Available online at www.sciencedirect.com South African Journal of Botany 76 (2010) 196 – 209 www.elsevier.com/locate/sajb Lemma micromorphological characters in the Chloridoideae (Poaceae) optimized on a molecular phylogeny Q. Liu a,b,⁎, D.X. Zhang a , P.M. Peterson b a Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, the Chinese Academy of Sciences, Guangzhou 510650, China b Department of Botany, Smithsonian Institution, Washington DC 20013-7012, USA Received 16 February 2009; received in revised form 2 September 2009; accepted 21 October 2009 Abstract Eight lemma micromorphological characters of 83 taxa representing 61 genera in the Chloridoideae have been investigated using Scanning Electron Microscopy, including long cells, cork cells, stomata, bicellular microhairs, papillae, silica cells, microprickles, and macrohairs. Five new types of lemma micromorphological characters were reported here. Data for 27 taxa representing 19 genera from previous publications were also supplied to access the homology of lemma micromorphological characters for different groups through optimization onto a molecular cladogram. Given the optimization, five characters including long cells, cork cells, stomata, papillae, microprickles are of phylogenetic significance for suprageneric groups. Seven characters including straight outline long cells, crescent-shaped cork cells, absent stomata, absent papillae, dumb-bellshaped silica cells, c-type microprickles, and papillate-base macrohairs may not be homologous, however, the enneapogonoid-type bicellular microhairs appeared as a synapomorphy for the Chloridoideae. © 2009 SAAB. Published by Elsevier B.V. All rights reserved. Keywords: Character; Chloridoideae; Lemma micromorphology; Poaceae; Synapomorphies 1. Introduction Chloridoideae comprises approximately 140 genera and 1420 species, whose adoption of efficient C4 photosynthesis has led to its successful proliferation in the tropics and subtropics (Watson and Dallwitz, 1992; Clayton et al., 2008). Taxonomic history clearly shows that it is difficult to recognize monophyletic supra-generic groups within the subfamily (Jacobs, 1986; Kellogg and Campbell, 1987; Davis and Soreng, 1993; Clark et al., 1995; Van den Borre and Watson, 1997; GPWG, 2001; Liu and Zhao, 2004; Columbus et al., 2007; Soreng et al., 2009). For example, the central issue has been whether to ⁎ Corresponding author. Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, the Chinese Academy of Sciences, Guangzhou 510650, China. Tel.: +86 20 37252567. E-mail address: liuqing@scib.ac.cn (Q. Liu). recognize the traditional tribes Cynodonteae and Eragrostideae (Clayton and Renvoize, 1986; Soreng et al., 2009; Peterson et al., in review) as distinct. Several attempts have been made to address the phylogenetic relationships in Chloridoideae based on morphological and molecular evidence (Hilu and Wright, 1982; Hilu and Alice, 2001; Liu et al., 2005a,b; Columbus et al., 2007; Peterson et al., 2007a), the phylogenetic patterns of these studies rejected the traditional circumscriptions of Cynodonteae and Eragrostideae. The importance of lemma micromorphological characters for assessing phylogenetic relationships from species level to tribal level, has been well established in some clades of Poaceae (Terrell and Wergin, 1981; Vignal, 1984; Thomasson, 1986; Peterson, 1989; Valdés-Reyna and Hatch, 1991; Snow, 1996; Mejia-Saules and Bisby, 2003; Bell and Columbus, 2008). While in Chloridoideae, except for descriptive studies of closely related genera in the tribe Eragrostideae (Valdés-Reyna and Hatch, 1991; Snow, 1996), little is known about the phylogenetic implications of lemma micromorphological characters 0254-6299/$ - see front matter © 2009 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2009.10.006 197 Q. Liu et al. / South African Journal of Botany 76 (2010) 196–209 Table 1 Chloridoideae material studied (following the classification of Soreng et al., 2009; Peterson et al., in review). Tribe/subtribe Species Country Voucher Figure(s) Tetrachaete elionuroides Chiov. Farrago racemosa Clayton Oropetium thomaeum (L. f.) Trin. Odyssea paucinervis (Nees) Stapf Halopyrum mucronatum (L.) Stapf Leptocarydion vulpiastrum (DeNot.) Stapf Drake–Brockmania somalensis Stapf Psilolemma jaegeri (Pilg.) S.M. Phillips Desmostachya bipinnata (L.) Stapf Daknopholis boivinii (A. Camus) Clayton Leptothrium senegalense (Kunth) Clayton Habrochloa bullockii C.E. Hubb. Chrysochloa hindsii C.E. Hubb. Tanzania Tanzania India Ethiopia Kenya Kenya Kenya Tanzania China Madagascan Kenya Tanzania Tanzania Peter 13516 (K) Anderson 1150 (K) Koenig s.n. (LINN) Schimper 1652 (P) Polhill & Paulo 766 (K) Gillett 13107 (K) Greenway 9201 (K) Jaeger 320 (B) Guoai Fu 6038 (IBSC) B. Oivin 2288 (P) Gillett 12992 (K) Webster T. 234 (K) Webster T. 39 (K) 52 50 – 20 – – 21 43 42 – – – – Triraphis schinzii Hack. Neyraudia reynaudiana (Kunth) Keng ex Hitchc. Tanzania USA China Semsei 1325 (K) Brooks 1045 (US) Binggui Li 5330 (IBSC) – 28 18 Enneapogon desvauxii P. Beauv. Schmidtia pappophoroides Steud. ex J.A. Schmidt Uniola paniculata L. Fingerhuthia africana Nees ex Lehm. Eragrostis atrovirens (Desf.) Trin. ex Steud. E. cilianensis (All.) Vignolo ex Janch. E. ciliata (Roxb.) Nees E. cylindrica (Roxb.) Nees ex Hook. & Arn. E. tenella (L.) P. Beauv. ex Roem. & Schult. Pogonarthria squarrosa (Roem. & Schult.) Pilg. Harpachne schimperi Hochst ex A.Rich. Cape Province Uganda USA Cape Province China China China China China South Africa Tanzania Smook L. 8570 (IBSC) Eggeling 5819 (K) Jackson E.N.S. 4256 (IBSC) Hayden & Johnson 136 (IBSC) Guoliang Shi 15328 (IBSC) Ceming Tan 99555 (IBSC) Shaoqing Chen 11076 (IBSC) Xuewen Wang 331 (IBSC) Shaoqing Chen 11076 (IBSC) Lichtenstein (B) Richards 25175 (K) 9,45,51 7,19 – 36 – 8 – – – – – Zoysia japonica Steud. Sporobolus tenuissimus (Mart.ex Schrank) Kuntze S. virginicus (L.) Kunth S. fertilis (Steud.) Clayton Crypsis aculeata (L.) Aiton C. schoenoides (L.) Lam. Spartina alterniflora Loisel. S. anglica C.E.Hubb. Pogoneura biflora Napper China France China China China Spain China China Tanzania Rui Wang 007 (IBSC) Homburg 80 (IBSC) Binghui Chen 355 (IBSC) Qing Liu 021 (IBSC) Fangdong Liu 10332 (IBSC) Löfling s. n. (LINN) Kinshen Hao 3437 (IBSC) Binghui Chen 773 (IBSC) Greenway 10620 (K) 25,49 40 41 – 57 – 17,27,58 48 11 Bewsia biflora (Hack.) Gooss. Ctenium newtonii Hack. Eragrostiella bifaria (Vahl) Bor Dactyloctenium aegyptium (L.)P.Beauv. D. australe Steud. Trichoneura ciliata (Peter) S.M.Phillips Orinus thoroldii (Stapf ex Hemsl.) Bor Cleistogenes longiflora Keng ex Keng f. e& L.Liou C. hackelii (Honda) Honda C. chinensis (Maxim.) Keng C. caespitosa Keng Acrachne racemosa (B.Heyne ex Roem. & Schult.) Ohwi Dignathia gracilis Stapf Triodia irritans R. Br. T. lanata J. M. Black Aeluropus littoralis (Gouan) Parl. A. littoralis Parl. subsp. sinensis (Debeaux) Tzvelev Apochiton burttii C. E. Hubb. Leptochloa fusca (L.) Kunth L. chinensis (L.) Nees Dinebra retroflexa (Vahl) Panz. Tanzania Uganda Kenya China Tanzania Tanzania China China China China China China Webster T. 59 (K) Buechner 129 (K) Bogdan 3360 (K) Qing Liu 042 (IBSC) Batianoff 1261 (IBSC) Greenway 10196 (K) Exp. Jinsha River 6444 (KUN) Anren Li 6250 (IBSC) Linhan Liu 16443 (IBSC) Yourun Lin 83–82 (IBSC) Exp. Huang Mt. 818 (IBSC) Xinqi Liu 27579 (IBSC) 23 – – 12,38 – – 30,47 2,13,46 55 56 – – Kenya Australia Australia China China Tanzania China China China Greenway 9497 (K) Jackson E. N. S. 4243 (IBSC) Jackson E. N. S. 4256 (IBSC) Smook L. 8500 (IBSC) Kejian Guan 868 (IBSC) Glover 1 (K) Fangdong Liu 11076 (IBSC) Exp. Longxi Mt. 2151 (IBSC) Zhihao Hu 7900749 (PE) – 14,31 – 37 33 32 10,29 60 – Incertae Sedis Triraphideae Triraphidinae Eragrostideae Cotteinae Uniolinae Eragrostidinae Zoysieae Zoysiinae Sporobolinae Cynodonteae Triodiinae Aluropodinae Eleusininiae (continued on next page) 198 Q. Liu et al. / South African Journal of Botany 76 (2010) 196–209 Table 1 (continued ) Tribe/subtribe Monanthochloinae Muhlenbergiinae Boutelouinae Traginae Species Country Voucher Figure(s) D. polycarpha S.M. Phillips Coelachyrum longiglume Napper Eleusine indica (L.) Gaertn. E. coracana (L.) Gaertn. Lepturus repens (G. Forst.) R.Br. Enteropogon dolichostachyus (Lag.) Keng ex Lazarides Microchloa indica (L.f.) P. Beauv. M. altera (Rendle) Stapf Cynodon arcuatus J. Presl Brachyachne patentiflora (Stent & Rattray) C.E. Hubb. Schoenefeldia transiens (Pilg.) Chiov. Distichlis spicata (L.) Greene Muhlenbergia japonica Steud. M. curviaristata (Ohwi) Ohwi Bouteloua curtipendula (Michx.) Torr. Tragus australianus S.T. Blake T. berteronianus Schult. Kenya Kenya China China China Australia China Tanzania China Kenya Bogdan 3918 (K) M. Senyimba 365 (K) Dingyi Peng 46843 (IBSC) Qing Liu 028 (IBSC) Zexian Li 5374 (IBSC) Thompson BUC 733 (IBSC) Heyun Mai 20246 (IBSC) Procter 2442 (K) Qing Liu 015 (IBSC) Bogdan 4075 (K) – 34 – – – 35 15,26,39 – 16 – Tanzania China China China China (Introduced) Australia China Vhlig 982 (B) Zhenwan Zhang 436 (IBSC) Tianlun Dai 103515 (IBSC) Yanchang Wang 785 (IBSC) Zhenwan Zhang 593 (IBSC) Badman 1848 (IBSC) Fangdong Liu 10377 (IBSC) – – – – 3 53,54 – for the supra-generic groups. Furthermore, the homology of synapomorphies supporting these groups needs to be explored through optimization of lemma micromorphological characters onto molecular cladograms of Chloridoideae (De Pinna, 1991). The present study aims to: (1) examine lemma micromorphological data for representatives of Chloridoideae, (2) discuss phylogenetic implications for the taxonomy of Chloridoideae, and (3) assess synapomorphies supporting supra-generic groups within the Chloridoideae. 2. Materials and methods The lemma abaxial surfaces from the proximal florets in spikelets of 83 taxa representing 61 genera of Chloridoideae were observed using a Jeol JSM-6360V scanning electron microscope (Table 1). Data of 27 taxa representing 19 genera from previous publications (Zuloaga, 1986; Valdés-Reyna and Hatch, 1991; Columbus, 1996; Snow, 1996) was reviewed. Sampling was aimed at covering the major supra-generic groups within Chloridoideae (Clayton and Renvoize, 1986; Soreng et al., 2009; Peterson et al., in review). Three specimens of most taxa were studied, while a scanning electron micrograph of each taxon was used to score relevant characters (Tables 2 and 3). Where possible, micrographs have the lemma tip towards the right. No attempt was made to measure quantify variation of the characters (except in the discussion of distribution frequency) because we only focused on presence or absence of characters. Terminology follows Amarasinghe and Watson (1988, 1991) and Snow (1996). The molecular matrix of 51 taxa was downloaded from TreeBASE web (http://www.treebase.org/treebase) (Columbus et al., 2007). The lemma micromorphological characters were optimized onto the strict consensus tree inferred from WinClada version 1.00.08 (Nixon, 2002) running NONA version 2.00 (http://www.cladistics.com) as a son process. In the NONA analysis, three sequential parsimony ratchet runs were chosen, and each replicate included 200 iterations with 20 trees held in memory. 3. Results 3.1. Lemma micromorphology Eight sets of diagnostic characteristics of the lemma micromorphology in the subfamily Chloridoideae are recognized: (1) long cell outline straight, U-shaped, V-shaped or Ω-shaped (Fig. 16); (2) cork cell semi-circle-shaped, scalariform, crescentshaped, nodular, or oblong (Figs. 7–11); (3) stomata subsidiary cells triangular, semi-circle-shaped, or rectangular (Figs. 12– 17); (4) bicellular microhairs panicoid-type, enneapogonoid- Table 2 Lemma micromorphological characters and character states used in this study. No. Char. Char. state 1 2 3 4 5 6 7 8 Long cell outline (LC) Cork cell (CC) Stomata subsidiary cell (ST) Bicellular microhairs (BM) Papillae (PA) Silica cell (SL) Microprickle (PR) Macrohair (MA) 0 = straight, 1 = U-shaped, 2 = V-shaped, 3 = Ω-shaped, 4 = ? 0 = absent, 1 = semi-circle-shaped, 2 = scalfariform, 3 = crescent-shaped, 4 = nodular, 5 = oblong, 6 = ? 0 = absent, 1 = triangular, 2 = semi-circle-shaped, 3 = rectangular, 4 = ? 0 = absent, 1 = long base-cell chloridoid, 2 = short base-cell chloridoid, 3 = enneapogonoid, P = panicoid 0 = absent, 1 = papillae on long cells, 2 = papillae on long and short cells, 3 = distal swellings on long cells, 4 = ? 0 = absent, 1 = dumbbell-shaped, 2 = oblong sinuous, 3 = cross-shaped, 4 = saddle-shaped, 5 = circular 0 = absent, 1 = a-type, 2 = b-type, 3 = c-type, 4 = d-type, 5 = ? 0 = absent, 1 = papillate base, 2 = sting-shaped, 3 = apiculate, 4 = geniculate, 5 = ? ? = Missing. 199 Q. Liu et al. / South African Journal of Botany 76 (2010) 196–209 Table 3 Data matrix with eight lemma micromorphological characters. Table 3 (continued ) Taxon\char no. 1 2 3 4 5 6 7 8 Aeluropus littoralis A. littoralis subsp sinensis Acrachne racemosa Aegopogon cenchroidesa Apochiton burttii Astrebla pectinata Bewsia biflora Blepharidachne tricholepisb Bouteloua aristidoidesa B. curtipendula B. dactyloidesa Brachyachne patentiflora Chloris barbata C. formosana C. gayana C. schoenoidesb C. virgata C. latisquamea C. paniculatac Chrysochloa hindsii Coelachyrum longiglume Crypsis aculeata Ctenium newtonii Cynodon arcuatus Dactyloctenium australe D. polycarpha D. aegyptium Daknopholis boivinii Desmostachya bipinatata Dignathia gracilis Dinebra retroflexa Distichlis spicata Drake–Brockmania somalensis Eragrostis atrovirens E. cilianensis E. ciliata E. coracana E. cylindrica E. tenella Ectrosia leporinac Eleusine indica Enneapogon desvauxii Enteropogon dolichostachyus Eragrostiella bifaria Erioneuron avenaceumb Eustachys tenera Farrago racemosa Fingerhuthia africana Gouinia latifoliab Gymnopogon ambiguusb Habrochloa bullockii Halopyrum mucronatum Harpachne schimperi Cleistogenes caespitosa C. chinensis C. hackelii C. longiflora Leptocarydion vulpiastrum Leptochloa chinensis L. dubiac L. fusca L. paniceac Leptothrium senegalense Lepturus repens 1 1 2 2 0 2 1 0 1 1 1 1 0 0 0 2 0 0 1 1 2 2 2 1 2 1 0 1 1 2 1 1 1 1 1 1 2 1 1 0 2 1 2 1 3 3 0 0 1 2 0 1 1 0 0 0 0 1 2 0 2 1 2 2 2 2 5 5 5 3 1 0 2 2 2 1 4 4 4 0 4 4 3 1 5 0 3 5 5 5 5 5 5 3 5 5 2 4 4 4 5 4 4 5 5 4 3 4 5 3 5 4 5 3 4 1 4 1 1 1 1 1 4 3 5 5 3 3 0 0 1 ? 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 3 0 1 0 3 1 0 1 1 0 1 0 ? 0 0 0 0 1 0 0 0 1 1 0 0 ? 0 0 1 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 4 4 1 1 1 1 2 1 1 1 1 2 2 2 2 1 2 2 2 2 4 1 2 2 1 1 1 2 1 2 1 4 1 1 1 1 1 1 1 4 1 3 2 1 2 2 2 1 2 2 4 1 4 0 0 0 0 1 1 1 1 1 2 1 1 1 1 1 1 1 0 0 1 3 3 0 0 0 0 3 0 0 0 0 3 3 0 0 1 2 1 0 0 0 2 2 1 0 0 0 1 0 0 0 1 0 3 0 ? 0 0 1 0 1 0 2 0 0 0 0 0 0 0 0 2 1 0 1 1 1 1 5 0 2 0 0 0 4 4 0 0 0 0 0 0 0 0 0 0 0 2 0 1 0 1 2 4 4 0 1 0 0 0 0 1 0 1 1 1 0 0 4 1 0 3 1 0 0 0 1 1 0 0 0 0 1 0 0 0 0 4 4 2 2 0 3 0 0 1 1 ? 1 1 0 3 3 3 1 3 3 3 0 0 1 0 3 0 1 0 3 3 0 1 2 2 1 1 1 0 1 1 1 0 1 0 0 3 0 0 0 1 0 0 0 2 2 2 2 2 0 0 2 2 1 0 0 0 0 0 2 0 2 0 0 2 3 2 0 0 0 0 3 0 0 2 0 0 3 0 3 0 0 0 2 0 0 2 2 0 0 0 0 0 0 0 0 0 2 0 2 ? 4 0 0 2 3 0 0 0 2 2 2 2 0 4 2 0 2 0 0 Taxon\char no. 1 2 3 4 5 6 7 8 Lintonia nutans Microchloa altera M. indica Melanocenchris abyssinicaa Muhlenbergia curviaristata M. japonica Munroa squarrosab Neobouteloua lophostachyaa Neyraudia reynaudiana Odyssea paucinervis Orinus thoroldii Oropetium thomaeum Pereilema crinitumb Pleuraphis jamesiia Pogonarthria squarrosa Pogoneura biflora Psilolemma jaegeri Redfieldia flexuosab Schaffnerella gracilisa Schmidtia pappophoroides Schoenefeldia transiens Scleropogon brevifoliusc Sohnsia filifoliac Spartina alterniflora S. anglica Sporobolus fertilis S. tenuissimus S. virginicus Tetrachaete elionuroides Tetrapogon tenellus Tragus australianus T. berteronianus Trichoneura ciliata T. elegansb Tridens muticusb Triodia irritans T. lanata Triplasis americanac Tripogon chinensis Triraphis schinzii Uniola paniculata Vaseyochloa multinervosab Zoysia japonica Outgroup Panicum hirticauled 2 2 1 0 1 1 2 0 0 0 2 0 2 3 1 2 0 0 2 2 1 ? ? 2 2 1 1 1 0 2 2 2 2 2 3 1 1 ? 0 0 3 0 2 3 3 5 1 5 5 0 3 4 4 2 0 0 5 4 5 1 0 1 3 0 ? ? 0 0 5 5 5 4 3 2 2 1 3 5 3 3 ? 4 4 4 5 3 0 0 3 3 0 0 0 0 0 0 0 0 0 ? 0 0 0 0 0 1 1 0 0 1 1 0 0 0 0 0 0 0 0 0 ? 1 1 0 0 1 1 0 0 1 2 2 1 1 1 2 1 4 1 0 1 1 1 1 1 1 2 2 3 2 1 1 2 2 4 4 4 2 1 2 2 1 2 2 1 1 1 1 4 1 2 2 0 0 0 1 1 1 2 2 0 1 1 1 1 1 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 1 2 2 0 0 1 1 1 0 0 0 1 1 0 0 0 2 3 3 3 0 ? 0 1 1 0 0 1 1 2 5 0 ? 0 2 ? ? 0 0 3 3 3 3 0 5 5 0 0 2 1 1 0 0 1 1 4 4 0 0 0 2 0 0 4 ? 2 2 2 2 3 3 0 0 0 4 2 1 0 ? 0 3 3 0 0 0 0 0 4 4 0 0 0 1 1 2 2 0 0 0 0 2 0 0 3 0 0 2 4 0 0 2 0 0 ? 0 0 0 2 2 4 0 2 0 3 2 0 0 0 2 0 1 1 0 1 0 0 0 2 0 2 0 4 0 1 5 1 4 1 1 0 1 Character number (char no.): 1 = long cell outline; 2 = cork cell; 3 = stomata; 4 = bicellular microhair; 5 = papillae; 6 = silica cell; 7 = microprickles; 8 = macrohairs. Previous publications: aFrom Columbus (1996); bFrom Valdés-Reyna and Hatch (1991); cFrom Snow (1996); dFrom Zuloaga 1986). Character states obtained in this study are in bold. Fig. 1. Basic patterns of anticlinal walls as seen in surface view (adapted from Metcalfe and Chalk, 1979). (a) Outline straight; (b) Outline curved, wide U-shaped curves of shallow amplitude; (c) Outline curved, V-shaped curves of deep amplitude; (d) Outline tightly curved, Ω-shaped curves of deep convolutions. 200 Q. Liu et al. / South African Journal of Botany 76 (2010) 196–209 Fig. 2–11. Long cells and cork cells in the lemma of Chloridoideae. (2) Cleistogenes longiflora, intercostal long cells outline straight, with semi-circle-shaped cork cells. (3) Bouteloua curtipendula, intercostal long cells outline curved, wide U-shaped, with scalfariform cork cells. 4–5 Intercostal long cells outline V-shaped (4) Spartina alterniflora. (5) Crypsis aculeata. (6) Eustachys tenera, intercostal long cells outline deep convolution of omega shape with crescent-shaped cork cells. (7) Schmidtia pappophoroides, with crescent-shaped cork cells. 8–9 Nodular cork cells. (8) Eragrostis cilianensis. (9) Enneapogon desvauxii. 10–11 Oblong cork cells. (10) Leptochloa fusca. (11) Pogoneura biflora. Scale bars: 2, 4–6, 11, 20 µm; 46, 7–10, 10 µm. Dotted line indicated long cell outline. Q. Liu et al. / South African Journal of Botany 76 (2010) 196–209 type or chloridoid-type (including long base-cell and short basecell subtypes) (Figs. 18–27); (5) papillae on long and short cells, on long cells, or distal swellings on long cells (Figs. 28–35); (6) silica cells dumb-bell-shaped, oblong-sinuous, cross-shaped, saddle-shaped or circular (Figs. 36–43); (7) microprickles a-, b-, c-, or d-type (Figs. 44–50); (8) macrohairs papillate-base, stingshaped, apiculate, or geniculate (Figs. 51–60). For comparison, the lemma microcharacters of an additional 27 taxa were supplied in Table 3. Full characterization of lemma micromorphology of a few large genera such as Eragrostis and Sporobolus awaits further study. 3.1.1. Long cells Intercostal long cell outlines fall into four types (Fig. 1a–d), following the classification of Metcalfe and Chalk (1979): (a) outline straight in Cleistogenes longiflora (Fig. 2); (b) outline curved, wide U-shaped in Bouteloua curtipendula (Fig. 3) the type occurs the most frequently here; (c) outline loose, V-shaped in Spartina alterniflora (Fig. 4) and Crypsis aculeata (Fig. 5); (d) outline tight, Ω-shaped only in Uniola paniculata and Eustachys tenera (Fig. 6). 3.1.2. Cork cells These cells were nearly isodiametric and shorter than long cells. They occurred in axial rows (Figs. 7–10) or were oc- 201 casionally solitary (Fig. 11). They were more frequent in the costal zones than in the intercostal zones. The five types of cork cell shapes were: semi-circle-shaped in C. longiflora (Fig. 2); scalariform in Bouteloua curtipendula (Fig. 3); crescent-shaped in Eustachys tenera (Fig. 6) and Schmidtia pappophoroides (Fig. 7); nodular in Eragrostis cilianensis (Fig. 8) and Enneapogon desvauxii (Fig. 9); oblong in Leptochloa fusca (Fig. 10) and Pogoneura biflora (Fig. 11). 3.1.3. Stomata Paracytic stomata occurs in the Chloridoideae (Carpenter, 2005). They were distributed throughout the intercostal zones of rows parallel to the long cells. Three stomata shapes were found: triangular in Dactyloctenium aegyptium (Fig. 12); semicircle-shaped in Cleistogenes longiflora (Fig. 13) and Triodia irritans (Fig. 14); rectangular in Microchloa indica (Fig. 15), Cynodon arcuatus (Fig. 16), and Spartina alterniflora (Fig. 17). 3.1.4. Bicellular microhairs These hairs were generally scattered in the intercostal zones near the lemma tip. Three of the microhair types were found in Chloridoideae (Amarasinghe and Watson, 1988): panicoid-type of relatively narrow cap cells in Neyraudia reynaudiana (Kunth) Keng ex Hitchc. (Figs. 18); enneapogonoid-type of relatively long base cells in Schmidtia pappophoroides Fig. 12–17. Stomata in the lemma of Chloridoideae. (12) Dactyloctenium aegyptium, triangular subsidiary cells of stomata. 13–14 Semi-circle-shaped subsidiary cells of stomata. (13) Cleistogenes longiflora. (14) Triodia irritans. 15–17 Rectangular subsidiary cells of stomata. (15) Microchloa indica. (16) Cynodon arcuatus. (17) Spartina alterniflora. Scale bars: 12, 14–17, 2 µm; 13, 1 µm. 202 Q. Liu et al. / South African Journal of Botany 76 (2010) 196–209 Fig. 18–27. Bicellular microhairs in the lemma of Chloridoideae. (18) Panicoid-type in Neyraudia reynaudiana. (19) Enneapogonoid-type in Schmidtia pappophoroides. 20–22 Long base-cell chloridoid-type. (20) Odyssea paucinervis. (21) Drake–Brockmania somalensis. (22) Tripogon chinensis. 23–27. Short basecell chloridoid-type. (23) Bewsia biflora. (24) Chloris latisquamea. (25) Zoysia japonica; (26) Microchloa indica. (27) Spartina alterniflora. Scale bars: 18, 20–25, 27, 2 µm; 19, 10 µm; 26, 20 µm. (Figs. 19); chloridoid-type of hemispherical cap cells in Odyssea paucinervis (Figs. 20), Drake-Brockmania somalensis (Fig. 21), Tripogon chinensis (Fig. 22), Bewsia biflora (Fig. 23), Chloris latisquamea (Fig. 24), Zoysia japonica (Fig. 25), Microchloa indica (Fig. 26), and Spartina alterniflora (Fig. 27). Chloridoid-type microhairs can also be classified into Q. Liu et al. / South African Journal of Botany 76 (2010) 196–209 two subtypes: long-base-cell subtype with the cap cell not immersed into the long base cell (Figs. 20–22) and short-basecell subtype with cap cell immersed into the short base cell (Figs. 23–27). 3.1.5. Papillae These are short, undifferentiated processes that arise from the outer cell wall. They occurred in three locations of the middle intercostal zones of lemmas in Chloridoideae: on long and short cells in Distichlis spicata (Fig. 28) and Leptochloa fusca (Fig. 29); on long cells in Orinus thoroldii (Fig. 30), Triodia irritans (Fig. 31), Apochiton burttii (Fig. 32), and Aeluropus 203 littoralis (Fig. 33); on the distal portion of long cells in Coelachyrum longiglume (Fig. 34) and Enteropogon dolichostachyus (Fig. 35). 3.1.6. Silica cells These cells were filled with a single silica body. They occurred more frequently in costal zones than in intercostal zones, and are single row (Figs. 36–39), or solitary (Figs. 40– 43). Five different shapes of silica cells were found: dumb-bell in Uniola paniculata (Fig. 36), Fingerhuthia africana Lehm. (Fig. 37), and Dactyloctenium aegyptium (Fig. 38); oblongsinuous in Microchloa indica (Fig. 39); cross-shaped in Fig. 28–35. Papillae in the lemma of Chloridoideae. 28–29 Papillae on long and short cells. (28) Distichlis spicata. (29) Leptochloa fusca. 30–33 Papillae on long cells. (30) Orinus thoroldii. (31) Triodia irritans. (32) Apochiton burttii. (33) Aeluropus littoralis. 34–35 Distal swellings on long cells. (34) Coelachyrum longiglume. (35) Enteropogon dolichostachyus. Scale bar: 10 µm. Abbreviations: PLC, papilla on long cell; PSC, papillae on long and short cells; DSLC, distal swelling on long cell; CC, cork cell. 204 Q. Liu et al. / South African Journal of Botany 76 (2010) 196–209 Fig. 36–43. Silica cells in the lemma of Chloridoideae. 36–38 Dumb-bell-shaped silica cells. (36) Uniola paniculata. (37) Fingerhuthia africana. (38) Dactyloctenium aegyptium. (39) Oblong sinuous silica cell in Microchloa indica. 40–41 Cross-shaped silica cells. (40) Sporobolus tenuissimus. (41) S. virginicus. (42) Saddleshaped silica cells in Desmostachya bipinnata. (43) Circular silica cells in Psilolemma jaegeri. Scale bars: 36–38, 41, 43, 10 µm; 39, 2 µm; 40, 20 µm. Abbreviations: SC, silica cells. Sporobolus tenuissimus (Fig. 40) and S. virginicus (Fig. 41); saddle-shaped in Desmostachya bipinnata (Fig. 42); and circular obscured by epicuticular waxes in Psilolemma jaegeri (Fig. 43). We found the dumb-bell-shaped silica cells in Leptocarydion (Valdés-Reyna and Hatch, 1991; Piperno and Pearsall, 1998) that were not observed by Snow (1996). 3.1.7. Microprickles These swollen processes arising from zones in both the costal and intercostal zones, increased in frequency from the base to the lemma tip. Four types of microprickles (Fig. 44) were observed: (a) straight sided (Fig. 45); (b) sides straight with tip Fig. 44. Microprickle diagrams in lemma of Chloridoideae (adapted from Ellis, 1979). (a) Barbs developed basally from the apex of the base; (b) Barb with direct point not developed from the apex of the base; (c) Barb with recurved point not developed from the base; (d) Barb with contrarily recurved point not developed from the apex of the base. Q. Liu et al. / South African Journal of Botany 76 (2010) 196–209 at the microprickle center and angle to the base (Figs. 46 and 47); (c) sides curved with tip deflected away from the microprickle center (Fig. 48); and (d) sides curved towards the microprickle center (Figs. 49 and 50). 3.1.8. Macrohairs These are unicellular structures visible in Enneapogon desvauxii (Fig. 51 and Tetrachaete elionuroides Chiov. (Fig. 52). They occurred in the intercostal zones, but were more commonly found over the costal zones or at the lemma margin with variation in length and flexibility. Four types of macrohairs are found: papillae-based in Tragus australianus S. T. Blake (Figs. 53 and 54); sting-shaped in C. hackelii (Fig. 55) and C. chinensis (Fig. 56); apiculate in C. aculeate (Fig. 57) and Spartina alterniflora (Fig. 58); geniculate in Eustachya tenera (Fig. 59) and Leptochloa chinensis (Fig. 60). 3.2. Phylogenetic implications of lemma micromorphological characters and homology assessment for synapomorphies Eight lemma micromorphological characters were informative when mapped onto a strict consensus tree of molecular data 205 (Columbus et al., 2007), genera were substituted for species in Fig. 61. Shared lemma micromorphological characters indicated that they are reliable phylogenetic markers at the generic level in Chloridoideae (Snow, 1996). The Chloridoideae was not defined by any lemma morphological characters, but seven supra-generic groups were newly defined by lemma micromorphological characters in Fig. 61: Enneapogon–Eragrostis was defined by straight outline long cells (char. 1:0), Fingerhuthia–Uniola was defined by semi-circle-shaped stomata (char. 3:2), Zoysia–Spartina was defined by V-shaped outline long cells (char. 1:2), Sporobolus–Spartina was defined by lacking cork cells and papillae (chars 2:0 and 5:0), Erioneuron–Pleuraphis was defined by c-type microprickles (char. 7:3), Triodia–Leptochloafus was defined by straight outline long cells (char. 1:0) and crescent-shaped cork cells (char. 2:3), Dactylocteniumaeg– Neobouteloua was defined by straight outline long cells (char. 1:0), Enteropogonmol–Microchloaind was defined by crescent-shaped cork cells (char. 2:3) and absent papillae (char. 5:0). Seven characters including straight outline long cells, crescent-shaped cork cells, absent stomata, absent papillae, dumb-bell-shaped silica cells, c-type microprickles, and Fig. 45–50. Microprickles of in the lemma of Chloridoideae. (45) a-type of microprickles in Enneapogon desvauxii. 46–47 b-type of microprickles. (46) Cleistogenes longiflora. (47) Orinus thoroldii. (48) c-type of microprickles in Spartina anglica. 49–50 d-type of microprickles. (49) Zoysia japonica. (50) Farrago racemosa. Scale bar: 20 µm. 206 Q. Liu et al. / South African Journal of Botany 76 (2010) 196–209 Fig. 51–60. Macrohairs in the lemma of Chloridoideae. 51–52 and 56–57 Sting-shaped macrohairs. (51) Enneapogon desvauxii. (52) Tetrachaete elionuroides. 53–54 Papilla-base macrohairs. (53) Tragus australianus. (54) Close-up of the papilla-base macrohair. (55) Cleistogenes hackelii. (56) Cleistogenes chinensis. 57–58 Apiculate macrohairs. (57) Crypsis aculeata. (58) Spartina alterniflora. 59–60 Geniculate macrohairs. (59) Eustachys tenera. (60) Leptochloa chinensis. Scale bars: 51, 500 µm; 52, 60, 50 µm; 53–59, 20 µm. papillate-base macrohairs (chars 1:0, 1:2, 2:3, 3:1, 5:0, 6:1, 7:3, and 8:2) are homologousious. However, the enneapogonoidtype bicellular microhairs appeared as nonhomoplasious in the Chloridoideae. 4. Discussion Our study provided two important findings: (1) five exhaustive lemma micromorphological characters are presented in Q. Liu et al. / South African Journal of Botany 76 (2010) 196–209 207 Fig. 10. Lemma micromorphological characters mapped onto the strict consensus tree of molecular data (Columbus et al., 2007). Numbers above branches are bootstrap percentages. Numbers above and below circles are character number and character state (see Tables 2 and 3), respectively. Black circles indicated nonhomoplasious synapomorphies, white circles indicated homoplasious synapomorphies. 208 Q. Liu et al. / South African Journal of Botany 76 (2010) 196–209 Chloridoideae; (2) the phylogenetic implications and homology for eight lemma micromorphological characters are discussed for supra-generic groups of Chloridoideae. The five exhaustive lemma micromorphological characters and their states are reported here: U-shaped, V-shaped, and Ω-shaped long cell outlines (chars 1:1, 1:2, and 1:3); long base-cell and short base-cell chloridoid bicellular microhairs (chars 4:1 and 4:2); papillae on long and short cells, on long cells, distal swellings on long cells (chars 5:1, 5:2, and 5:3); a-, b-, c-, and d-types microprickles (chars 7:1, 7:2, 7:3, and 7:4); papillate base and sting-shaped macrohairs (chars 8:2 and 8:3). The optimization of lemma micromorphological characters suggests that these five lemma micromorphological characters (long cells, cork cells, stomata, papillae, and microprickles) are of phylogenetic significance for some supra-generic groups. Three types of long cells occurred universally as phylogenetic markers in the Chloridoieae. The supra-generic group of the Enneapogon (Cotteinae) sister to Fingerhuthia and Uniola (Uniolinae) and Ectrosia and Eragrostis (Eragrostidinae), circumscribed as a smaller Eragrostideae (Peterson et al., 2007a; Soreng et al., 2009), was supported by straight outline long cells (char.1:0). The supra-generic group of Zoysia–Crypsis– Sporobolus–Spartina was positioned in a single tribe in Soreng et al. (2009) and Peterson et al. (in review). Crypsis and Sporobolus were placed in subtribe Sporobolinae based on rebranched inflorescences and spikelets with single florets (Clayton and Renvoize, 1986); and Spartina was placed in Cynodonteae subtribe Chloridinae based on spikelets having single fertile nondeciduous florets arranged along one side and Zoysia was positioned in Cynodonteae subtribe Zoysiinae based on its spiciform inflorescence and spikelets having single florets and falling as a single unit. The V-shaped outline long cell (char. 1:2) of lemma supported their close relationship among genera exhibiting morphologically diverse inflorescences. The V-shaped outline long cells (char. 1:2) also occurred in the Eleusine–Bouteloua group (Fig. 61), whose genera were positioned in the large tribe Cynodonteae of Soreng et al. (2009): Distichlis was placed in subtribe Monanthochloinae based on the possession of several disarticulate fertile florets; Eleusine was placed in subtribe Eleusininiae; Blepharidachne and Scleropogon were placed in the tribe Cynodonteae, while Muhlenbergia, Aegopogon, Pereilema, Redfieldia, and Schaffnerella were placed in subtribe Muhlenbergiinae (Peterson et al., 2007b); Bouteloua was placed in Cynodonteae based on one fertile floret with additional sterile florets (Peterson et al., 2007b; Peterson et al., in review). The straight outline long cells (char 1:0) also occurred twice in supra-generic groups of Triodia–Dinebra–Leptochloa and Dactyloctenium–Neobouteloua. Phillips (1973) stated that Dinebra is closely related to Leptochloa differing in part by its deciduous inflorescence branches. Here we show the character ‘straight long cell outline’ (char 1:0) and ‘crescent-shaped cork cells’ (char 2:3) support her opinion. The crescent-shaped cork cell (char 2:3) and absent papillae (char 5:0) occurred in Enteropogon–Microchloa clade. Peterson et al. (in review) placed these genera in the tribe Cynodonteae subtribe Eleusininae based on seven molecular markers. Inflorescences of the clade bear only nondeciduous primary branches, spikelets arranged along one side, and three-nerved lemmas. With the exception of Leptochloa dubia which has multiple fertile florets per spikelet, the clade members share a single fertile floret per spikelet, usually accompanied by one or more sterile upper florets but further sampling of allied genera is required. This distinction led Clayton and Renvoize (1986) to place Leptochloa in Eleusininae away from the other members of the subtribe. Another species Leptochloa fusca forms a well-supported clade with Dinebra as a sister taxon. Additional data are required to critically evaluate relationships between Chloris and its near relatives. The subtribe Uniolinae (Soreng et al., 2009) is a monophyletic assemblage in the matK, trnL-F, and ITS phylogenies of Hilu and Alice (2001). Two genera Fingerhuthia and Uniola formed a supra-generic group with support of semi-circular stomata subsidiary cells (char 3:2). Snow (1996) stated that stomata appeared as atavisms from the transformationally antecedent leaves; ontogenetic studies of stomata for Chloridoideae will be needed to resolve this question. Mapping lemma micromorphological characters on a molecular phylogeny is a valuable approach that can provide important information on character evolution. Our morphological data often gave poorly resolved consensus trees with clades of low support. Moreover, the single data set often resulted in artificial groupings which have been alluded to by Hilu and Wright (1982) in their phenetic study of grasses. These factors suggest that mapping morphological character distribution within a phylogenetic context might lead to a more accurate estimation of synapomorphies. From the phylogenetic distribution of characters, seven lemma micromorphological characters were homoplasious in the Chloridoideae, and only the enneapogonoid-type bicellular microhairs were synapomorphic in Enneapogon and Schmidtia. This may reflect strong directional selection under environmental control. With respect to lemma homoplasy, microprickles and macrohairs have been reported to accumulate silica which has been associated with a range of effects (Valdés-Reyna and Hatch, 1991). From the data presented, five characters (long cells, cork cells, stomata, papillae, microprickles) were of phylogenetic significance for the supra-generic groups within the Chloridoideae. Seven characteristics (straight outline long cell, crescent-shaped cork cell, absent stomata, absent papillae, dumb-bell-shaped silica cell, c-type microprickle, and papillate-base macrohair) support the majority of species in the Chloridoideae. Further investigations are needed to explore the ontogeny of lemma micromorphological characters within this subfamily. Acknowledgements This study was supported by the National Natural Science Foundation of China (30700043), Q. Liu was supported to visit to the Department of Botany, Key Laboratory of Plant Resources Conservation and Sustainable Utilization, Chinese Academy of Sciences (200922), and Smithsonian Institution by Chinese Government Scholarships (2008491004). We are indebted to staff of B, IBSC, K, KUN, LINN, P, PE, and US for loan specimens, to H.L. Bell and G. Hao for the discussion, and to Ms. X.Y. Hu for her help with SEM. The authors also Q. 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