendle not represented D. abyssinica (Hochst. ex A. Rich.) Stapf D. heterantha (Hook. f.) Merr. D. horizontalis Willd. D. pearsonii Stapf D. perrottetii (Kunth) Stapf D. brownii (Roem. & Schult.) Hughes D. leiantha (Hack.) Parodi D. katangensis Robyns not represented D. monodactyla (Nees) Stapf D. argillacea (Hitchc. & Chase) Fernald D. fragilis (Steud.) Luces D. cayoensis Swallen D. lecardii (Pilg.) Stapf D. parviflora (R. Br.) Hughes D. gymnostachys Pilg. D. ammophila (F. Muell.) Hughes D. coenicola (F. Muell.) Hughes D. divaricatissima (R. Br.) Hughes D. pennata (Hochst.) T. Cooke not represented D. sanguinalis (L.) Scop. D. ciliaris (Retz.) Koeler D. junghuhniana (Nees ex Steud.) Henrard not represented D. similis Beetle ex Gould D. californica (Benth.) Henrard D. swalleniana Henrard D. sacchariflora (Nees) Henrard D. insularis (L.) Fedde D. tenuis (Nees) Henrard D. hitchcockii (Chase) Stuck. D. laxa (Rchb.) Parodi D. patens (Swallen) Henrard D. catamarcensis Rúgolo (Continued) 314 Table 1. SYSTEMATIC BOTANY [Volume 34 Continued Digitaria Sect. Tricholaenoides Henrard (4 spp.) Sect. Trichophorae Henrard (3 spp.) Sect. Verrucipilae (Stapf) Henrard (24 spp.) Sect. Xanthotrichae Henrard (1 spp.) family. A DNA sequence data analysis from the chloroplast gene ndhF (Giussani et al. 2001) suggests that Panicoideae is divided into three well supported clades: two corresponding to the long-recognized tribe Paniceae and the remainder corresponding to the Andropogoneae. The splitting of Paniceae into two clades correlates with base chromosome numbers x = 9 and x = 10. Digitaria belongs to the x = 9 Paniceae, and appears as sister to the remaining genera within this clade. A second analysis using sequences from the rpoC2 insert (Duvall et al. 2001) also supports the monophyly of Digitaria and its relationship with the remaining x = 9 Paniceae. Thus, the hypothesis of affinity of Digitaria with Paspalum, Axonopus, and other genera with chromosome base number x = 10 proposed by earlier authors (Henrard 1950; Butzin 1970; Rúgolo de Agrasar 1974) would be rejected on the basis of molecular and chromosome base number. However, the position of Digitaria is still uncertain, whether sister to all x = 9 Paniceae or sister to the Setaria/Urochloa/Panicum clade (Giussani et al. 2001). Since no hypothesis regarding the phylogeny within Digitaria is currently available, the aim of the present paper is to explore phylogenetic relationships within the genus, and to test the monophyly of the currently recognized subgeneric entities on the basis of morphological evidence. This analysis will be complementary to an ongoing study based on molecular data (Vega et al. unpubl.). Materials and Methods Ingroup Taxa—A set of 67 species of Digitaria was considered in our analysis, including representatives of subgenera Leptoloma, Setariopsis, Solitaria, and 26 of the 32 sections of subgenus Digitaria (Table 1). Outgroup Taxa—Three further representatives of the x = 9 clade of Paniceae were included in our analysis: Echinochloa pyramidalis (Lam.) Hitchc. & Chase, Urochloa brizantha (Hochst. ex A. Rich.) R. D. Webster, and Panicum repens L. Anthaenantia lanata (Kunth) Benth., a member of the x = 10 Paniceae, was also included and used for the purpose of rooting. Because “the resolution among multiple outgroup terminals […] may affect both the position of the ingroup relative to outgroups and the topology of relationships within the ingroup” (Nixon and Carpenter 1993: 422), some characters were included in our analysis to resolve outgroup relationships. Material—Morphological characters (vegetative, reproductive, and anatomical) were scored from herbarium material. Specimens belonging to the following herbaria were examined (acronyms after Holmgren et al. 1990): AAU, BAA, C, CANB, CTES, G, K, L, LIL, MO, P, PRE, SI, SJ, UB, US, VEN, and WU. Only one or two representative specimens of each taxon are listed in Appendix 1. Whenever possible, morphological data were corroborated with living material, through field observations as well as examination of plants cultivated in the “Lucien Hauman” Botanical Garden, Facultad de Agronomía, Universidad de Buenos Aires, Argentina for D. aequiglumis, D. argyrotricha, D. bicornis, D. californica, D. catamarcensis, D. ciliaris, D. diagonalis, D. eriantha, D. fuscescens, D. insularis, D. laxa, D. phaeotrix, D. similis, D. swalleniana, D. ternata, and D. sanguinalis. Morphological characters related to inflorescence structure and growth habit were scored according to Rua (2003). not represented D. pittieri (Hack.) Henrard D. eggersii (Hack.) Henrard D. argyrotricha (Andersson ex Peters) Chiov. D. angolensis Rendle D. violascens Link D. mollicoma (Kunth) Henrard not represented Anatomical Studies—Segments of the middle portion of the penultimate leaf blade of a fertile innovation were used in anatomical studies. Leaf blades of all studied species were taken from living plants when available, or from herbarium specimens and soaked in a nonionic detergent solution at 70°C for 1 hr. Materials were either hand sectioned and stained with safranin, or embedded in paraffin and cut with a rotary microtome, dehydrated in an ethanol series, and double stained with safranin-fast green (D’Ambrogio de Argüeso 1986). Transverse sections were studied under a Wild M20 light microscope. Observations and measurements of epidermal cells of the upper lemma were made with an optical microscope (Zeiss Axioplan, Zeiss, Oberkochen, Germany) connected to an image analyzer (Imagenation Px, Imagenation Corp., Beaverton, Oregon). Upper lemmas were dissected from the spikelets and mounted in gelatine-glycerine. Measurements of cell width and relation between the maximum (outer) and minimum (inner) distance of undulations in horizontal anticlinal walls (Ellis 1979) were taken from three cells of each specimen. Longitudinal sections of the spikelets as well as different types of pilose indumentum were selected, mounted and coated with a goldpalladium (40% - 60%) alloy by a Thermo VGScientific, and observed using a ZEISS DSM 940A Scanning Electron Microscope (SEM) at the Instituto de Botánica Darwinion, Argentina, and a Phillips XL 30 (Phillips, The Netherlands) SEM at the Museo Bernardino Rivadavia, Argentina. Characters—The matrix included 113 discrete plus six continuous morphological characters (Appendix 2); 105 of these showed variation at the ingroup level. The remaining 14 characters were included either as possible synapomorphies joining the genus Digitaria with outgroup terminals, or as informative characters for the outgroups. Autapomorphies were not included in our analysis. Polymorphic characters were scored as such, as recommended when the polarity of the characters are unknown from previous analysis (Kornet and Turner 1999). Missing data (including unavailable as well as inapplicable data) represent 5.7% of the entries in the data matrix. Continuous characters were analized as such, using the methodology implemented in TNT (Goloboff et al. 2005). Data Analysis—The data matrix (Supplemental Appendix 3) was analized using TNT (Goloboff et al. 2003a). Data matrix and trees were submitted to TreeBASE (Study number S2186). A heuristic search strategy was adopted, consisting of 100 random addition sequences followed by TBR swapping, using Wagner trees as starting trees and holding a maximum of two trees each time. The trees obtained were submitted to a round of TBR swapping, then to 1,000 iterations of Parsimony Ratchet (Nixon 1999), and then to an additional round of TBR. Branches with ambiguous support (min. length = 0) were collapsed. Group support was quantified through: (1) the decay index of Bremer (BS, Bremer 1994), and (2) the symmetric jackknife group frequency (SJF, Goloboff et al. 2003b). Because of the high homoplasy of the data set, tree searches were performed using implied weights (Goloboff 1993, Goloboff et al. 2008). When using implied weights, TNT downweights homoplastic characters in proportion to their amount of extra steps (homoplasy), and saves trees that minimize ‘distortion’ (D), which is an increasing function of the homoplasy (Goloboff et al. 2003a). Distortion is quantified with the equation, D = e/(e + k), where e = extra steps, and k = constant of concavity. The strength with which a homoplastic character is downweighted depends on the concavity value (k) of the weighting function: the lower the k value the stronger the weighting function. To explore the stability of the results, analyses were performed under 33 different k values. Since distortion is not a linear function of concavity, k values were selected in such a way that they produce regular distortion increments of 1.25%, within a range of 50–90% related to an average nonhomoplastic character (Mirande 2007). To test tree stability related to variations of k, comparisons between pairs of contiguous trees (i.e. between trees obtained using kn and kn-1) 2009] VEGA ET AL.: PHYLOGENY OF DIGITARIA 315 Fig. 1. Majority rule consensus of cladograms resulting from parsimony analysis using implied weighting, with k = 4.2–37.9 (for details see text). Numbers above branches indicate the branch frequency (higher than 50%) in all trees. 316 SYSTEMATIC BOTANY were performed through calculation of (1) SPR-difference, i.e. the number of SPR-swaps required to convert tree n into tree n-1; (2) number of shared taxa (= nodes in agreement subtree), and (3) number of shared groups (= nodes in strict consensus tree). Calculation of k values, tree searches, and calculation of stability measures were all performed using a TNT script written by J. Marcos Mirande (unpublished), who kindly made it available to us. Since support measures are not comparable when using different weighting functions, BS and SJF values were independently calculated for each concavity. Results Thirty-three trees were found, using 33 k values ranging between 4.2 and 37.9. These results are summarized in the majority rule consensus tree presented in Fig. 1, which shows the groups being more stable across the range of k values tried. Sixteen of the obtained topologies were different. Values of k ranging between 12 and 17 yielded the more stable topology (Fig. 2). Support measures shown in Fig. 2 were calculated under k = 15. In all trees Digitaria was monophyletic (Fig. 1) and relatively well supported by several synapomorphies. In most cases, clades that were more robust in relation to weighting intensity (i.e. having higher frequencies in the majority rule consensus, Fig. 1) were also the better supported (Fig. 2). The following clades (Figs. 1, 2) merit some comments: [Volume 34 1) A clade including representatives of sect. Trichachne and Trichophorae (hereafter the [Trichachne + Trichophorae] clade), which splits into two subclades, one of them comprising South American species of the sect. Trichachne characterized by large, acuminate spikelets (D. laxa, D. swalleniana, D. similis, D. catamarcensis, D. insularis, and D. sacchariflora, hereafter the ‘core-Trichachne’ clade), the other one including the Trichophorae [D. pittieri + D. eggersii] plus D. californica and D. tenuis. 2) A clade characterized by ‘tumbleweed’ inflorescences composed of primary branches with a spikelet-free proximal portion, the basalmost [sub]verticillate (hereafter, the ‘tumbleweed’ clade); this clade includes D. gymnostachys as sister to a subclade which splits into two monophyletic groups: [D. pubiflora + D. arenicola + D. cognata] (i.e. Digitaria subg. Leptoloma) and [D. pennata + D. ammophila + D. divaricatissima + D. coenicola] (D. sect. Pennatae). In most topologies, the ‘tumbleweed’ clade is sister to [D. brownii + D. patens], two “peripheral” members of the sect. Trichachne native to Australia and North America respectively. 3) A large clade characterized by having spikelets with glumes and lemmas approximate, i.e. not separated by conspicuous internodes (hereafter, the [Digitaria + Ternatae] clade). Most topologies favored the splitting of this clade into two subclades, one including species with decumbent to creeping Fig. 2. Most parsimonious tree obtained under k = 12.6–16.8. Numbers above branches represent Bremer support, number below branches refer to symmetric jackknife group frequencies; both support measures calculated under k = 15. 2009] VEGA ET AL.: PHYLOGENY OF DIGITARIA 317 culms allied to the type species D. sanguinalis (hereafter the ‘core-Digitaria’ clade), the other subclade including most species with ternate short paraclades (hereafter the ‘Ternata’ clade). Species of Digitaria sections Atrofuscae and Verrucipilae, which have ternate short paraclades, were intermingled and separate into two clades, one of them nested within the coreDigitariae (D. fuscescens, D. violascens, D. argyrotricha, D. mollicoma) and the other one within the Ternatae (D. angolensis, D. curvinervis). Digitaria mariannensis, the only species of the subgenus Solitaria included in our analysis, was also placed within the core-Digitaria clade. Digitaria subgenus Setariopsis and a clade including most species of the section Clavipilae were nested within the Ternatae. Discussion Monophyly of Digitaria—The present analysis supports the monophyly of Digitaria which is unambiguously supported by several morphological synapomorphies. Our results are in agreement with previous analyses based on molecular data (Giussani et al. 2001; Duvall et al. 2001). Subgeneric Classification—Chase distinguished the genera Trichachne (sub. nom. Valota Adans.) and Leptoloma from Digitaria (sub. nom. Syntherisma Walt.), but her concept was disregarded by Henrard (1950), who defined Digitaria in a comprehensive way by including Leptoloma and Trichachne. Instead, Henrard subdivided Digitaria into four subgenera (Leptoloma, Setariopsis, Solitaria, and Digitaria). The species of the former genus Trichachne were divided by Henrard into two sections, D. sect. Trichachne and sect. Trichophorae, whereas the Australian species of Leptoloma were included in D. sect. Pennatae. The North American Digitaria cognata was grouped together with the Indian D. tomentosa (Schult.) Pilg. into D. subg. Leptoloma. Our analysis is more consistent with Chase’s rather than Henrard’s concept. Indeed, species of both Digitaria sect. Trichachne and D. subg. Leptoloma form a clade outside the core Digitaria clade. Nevertheless, a clade containing both Trichachne and Leptoloma species was recovered under k = 7 (tree not shown). Henrard’s Digitaria sect. Trichophorae, formerly placed within Valota (Chase 1906), and D. sect. Laniflorae, also included by Webster (1983) in the D. sect. Trichachne, were also aligned outside the core Digitaria, as well as the D. sect. Pennatae, which is sister to Leptoloma. According to our data Digitaria appears to be composed of a major clade corresponding to a core Digitaria, and a doubtfully resolved portion including the D. subg. Leptoloma and D. sections Trichachne, Trichophorae, Laniflorae, and Pennatae. Clearly, the subgeneric scheme of Henrard must be rejected, since the three minor subgenera Leptoloma, Setariopsis, and Solitaria appear nested within a paraphyletic D. subg. Digitaria. The validity of some of Henrard’s sections will be discussed below. Our analysis is the first preliminary cladistic contribution to the understanding of the phylogeny within Digitaria. Nevertheless, taxonomic decisions seem premature at this time until molecular analyses are completed (A. S. Vega et al. unpubl.). Evolution of Selected Characters within Digitaria— Spikelet Indumentum—Stapf (1919) was the first agrostologist to use the varied nature of the hairs in the spikelets as a guide to the infra-generic taxonomy of Digitaria. Henrard Fig. 3. A–E. Types of pilose indumentum of the spikelets. SEM photographs. A–B. Linear hairs with acute or obtuse apex. A. Smooth walls [D. aequiglumis, Schinini et al. 18993 (CTES)]. Bar = 1 µm. B. Verrucose walls [D. violascens, Klein 11249 (SI)]. Bar = 10 µm. C. Hairs with a slightly dilated and mucronate apex [D. phaeotrix var. adusta, Stuckert 18615 (G)]. Bar = 20 µm. D–E. Claviform hairs. D. Type Clavipilae [D. filiformis, Bittmore 799a (L)]. Bar = 10 µm. E. Type Corynotrichae [D. gerdesii, Hassler 8384 (G)]. Bar = 20 µm. (1950) based several sections on this feature, in combination with other, more general characters. Spikelets of Digitaria are typically pilose, although glabrous spikelets occur as well, and a few sections are characterized by glabrous spikelets. Indumentum is varied, usually restricted to upper glume and lower lemma, and occasionally present on the back of the reduced lower palea. Pubescence on the back of a cartilaginous upper lemma had mistakenly been reported in D. atra (Luces 1942); however, the cartilaginous bract corresponds to the lower lemma (Vega and Rúgolo de Agrasar 2001). The distribution of indumentum on the upper glume and lower lemma has been considered a taxonomically valuable character due to its constancy in each taxon with the exception of D. ischaemum, where two types of hair indumentum were reported (Veldkamp 1973). Nevertheless, distribution of the indumentum on the spikelet bracts is highly homoplasious and carries poor phylogenetic information. Two general types of hairs were recognized in Digitaria species: linear hairs, with smooth or verrucose walls with acute or obtuse apices (Fig. 3A, B) and hairs with a dilated apex and smooth walls (Fig. 3C–E). Within this last type, two morphological subtypes can further be recognized: hairs with slightly dilated apices, sometimes provided with an acumen (Fig. 3C), and claviform hairs which are dark at maturity (Fig. 3D, E). Claviform hairs have different forms, which have been used by Henrard (1950) to characterize different sections within the genus: Clavipilae (Fig. 3D), Corynotrichae (Fig. 3E), and Capitipilae. Hairs with more or less dilated apices are synapomorphic for a large group within the ‘Ternatae’, and species with plainly dilated hairs form a clade nested within a more general group with slightly dilated hairs (Fig. 4A). Section Clavipilae, including species distributed in temperate and tropical regions of both hemispheres, forms a well supported 318 SYSTEMATIC BOTANY [Volume 34 Fig. 4. A–D. Mapping of the apices of spikelet hairs (character (ch.) 6, cladogram (cl.) A), walls of spikelet hairs (ch. 8, cl. B), elongation of the first rachilla internode (ch. 13, cl. C), and life cycle (ch. 79, cl. D) onto the topology of Fig. 2. 2009] VEGA ET AL.: PHYLOGENY OF DIGITARIA clade. Under some concavity values (i.e. 4.2, 6.0, and 6.7), the Clavipilae are nested within a clade composed of all species sharing clavate hairs (trees not shown), which also includes species belonging to sections Capitipilae and Corynotrichae, endemic to tropical Africa and South America respectively, as well as the African D. monodactyla, a species with nondilated hairs. Due to difficulties in placing taxa with glabrous spikelets in the current subgeneric classification, Henrard (1950) created some sections exclusively based on this character, e.g. D. sect. Atrofuscae and sect. Glabratae. Species with glabrous spikelets occur together with species having pilose spikelets in other sections. According to our analysis, species of D. section Atrofuscae appear intermingled with representatives of D. sect. Verrucipilae, characterized by verrucose hairs. Species belonging to D. sections Verrucipilae and Atrofuscae formed a clade under stronger weighting functions (trees not shown). Digitaria mollicoma and D. fuscescens, in D. sect. Verrucipilae and sect. Atrofuscae, respectively, form a highly supported clade under all concavity values explored (Figs. 1, 2). Digitaria abyssinica, the only species of D. sect. Glabratae included in our analysis, floats across the core Digitaria as the weighting constant varies. Spikelet hairs seem to have been lost in several independent lineages during the evolution of this character in Digitaria. On the other hand, verrucose hairs could have evolved once or twice (Fig. 4B) within the genus. Rachilla Internodes—Digitaria section Trichachne was distinguished from other groups of Digitaria based on elongate spikelets with conspicuous internodes (Henrard 1950; Rúgolo de Agrasar 1974) [Fig. 5A]. Elongated rachilla internodes also occur in D. subg. Leptoloma and in D. sections Trichophorae, Laniflorae, and Pennatae, where at least the first rachilla internode is conspicuously elongated. Elongated rachilla internodes occur frequently among the x = 9 Paniceae, and seem to be plesiomorphic for Digitaria. Inconspicuous rachilla internodes (Fig. 5B) are synapomorphic for a large clade which includes the typical species D. sanguinalis and other common weeds like D. ciliaris (Fig. 4C). An elongate first rachilla internode is autapomorphic for D. debilis (Fig. 5C, D), a species probably related to D. heterantha and clearly nested within the ‘core Digitaria’ clade. Upper Lemma Shape and Epidermal Features—In Digitaria, the upper floret is cartilaginous, composed of an upper lemma with membranous flat margins that embrace a similar, subequal upper palea (Fig. 6A). The apex of the upper florets can be obtuse, acute or acuminate (Fig. 6B, C) and both bracts are visibly striate due to the presence of rows of epidermal cells, each one containing an excentric papilla (Fig. 6D). The texture of the upper floret depends on the characteristics of these epidermal cells, which can show differences in length and width as well as in the number and depth of lateral indentations. Although acuminate upper florets seem to have been acquired several times during spikelet diversification in Digitaria, they are possibly synapomorphic of a clade including most species of sect. Trichachne. On the other hand, width of the epidermal cells and depth of the cell wall indentations, as well as the other continuous characters, were informative at several levels of the tree topologies. Caryopsis Dispersal—Many dispersal mechanisms have been described among the Paniceae, including endozoochory, adhesive dispersal by animals, ant dispersal, and wind 319 Fig. 5. Spikelets showing rachilla internodes and bract insertion. SEM photographs. A–B, D. Spikelet view in longitudinal section showing rachilla internodes: A. Conspicuous internodes [D. similis, Hitchcock 9008 (US)]. Bar = 200 µm. B. Inconspicuous internodes [D. ciliaris, Johnston 683 (US)]. Bar = 200 µm. C. Spikelet showing bracts distantly inserted [D. debilis, Simon & Williamson 1595 (US)]. Bar = 500 µm. D. Conspicuous internodes [D. debilis, Simon & Williamson 1595 (US)]. Bar = 200 µm. References: lg. Lower glume; ll. Lower lemma; lp. Lower palea; pe. Pedicel; ra. Rachilla; uf. Upper flower; ug. Upper glume; ul. Upper lemma; up. Upper palea. dispersal (Davidse 1987). One syndrome favoring wind dispersal is the production of tumbleweeds through detachment of the entire inflorescence, as occurring in species of Digitaria, Panicum, and some less species-rich genera. Inflorescence tumbleweed species define a well supported clade composed of members of D. sections Parviglumae and Pennatae, and D. subg. Leptoloma. In the remaining taxa, the unit of dispersal is the spikelet. The small size of Digitaria spikelets and the different hair types and hair distribution patterns on the bracts seem to be adaptations for wind and adhesive dispersal (Davidse 1987). Inflorescence Diversity—The synflorescence of Digitaria usually consists of a main axis and a variable number of primary branches (“long paraclades”) along the proximal portion (Rua 2003). Both the primary branches and the distal portion of the main axis bear grouplets of two, three or more spikelets or, more rarely, solitary spikelets (‘short paraclades’, see below). The number of long paraclades and their length relative to that of the main axis is responsible for the overall appearance of the inflorescence, which can vary from a more or less paniculate aspect to a typically digitate one. Therefore, the genus is appropriately named. Some evolutionary patterns can be traced upon the tree topologies, e.g. diverse 320 SYSTEMATIC BOTANY Fig. 6. Upper floret. SEM photographs. A. Middle portion, view from the back of the upper palea. Note membranous and flat margins of the upper lemma. [D. bonplandii, Hassler 11927 (G)]. Bar = 100 µm. B–C. Upper middle, view from the back of the upper lemma: B. Apex acute. [D. ternata, Nicora 8746 (SI)]. Bar = 250 µm. C. Apex acuminate. [D. californica var. villosissima, Parodi 14052 (BAA)]. Bar = 500 µm. D. Upper lemma epidermis [D. violascens, Dusén 15176 (G)]. Bar = 10 µm. References: pa. Papilla; ul. Upper lemma; up. Upper palea. degrees of truncation of the main axis occur in the clade of D. sanguinalis and relatives, elongation of internodes and pedicels occur among the Pennatae and D. subg. Leptoloma, and solitary as well as ternate spikelets arose from plesiomorphic binate short paraclades. The morphological diversification of inflorescences in Digitaria will be extensively revisited in an ongoing paper (G. H. Rua and A. S. Vega, unpubl. results). Spikelet Grouping—The spikelets of Digitaria can be solitary (Fig. 7A) or, more usually, grouped in pairs (Fig. 7B) or triads/tetrads (Fig. 7C), arranged as ‘short paraclades’ (Weberling et al. 1993) along the main axis and the primary branches of the inflorescences. As stated above, Hackel (1901) divided the genus in three series, according to spikelet grouping. Binate spikelets are plesiomorphic within Digitaria. Single, sessile spikelets occur in a few species (Henrard’s subgenus Solitaria), of which only one species was included in our analysis. They are clearly apomorphic within the genus and they seem to have evolved from binate spikelets through reduction. Short paraclades composed of three or more spikelets are also apomorphic, and they seem to have arisen at least two times during inflorescence diversification, although a unique acquisition of this character was favored under k = 18.2–19.5. Leaf Anatomy Features—Leaf anatomy has been fragmentarily studied in the genus Digitaria (Rúgolo de Agrasar and Sánchez 1989). Anatomical studies have referred to leaf blades in transverse section, epidermis in paradermal view, and features related to the type of photosynthetic pathway (Metcalfe 1960; Ellis 1977; Brown 1977; Webster 1983, 1987; Renvoize 1987; Rúgolo deAgrasar and Sánchez 1989). The typical photosynthetic pathway among Digitaria species is C4 NADP- [Volume 34 Fig. 7. A–C. Spikelet grouping. Portion of the main axis bearing short paracladia composed of 1–4 axes of successive branching order. SEM photographs. A. Solitary spikelets [D. mariannensis, Metzner 45 a (L)]. Bar = 0.3 mm. B. In pairs, composed of one subsessile and other pedicellate young spikelets [D. sacchariflora, Rua et al. 34 (BAA)]. Bar = 0.5 mm. C. In triads or more: one subsessile and three pedicellate young spikelets. [D. phaeotrix, Rua s.n. (BAA 22205)]. Bar = 0.25 mm. References: ss. Solitary spikelet; su. Subsessile spikelet; ps. Pedicellate spikelet. ME. Ellis (1977) doubtfully mentioned two African species representative of D. sect. Flaccidulae as possible PCK species (one, D. nitens, was included here), based on the absence of Kranz sheaths in lateral primary bundles and their progressive development toward the median bundle. This characteristic is not typical of PCK type species (Hattersley 1987). The PCK photosynthetic pathway is apparently restricted to a clade of Paniceae containing Urochloa and related genera (Giussani et al. 2001), and Digitaria sect. Flaccidulae is probably not closely related. Other anatomical leaf characters included in our analysis were highly homoplastic. Growth Form Features—The growth form of a plant is determined by a particular combination of vegetative features (Mühlberg 1967; Meusel 1970; Rua and Gróttola 1997), some of which are frequently underscored in taxonomic treatments. Ancestral growth form reconstruction includes perenniality, short rhizomes sheltered by cataphylls, and erect culms, i.e. a character syndrome for Digitaria sect. Trichachne (Rua 2003). Annuality is apomorphic in Digitaria, and restricted to members of the [Digitaria + Ternatae] clade (Fig. 4D), as well as lacking cataphylls, whereas decumbent or creeping culms are synapomorphic for the core-Digitaria. Although our analysis allow us to gain some insight into the evolution of morphological characters in Digitaria, character evolution hypotheses should be considered cautiously until they can be tested by a phylogeny using molecular data. Acknowledgments. We are indebted to the curators of the herbaria for making the material available, to J. Marcos Mirande for kindly making available his unpublished TNT script, to Florencia Agrasar for language assistance, and to Gabriela Zarlavsky for anatomical preparations. This work received financial support through grants UBACyT G027/2001-2002 2009] VEGA ET AL.: PHYLOGENY OF DIGITARIA and G105/2004-2007. ASV, GHR, and ZERA are members of the “Carrera del Investigador” of the “Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)”, Argentina. Literature Cited Bentham, G. 1878. Panicum. Pp. 463–491 in Flora Australiensis: a description of the plants of the Australian territory, vol. 7. London: L. Reeve and Co. Bremer, K. 1994. 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Brunonia 6: 131–216. Webster, R. D. 1987. Taxonomy of Digitaria section Digitaria in North America (Poaceae: Paniceae). Sida 12: 209–222. Wipff, J. K. and S. L. Hatch. 1994. A systematic study of Digitaria sect. Pennatae (Poaceae: Paniceae) in the New World. Systematic Botany 19: 613–627. Appendix 1. Taxa of Digitaria and outgroups considered in this study including at least one representative specimen used for anatomical observations. Anthaenantia lanata (Kunth) Benth., G. H. Rua & Boccaloni 151 (BAA 22785). Echinochloa pyramidalis (Lam.) Hitchc. & Chase, G. H. Rua s.n. (BAA 22207). Panicum repens L., G. H. Rua et al. 270 (BAA 22959). Urochloa brizantha (Hochst. ex A. Rich.) R. D. Webster, G. H. Rua et al. 68 (BAA 22682). Digitaria abyssinica (Hochst. ex A. Rich.) Stapf, R. Pohl 11386 (US). D. aequiglumis (Hack. & Arechav.) Parodi, A. Da Silva 2520 (BAA). D. ammophila (F. Muell.) Hughes, Thompson GAL191 (CANB). D. angolensis Rendle, M. Reekmans 9850 (BAA). D. arenicola (Swallen) Beetle, J. Swallen 10588 (US). D. argillacea (Hitchc. & Chase) Fernald, J. Reeder & C. Reeder 4445 (US). D. argyrostachya (Steud.) Fernald, W. Harris 11413 (US 755154). D. argyrotricha (Anderss.) Chiov., G. H. Rua 526 (BAA). D. atra Luces emend A. S. Vega & Rúgolo, Müller s.n. (VEN 222492). D. badia (Scribn. & Merr.) Fernald, G. & J. Davidse 9904 (BAA). D. balansae Henrard, G. Davidse et al. 11001 (MO). D. bicornis (Lam.) Roem. & Schult., G. H. Rua et al. 272 (BAA); G. H. Rua et al. 561 (BAA). D. bonplandii Henrard, Gottsberger 1021-80R-16371 (MO). D. botriostachya Stapf, A. McKinnon S. 65 (UB). D. brownii (Roem. & Schult.) Hughes, R. Roe 502 (CANB); R. Pullen & Galore 4688 (CANB). D. californica (Benth.) Henrard, L. R. Parodi 13982 (BAA). D. catamarcensis Rúgolo, T. Killeen 1571 (SI). D. cayoensis Swallen, C. L. Lundell 6670 (K, US). D. chaseae Henrard, A. Chase 10764 (US). D. ciliaris (Retz.) Koeler, Vera Santos 7561 (L). D. coenicola (F. Muell.) Hughes, W. Moir s.n. (CANB). D. cognata (Schult.) Pilg., J. Lerew Kan-2-175 (US). D. curvinervis (Hack.) Fernald, E. Ekman 1049 (AAU, C, K, P). D. debilis (Desf.) Willd., Sampaio s.n. (WU 2071). D. diagonalis (Nees) Stapf, G. H. Rua 536 (BAA). D. divaricatissima (R. Br.) Hughes, C. Hubbard 5350 (CANB); C. Hubbard 5071 (CANB). D. eggersii (Hack.) Henrard, Proctor 48361 (SJ, US). D. eriantha Steud., [Volume 34 Z. Rúgolo 2118 (SI). D. eriostachya Mez, B. Rosengurtt 5461 (BAA). D. fragilis (Steud.) Luces, A. Chase 10833 (MO). D. fuscescens (J. Presl) Henrard, Sarmento s.n. (BAA). D. gazensis Rendle, G. Davidse et al. 6515 (BAA). D. gerdesii (Hack.) Parodi, G. Davidse et al. 11385 (MO). D. gymnostachys Pilg., K. L. Tinley 598 (PRE); R. P. Ellis 3644 (PRE). D. heterantha (Hook. f.) Merr., Kondo & Edaño s.n. Phil. Nat. Herb. 36605 (L). D. hitchcockii (Chase) Stuck., J. Roybal 48 (US 1935901). D. horizontalis Willd., G. H. Rua et al. 72 (BAA). D. insularis (L.) Fedde, Krapovickas & Schinini 31450 (BAA). D. junghuhniana (Nees ex Steud.) Henrard, Kievih 1701 (L). D. katangensis Robyns, M. Reekmans 5954 (BAA). D. laxa (Rchb.) Parodi, T. Meyer 2562 (BAA). D. lecardii (Pilg.) Stapf, Meinzingen & Conert 549 (LIL). D. leiantha (Hack.) Parodi, A. Schinini et al. 8322 (BAA, LIL). D. maitlandii Stapf & C. E. Hubb., M. Reekmans 6778 (BAA). D. mariannensis Merr., J. Metzner 45 a (L). D. melanochila Stapf, H. Schlieben 4644 (UB). D. minutiflora Stapf, J. Lambinon 78/234 (BAA). D. mollicoma (Kunth) Henrard, Lugd. Batav. s.n. (L). D. monodactyla (Nees) Stapf, E. van Jaarsveld 83 (PRE). D. nitens Rendle, R. Davies 2951 (SI). D. nodosa Parl., J. Duvigneaud 77 (BAA). D. parviflora (R. Br.) Hughes, C. Hubbard 5923 (L). D. patens (Swallen) Henrard, E. Bongsch S-214 (US). D. pearsonii Stapf, P. Ndabaneze 3 (BAA). D. pennata (Hochst.) T. Cooke, M. Gilbert 1649 (UB). D. perrottetii (Kunth) Stapf, P. Ndabaneze 48 (BAA). D. phaeotrix (Trin.) Parodi var. hackelii (Arechav.) Henrard, G. H. Rua & I. B. Boccaloni 159 (BAA). D. pittieri (Hack.) Henrard, P. Standley 35968 (US); O. Jimenez L. 707 (US). D. pubiflora (Vasey) Wipff, A. S. Hitchcock 5157 (US). D. sacchariflora (Nees) Henrard, Seidel 2704 (SI); G. H. Rua et al. 34 (BAA). D. sanguinalis (L.) Scop., G. H. Rua s.n. (BAA 24590). D. similis Beetle ex Gould, Seidel & Vargas 2119 (SI). D. swalleniana Henrard, A. Schulz 4013 (CTES). D. tenuis (Nees) Henrard, R. Smith 5313 (VEN). D. ternata (A. Rich.) Stapf, G. H. Rua 525 (BAA). D. thouaresiana (Flüggé) Camus, M. Reekmans 9945 (BAA). D. violascens Link, A. Chase 8520 (BAA). Appendix 2. analysis. Morphological characters used for phylogenetic 1. Inflorescence primary branches. 2. Depth of epidermal cell indentations. 3. Epidermal cell width. 4. Spikelet length [mm]. 5. Spikelet-width [mm]. 6. Anther length [mm]. 7. Spikelet, hair apex: not dilated [0], slightly dilated [1], dilated (claviform hairs) [2]. 8. Spikelet, hair apex: acute [0], apiculate-rounded [1], rounded [2]. 9. Spikelet, hair walls: smooth [0], verrucose [1]. 10. Spikelet, hair apex: straight [0], recurved [1]. 11. Spikelet, hair length: up to 2 mm [0], 3-5 mm [1]. 12. Spikelet, hair pigmentation: silver-white [0], isabelline [1], brownish [2]. 13. Spikelet, purple-tinged hairs: wanting [0], present [1]. 14. Spikelet, first rachilla internode: elongated [0], inconspicuous [1]. 15. Spikelet, second rachilla internode: elongated [0], inconspicuous [1]. 16. Spikelet position: Upper lemma abaxial [0], Upper lemma adaxial [1]. 17. Spikelet compression: biconvex [0], plano-convex [1], dorsiventrally flattened [2]. 18. Lower glume, whether present or not: wanting [0], present [1]. 19. Lower glume, shape: truncate, reduced to a rim [0], ovate, acute [1], obtuse [2], bi-lobed, very short and tiny [3]. 20. Lower glume, margins: not clasping [0], stem-clasping at the base [1], clasping, with overlapping margins [2]. 21. Lower glume texture: membranous [0], hyaline [1]. 22. Lower glume, number of nerves: manynerved [0], 3-nerved [1], without nerves [2]. 23. Upper glume, number of lateral veins on each glume half: 0 [0], 1 [1], 2 [2], 3 or more [3]. 24. Upper glume, apex: acuminate [0], acute [1], obtuse [2]. 25. Upper glume, relative length: obsolete [0], shorter than the lower lemma [1], (sub)equal to the lower lemma [2], longer than the lower lemma [3]. 26. Upper glume, relative width: wider than the upper floret, the glume covers the upper floret sides [0], narrower than the upper floret (upper floret laterally visible) [1]. 27. Upper glume indumentum: wanting [0], present [1]. 28. Upper glume, distribution of indumentum: pilose throughout [0], alternate, inner internervial spaces pilose [1], pilose between margins and outer veins, otherwise glabrous [2], alternate, inner internervial spaces glabrous [3], distal portion pilose [4]. 29. Upper glume, scabrousness of nerves: smooth [0], scabrous [1]. 30. Upper glume, distribution of nerves: equidistant [0], unequidistant, lateral nerves contiguous [1]. 31. Upper glume, distal convergence of lateral nerves: not convergent [0], convergent [1]. 32. Lower lemma, number of lateral veins on each lemma half: 1 [0], 2 [1], 3 [2], 4 or more [3]. 33. Lower lemma, apex: acuminate [0], acute [1], obtuse [2], apiculate [3]. 34. Lower lemma, texture: papyraceous [0], membranous [1], hyaline [2], cartilaginous [3]. 35. Lower lemma, indumentum: wanting [0], present [1]. 36. Lower lemma, distribution of indumentum: pilose between margins and outer veins, otherwise glabrous [0], alternate, inner internervial space glabrous [1], inner internervial space glabrous, otherwise pilose [2], all internervial spaces fringed with hairs [3], pilose throughout [4]. 37. Lower lemma, scabrousness of nerves: smooth [0], scabrous [1]. 38. Lower 2009] VEGA ET AL.: PHYLOGENY OF DIGITARIA lemma, pectinate bristles: lacking [0], present in subsessile and pedicellate spikelets [1], present in pedicellate spikelets only [2]. 39. Lower lemma, vein distribution: equidistant [0], unequidistant, lateral nerves contiguous [1], unequidistant, three central nerves contiguous [2]. 40. Lower lemma, distal convergence of lateral veins: not convergent [0], convergent [1]. 41. Lower lemma, axillary flower: lacking [0], reduced to a palea [1], malefertile [2]. 42. Upper floret, abscission: not occurring [0], occurring [1]. 43. Upper floret, lemma apex: closed [0], open [1]. 44. Upper floret, lemma texture: membranous [0], cartilaginous with membranous margins [1], crustaceous [2]. 45. Upper floret, lemma and palea surface: smooth [0], papillose [1]. 46. Upper floret, disposition of lemma margins: enrolled around the palea [0], folded upon the palea [1]. 47. Upper floret, short, stiff prickles towards lemma apex: wanting [0], present [1]. 48. Upper floret, pigmentation at maturity: stramineous to ochraceous [0], brown [1], dark purple [2]. 49. Upper floret, relative length: longer than the lower lemma [0], (sub)equal to the lower lemma [1], shorter than the lower lemma [2]. 50. Upper floret, apex: acute [0], acuminate [1], obtuse [2], apiculate [3]. 51. Upper floret, nerves of the upper lemma: 3 nerves distinguishable [0], 5 nerves distinguishable [1], 7 nerves distinguishable [2]. 52. Flower, pigmentation of anthers: yellow [0], purplish [1]. 53. Flower, pigmentation of stigmata: pale [0], purplish [1]. 54. Caryopsis, outline: elliptical/oblong [0], obovate [1], orbicular [2]. 55. Caryopsis, pigmentation: pale [0], brown [1]. 56. Reproduction, cleistogamy: none [0], occurring [1]. 57. Inflorescence, distribution of LPc along the main axis: racemes always alternate [0], proximal racemes (frequently) verticillate [1], distal racemes conjugate, otherwise alternate [2]. 58. Inflorescence, relative length of primary order branches: decreasing towards apex [0], about the same length throughout [1], longer at the middle, inflorescence fusiform [2]. 59. Inflorescence, length of racemes relative to main axis: racemes not reaching the apex [0], the lowermost racemes (almost) reaching the apex [1], racemes overtopping the apex [2]. 60. Inflorescence, orientation of LPc: ascending [0], spreading [1], rigidly horizontal [2]. 61. Inflorescence abscission as tumbleweed: not occurring [0], occurring [1]. 62. Inflorescence, homogenization: paniculate [0], proximally paniculate, homogenized distally [1], kPc homogeneous throughout [2]. 63. Inflorescence, main florescence (= terminal spikelet) and distal kPc: wanting [0], present [1]. 64. Inflorescence, long paraclades: wanting [0], present [1]. 65. Inflorescence, phyllotaxis: distichous [0], polystichous [1]. 66. Inflorescence, second order long paraclades: wanting (occasionally a reduced secondary LPc on the proximal raceme) [0], regularly present [1]. 67. Inflorescence, pulvini at the base of racemes: inconspicuous (lacking?) [0], conspicuous, bulky [1]. 68. Inflorescence, pulvini at the base of pedicels: lacking [0], present [1]. 69. Inflorescence, peduncle indumentum: glabrous [0], distally hirsute [1], hirsute throughout [2]. 70. Inflorescence, rachis of racemes: trichetrous [0], narrowly winged [1]. 71. Inflorescence, rachis indumentum: glabrous to scabrous [0], hirsute [1], ventrally pubescent [2]. 72. Inflorescence, pedicel development: very brief, spikelets subsessile [0], developed, normal [1], extraordinarily developed [2]. 73. Inflorescence, pedicel indumentum: glabrescent [0], scabrous to antrorse-echinulate (more-than-scabrous) [1], puberulous [2], hirsute/ciliolate [3]. 74. Inflorescence, pedicel apex (sensu Webster 1983): truncate [0], cupuliform [1], discoid [2]. 75. Inflorescence, coronula at the apex of pedicels: lacking [0], composed of short hairs [1], composed of long cilia [2]. 76. Inflorescence, (the more frequent) number of spikelets per node: one [0], two [1], three or more [2]. 77. Inflorescence, pedicel con- 323 crescence: pedicels free [0], pedicels concrescent with the rachis [1]. 78. Inflorescence, whether (sub)sessile or pedunculate racemes: (sub)sessile [0], with long peduncles [1]. 79. Inflorescence, relative length of raceme internodes: short (less than twice the length of the spikelets) [0], long (more than twice the length of the spikelets) [1]. 80. Growth form, life cycle: annual [0], perennial [1]. 81. Growth form, branching of culms: unbranched [0], branched [1]. 82. Growth form, growth direction of culms: orthotropous to geniculate [0], decumbent [1], plainly plagiotropous, stoloniferous [2]. 83. Growth form, internode indumentum: glabrous [0], distally hirsute [1]. 84. Growth form, stypochitum: wanting [0], present [1]. 85. Growth form, leaf sequence along tillers: the prophyll is followed by cataphylls [0], the prophyll if followed by foliage leaves [1]. 86. Leaves, pubescence of cataphylls: glabrous [0], pubescent [1]. 87. Growth form, direction of growth in the innovation zone: orthotropous [0], mainly orthotropous, but shortly plagiotropous proximally [1], plainly plagiotropous [2]. 88. Growth form, rhizome internodes: all internodes short [0], elongated in the plagiotropous portion, distally shortened [1], all elongated [2]. 89. Growth form, accessory innovation zones along the culm: lacking [0], present [1]. 90. Growth form, transition from short to long internodes: abrupt [0], gradual [1]. 91. Growth form, culm internodes: hollow [0], solid [1]. 92. Leaves, leaf sheath indumentum: glabrous [0], pubescent/pilose [1]. 93. Leaves, prefoliation: convolute [0], conduplicate [1]. 94. Leaves, indumentum of the ligular region: glabrous [0], hirsute/pubescent [1]. 95. Leaves, ligule outline: truncate [0], acute [1], obtuse [2]. 96. Leaves, ligule margin: entire [0], irregular, erose [1]. 97. Leaves, type of ligule: entirely membranous [0], membranous with a ciliate margin [1], ciliate, without a conspicuous membranous portion [2]. 98. Leaves, ligule development: well developed, conspicuous [0], reduced to an inconspicuous ridge [1]. 99. Leaves, leaf blade outline: narrowly lanceolate, acuminate [0], lanceolate [1], filiform [2]. 100. Leaves, leaf blade consistence: papiraceous [0], coriaceous [1]. 101. Leaves, mature leaf blade exposition: plane [0], involute [1]. 102. Leaf anatomy, midrib: differentiate, distinct [0], undifferentiate [1]. 103. Leaf anatomy, adaxial papillae: lacking [0], present [1]. 104. Leaf anatomy, adaxial prickles on rib zones: lacking [0], present [1]. 105. Leaf anatomy, abaxial prickles on rib zones: lacking [0], present [1]. 106. Leaf anatomy, differentiation of costal and intercostal adaxial regions: undifferentiate [0], slightly noticeable [1], noteworthy [2]. 107. Leaf anatomy, differentiation of costal and intercostal abaxial regions: undifferentiate [0], slightly noticeable [1], noteworthy [2]. 108. Leaf anatomy, location of bulliform cells: only adaxial [0], both adaxial and abaxial [1]. 109. Leaf anatomy, colorless parenchyma associated to midrib: adaxial [0], both adaxial and abaxial [1], none [2]. 110. Leaf anatomy, macrohairs: lacking [0], present [1]. 111. Leaf anatomy, crystals in chlorenchymatous cells: lacking [0], present [1]. 112. Leaf anatomy, abaxial microhairs: lacking [0], present [1]. 113. Leaf anatomy, adaxial microhairs: lacking [0], present [1]. 114. Leaf anatomy, chlorenchyma arrangement: diffuse [0], radiate [1]. 115. Leaf anatomy, cell layers forming radiate chlorenchyma: one layer [0], more than one [1]. 116. Leaf anatomy, shape of chlorenchymatous cells: tabular [0], isodiametric [1]. 117. Leaf anatomy, chlorenchymatous cells between radiate chlorenchyma: lacking, radiate chlorenchyma of contiguous bundles in contact [0], present, separating radiate chlorenchyma of contiguous bundles [1]. 118. Leaf anatomy, abaxial papillae: lacking [0], present [1]. 119. Leaf anatomy, photosynthetic pathway: C3 [0], NADPme [1], PCK or PCK-like NADme [2].