Anatomia foliar de espécies de Poaceae (Poales) e sua importância na sistemática e filogenia THALES HENRIQUE DIAS LEANDRO Novembro - 2016 UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” INSTITUTO DE BIOCIÊNCIAS - RIO CLARO PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS BIOLOGIA VEGETAL Anatomia foliar de espécies de Poaceae (Poales) e sua importância na sistemática e filogenia Tese apresentada ao Instituto de Biociências do Campus de Rio Claro, Universidade Estadual Paulista, como parte dos requisitos para obtenção do título de Doutor em Ciências Biológicas (Biologia Vegetal). Orientação: Profa. Dra. Vera Lúcia Scatena Universidade Estadual Paulista – UNESP, Rio Claro Coorientação: Profa. Dra. Lynn G. Clark Iowa State University – ISU, Ames, EUA THALES HENRIQUE DIAS LEANDRO Novembro - 2016 UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” INSTITUTO DE BIOCIÊNCIAS - RIO CLARO PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS BIOLOGIA VEGETAL Leandro, Thales Henrique Dias Anatomia foliar de espécies de Poaceae (Poales) e sua importância na sistemática e filogenia / Thales Henrique Dias Leandro. - Rio Claro, 2016 170 f. : il., figs., tabs. Tese (doutorado) - Universidade Estadual Paulista, Instituto de Biociências de Rio Claro Orientador: Vera Lucia Scatena Coorientador: Lynn G. Clark 1. Anatomia vegetal. 2. Folha. 3. Gramíneas. 4. Ontogenia. 5. Desenvolvimento. I. Título. 581.4 L437a Ficha Catalográfica elaborada pela STATI - Biblioteca da UNESP Campus de Rio Claro/SP Às conselheiras da construção do meu eu Lêda Dias, Nilma Dias, Rebeca Dias, Edna Scremin e Vera Scatena, dedico. AGRADECIMENTOS À Universidade Estadual Paulista – UNESP, Rio Claro e ao Programa de Pós- Graduação em Ciências Biológicas (Biologia Vegetal) por viabilizarem a execução deste trabalho. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e à Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pelas bolsas e auxílios financeiros concedidos. À Dra. Vera Lúcia Scatena pela confiança e aceite em orientar este trabalho, mas também por todos os ensinamentos profissionais e pessoais. À Dra. Lynn G. Clark pelo aceite em coorientar este trabalho, assim como por me receber em seu laboratório em Iowa State University (ISU), EUA, onde tive o privilégio de aprender muito sobre Poaceae e outras graminídeas. Ao Dr. Pedro Lage Viana pelo aceite em emitir o parecer referente ao processo de concessão da bolsa sanduíche, posteriormente implementada pela CAPES. Ao Dr. Tarciso S. Filgueiras pelos ensinamentos e pela parceria firmada durante a execução deste trabalho. À Dra. Tatiate Maria Rodrigues pela parceria firmada durante a execução deste trabalho, assim como pelo auxílio com as técnicas para análise em microscopia eletrônica de transmissão. À Maria de Fátima Scaff, Diretora do Núcleo de Pesquisa Reservas Paranapiacaba, pela concessão da autorização para coletas no Parque Estadual Fontes do Ipiranga (PEFI), São Paulo. À Ma. Regina T. Shirashuna pelo auxílio nas coletas de espécies de bambus no PEFI, mas também pela parceria firmada durante a execução deste trabalho. Ao Me. Rodrigo Sampaio Rodrigues pelo auxílio nas coletas no PEFI e ao Me. Christian da Silva pela coleta e envio de material botânico do Estado da Bahia. Aos colegas do grupo de pesquisa da UNESP, Rio Claro, pela amizade e discussões científicas, em especial Dra. Elaine Lopes Pereira Nunes, Dra. Blanca Auxiliadora Dugarte Corredor, Ma. Kaire de Oliveira Nardi, Ma. Mariana Maciel Monteiro e Ma. Leticia Peres Poli. Aos colegas do grupo de pesquisa da ISU pela troca de experiências e ensinamentos durante meu período em Iowa, em especial Dr. Timothy J. Gallaher e Dra. Lakshmi R. Attigala. Aos professores do Programa de Pós-Graduação em Ciências Biológicas (Biologia Vegetal) que de alguma forma contribuíram com minha formação, em especial Dra. Alessandra Ike Coan, Dra. Adelita Aparecida Sartori Paoli, Dra. Aline Oriani e Dra. Aline Redondo Martins. Aos funcionários do Centro de Microscopia Eletrônica (CME), UNESP, Botucatu e ao Me. Luiz Ricardo dos Santos Tozin pelo auxílio e processamento das amostras em microscopia eletrônica de transmissão. Ao Dr. Harry T. Horner, diretor do Microscopy and Nano-Imaging Facility (MNIF), ISU, pela autorização para acesso e uso de equipamentos do Centro de Microscopia. Aos funcionários do MNIF, ISU, pelo auxílio no processamento das amostras em microscopia eletrônica de varredura e transmissão, em especial Ma. Tracey M. Stewart. Aos funcionários do Departamento de Botânica, UNESP, Rio Claro, em especial Célia Maria Hebling (secretária). Ao Dr. Carlos Otávio Araújo Gussoni pela amizade e trocas científicas. À Vinícius Manvailer Gonçalves pelas inestimáveis discussões sobre as células fusoides, amizade e companheirismo. Aos amigos da Geologia por propiciarem experiências multidisciplinares essenciais para minha formação enquanto botânico, em especial Vanderlei de Farias. À Nilma e Rebeca Dias pelo apoio e confiança durante toda minha caminhada pessoal e profissional. “[...] Em um momento eu era folha, no outro eu era o que mais se apreciava naquele verde sagrado. [...]” (Lucas M. Furlan) ÍNDICE 1. RESUMO 1 2. ABSTRACT 2 3. INTRODUÇÃO GERAL 3 4. LITERATURA CITADA 6 ILUSTRAÇÕES 11 CAPÍTULO I. The utility of Bambusoideae (Poaceae, Poales) leaf blade anatomy for identification and systematics 16 Abstract 17 Resumo 18 Introduction 19 Material and Methods 20 Sampling area 20 Anatomical analysis 21 Results 21 Surface view 21 Cross section 23 Taxonomic treatment 24 Discussion 26 Conclusion 28 Acknowledgments 29 References 29 Tables 35 Figures 39 CAPÍTULO II. The contribution of foliar micromorphology and anatomy to the circumscription of species within Chusquea ramosissima Clade (Poaceae, Bambusoideae, Chusqueinae) 44 Abstract 45 Introduction 46 Material and Methods 48 Taxon sampling 48 Micromorphology (scanning electron microscopy) 49 Anatomy (light microscopy) 50 Images and terminology 50 Results 51 Epidermal surfaces (SEM and clearings) 51 Cross section 53 Discussion 54 Micromorphological and anatomical data 54 Implications for systematics 55 Taxonomic treatment 58 Acknowledgments 62 References 62 Tables 67 Figures 70 CAPÍTULO III. Fusoid cells in the grass family Poaceae (Poales): a developmental study reveals homologies and suggests insights into their functional role in young leaves 79 Abstract 80 Introduction 82 Material and Methods 85 Sampling 85 Developmental study (young leaves) 85 Anatomical study (mature leaf blades) 86 Description, observation, images, and tridimensional reconstruction 87 Results 87 Discussion 91 Fusoid cell origin and development 91 Phylogenetic implications of fusoid cells for the Graminid clade 94 Some evolutionary and functional insights on fusoid cells based on TEM data 96 Conclusions 98 Acknowledgments 99 Literature Cited 99 Tables 106 Figures and Supplementary Documents 110 CAPÍTULO IV. An update on comparative leaf blade anatomy in the systematics of Poaceae (Poales): the past thirty years since Ellis 123 Abstract 124 Introduction 125 Leaf blade anatomical features revisited 128 Plicate mesophyll parenchyma: arm and rosette cells 128 Midrib vasculature 130 Fusoid cells 131 Intercostal fibers 133 Refractive papillae 133 Stomatal apparatus bearing papillae on the subsidiary cells 134 Trichomes 135 Subfamilial descriptions and general notes based on leaf blade anatomy data 137 I. Anomochlooideae 137 II. Pharoideae 138 III. Puelioideae 138 IV. Oryzoideae 139 V. Bambusoideae 139 VI. Pooideae 140 VII. Aristidoideae 141 VIII. Panicoideae 142 IX. Arundinoideae 142 X. Micrairoideae 143 XI. Danthonioideae 143 XII. Chloridoideae 144 Future prospects for investigating grass leaf blade anatomical anatomical features 144 Final considerations 145 Literature Cited 146 Tables 161 Figure 166 CONSIDERAÇÕES FINAIS 169 1 1. RESUMO Buscando levantar caracteres da lâmina foliar que auxiliem na sistemática e filogenia de Poaceae foram realizados estudos anatômicos e ultraestruturais. Em Bambusoideae, caracteres como papilas dispersas na face abaxial e células buliformes organizadas paralelamente são úteis para o reconhecimento de Olyreae; enquanto papilas organizadas em colunas centrais e células buliformes em forma de leque são úteis para o reconhecimento de Bambuseae. Em Bambuseae, fibras intercostais e nervura central simples são caracteres diagnósticos de Arthrostylidiinae; enquanto complexo estomático portando duas papilas por célula subsidiária e nervura central complexa são caracteres diagnósticos de Chusqueinae. O levantamento de caracteres da lâmina foliar de espécies de Chusquea pertencentes ao clado II “Chusquea ramosissima” indica estruturas úteis para sua delimitação, tais como: complexo estomático, tipo de tricomas, tipo e arranjo de células silicosas, e tipo e número de camadas de células invaginantes; bem como sustenta o reconhecimento deste clado e de uma nova espécie para a Bolívia. O estudo do desenvolvimento foliar com ênfase nas células fusoides mostra sua origem proveniente do meristema fundamental e que a cavidade observada em secções transversais de lâminas foliares maduras é resultado do colapso de várias células fusoides. A origem proveniente do meristema fundamental é confirmada para toda a família e homologias são observadas dentre diferentes tipos de células parenquimáticas do mesofilo. O estudo ainda sugere que células fusoides evoluíram das células incolores observadas em Joinvilleaceae. São fornecidas descrições anatômicas gerais da lâmina foliar para as 12 subfamílias reconhecidas para Poaceae, bem como a definição estrutural e a reavalição da importância de alguns caracteres na sistemática do grupo. PALAVRAS-CHAVE: anatomia, clado BOP, clado PACMAD, clado Graminídeo, folha, gramíneas, linhagens basais, ontogenia. 2 2. ABSTRACT To seek leaf blade features useful for systematics and phylogenetics of Poaceae, anatomical and ultrastructural studies were carried out. Within Bambusoideae, features such as papillae scattered on the abaxial surface and parallel-sided arrays of bulliform cells are useful for recognizing Olyreae; whereas centrally organized papillae and fan-shaped arrays of bulliform cells are useful for recognizing Bambuseae. Within Bambuseae, intercostal fibers and simple midrib are diagnostic features of Arthrostylidiinae; whereas stomatal apparatus bearing two papillae per subsidiary cell and complex midrib are diagnostic features of Chusqueinae. The survey of leaf blade anatomical features of Chusquea species that comprise the clade II “Chusquea ramosissima” shows structures for delimiting species, such as: stomatal apparatus, type of trichomes, type and arrangement of silica bodies, and type and number of layers of arm cells; as well as supports the recognition of this clade and of a new species from Bolivia. The foliar developmental study with emphasis on fusoid cells shows their meristematic origin from the ground meristem and that the cavity as seen in cross section in mature leaves is resulting from the collapse of several fusoid cells. Their origin from the ground meristem is confirmed throughout the family and homologies are observed among different types of mesophyll parenchymatous cells. The study also suggests that fusoid cells evolved from colourless cells of Joinvilleaceae. General leaf blade anatomical descriptions of the 12 recognized subfamilies of Poaceae are provided, as well as structural definition and reevaluation of the relevance of anatomical features in grass systematics. KEY WORDS: anatomy, early-diverging lineages, BOP clade, Graminid clade, leaf, grasses, ontogeny, PACMAD clade. 3 3. INTRODUÇÃO GERAL Todas as espécies estudadas nesta tese pertencem a Poales, que está inserida no clado das comelinídeas e atualmente compreende 14 famílias que compartilham características estruturais, embriológicas e moleculares (APG IV 2016; Stevens, 2001 onwards) (Fig. 1). São reconhecidos seis clados para o grupo, dentre eles o clado graminídeo, que compreende [Flagellariaceae (Joinvilleaceae + Ecdeicoleaceae) Poaceae] baseado no compartilhamento de características morfológicas e embriológicas (Linder e Rudall 2005) (Figs. 2 e 3). Poaceae engloba aproximadamente 12.000 espécies que se caracterizam por apresentarem, de maneira geral, inflorescências bracteadas, perianto reduzido ou ausente, polén com exina ornamentada, embrião lateral diferenciado e cariopse (GPWG 2001; GPWG II 2012). Atualmente a circunscrição da família está estabilizada em 12 subfamílias (Soreng et al. 2015) (Fig. 4): Anomochlooideae, Pharoideae e Puelioideae, que constituem as linhagens basais; Oryzoideae, Bambusoideae e Pooideae, que compreendem o clado BOP; e Aristidoideae, Panicoideae, Arundinoideae, Micrairoideae, Danthonioideae e Chloridoideae, linhagens filogeneticamente mais derivadas que constituem o clado PACMAD. Embora seja uma família diversa e amplamente distribuída pelo mundo (Watson and Dallwitz 1992), sua circunscrição é bem sustentada por evidências estruturais, embriológicas, fisiológicas e moleculares (GPWG 2001; Stevens 2001 onwards; GPWG II 2012; Soreng et al. 2015). No Brasil, a diversidade da família está estimada em cerca de 1.478 espécies distribuídas em 11 subfamílias (Filgueiras et al. 2015) (e.g., Figs. 5-15), com exceção de Puelioideae, grupo que engloba espécies nativas de florestas tropicais da África (Clark et al. 2000). Poaceae desempenha relevante papel econômico e ecológico, contudo, a ampla diversidade de espécies torna sua sistemática por vezes um fator limitante. Como a classificação da família é tradicionalmente baseada principalmente em características morfológicas de estruturas reprodutivas (Longhi-Wagner 2012), a dificuldade 4 na obtenção de material fértil ou a incongruência de evidências morfológicas e moleculares tornam a sistemática e filogenia do grupo ainda mais desafiadora. Nesse contexto, um clássico exemplo são as espécies de bambus lignificados, que apresentam ciclo de vida monocárpico plurianual e, portanto, florescem após vários anos investidos apenas em propagação vegetativa e morrem logo após a dispersão dos frutos. Dessa forma, a busca por características vegetativas que auxiliem na taxonomia de Poaceae tornou-se essencial como subsídio à identificação de espécies e à classificação mais natural do grupo, onde, historicamente, a anatomia da lâmina foliar ganhou destaque (Fig. 16). Trabalhos como os de Duval-Jouve (1875), Schwendener (1890) e Avdulov (1931) foram pioneiros nesta linha e forneceram a base para estudos comparativos sobre a anatomia da lâmina foliar em Poaceae. Na década de 50, Brown (1958) estabeleceu características sistematicamente úteis para a família e determinou seis grandes grupos baseado em uma combinação única de caracteres da lâmina foliar examinados em secções transversais. Já Metcalfe, em 1960, forneceu descrições anatômicas gerais e diagnósticas da epiderme e mesofilo de vários táxons (e.g., Figs. 16-19), bem como ilustrações que foram um dos subsídios para a padronização terminológica estabelecida mais tarde por Ellis (1976, 1979). Anos depois, em 1987, Ellis apresentou uma revisão sobre a aplicabilidade das estruturas anatômicas da lâmina foliar na sistemática de Poaceae, demonstrando a importância desses caracteres para definir e delimitar as cinco subfamílias reconhecidas na época (Clayton e Renvoize 1986). Desde então, estudos anatômicos na família têm sido concentrados principalmente na lâmina foliar e são, em sua maioria, de cunho aplicado à sistemática e filogenia do grupo. Estruturas anatômicas de Poaceae como mesofilo com células invaginantes (arm cells) e fibras intercostais são exemplos de caracteres anatômicos taxonomicamente informativos (ver Judziewicz et al. 1999; Viana et al. 2011, 2013; Leandro et al. 2016a) e frequentemente considerados em estudos filogenéticos (e.g., Clark et al. 2000; GPWG 2001; Sánchez-Ken et 5 al. 2007; GPWG II 2012). Por outro lado, algumas estruturas ainda apresentam dados conflitantes apesar do constante crescimento de trabalhos anatômicos envolvendo espécies do grupo. Este é o exemplo das células fusoides, consideradas características de espécies de Bambusoideae, mas também observadas em linhagens basais e em algumas espécies de Oryzoideae, Pooideae e Panicoideae (Tateoka 1963; GPWG 2001, Leandro et al. 2016b). Embora seja considerado um caráter sinapomórfico e importante para a definição da família, até então não há dados na literatura sobre sua origem, limitando, portanto, interpretações filogenéticas mais consistentes. Considerando a importância da continuidade de estudos anatômicos da lâmina foliar em espécies de Poaceae, esta tese se apresenta estruturada em quatro capítulos: (i) O primeiro capitulo “The utility of Bambusoideae (Poaceae, Poales) leaf blade anatomy for identification and systematics” abrange dados anatômicos sobre 13 espécies de bambus lignificados (Bambuseae) e de três espécies de bambus herbáceos (Olyreae), totalizando 16 espécies nativas estudadas. Este capítulo visa levantar estruturas anatômicas da lâmina foliar que auxiliem na identificação destas espécies simpátricas, principalmente aquelas monocárpicas plurianuais, ocorrentes no Parque Estadual das Fontes do Ipiranga (PEFI), São Paulo. (ii) O segundo capítulo “The contribution of foliar micromorphology and anatomy to the circumscription of species within Chusquea ramosissima Clade (Poaceae, Bambusoideae, Chusqueinae)” visa fornecer dados não só para a diferenciação das espécies estudadas, mas também levantar características úteis ao reconhecimento do clado II “Chusquea ramosissima”, que compreende Chusquea ramosissima Lindman, C. tenella Nees e C. longispiculata L.G. Clark (Fisher et al. 2014). Chusquea Kunth é o grupo mais diverso de bambus lignificados e sua monofilia é sustentada principalmente por dados moleculares (Fisher et al. 2009; Kelchner 6 and BPG 2013; Fisher et al. 2014). O grupo é um exemplo da incongruência de evidências morfológicas e moleculares, reforçando a importância deste estudo. (iii) O terceiro capítulo “Fusoid cells in the grass family Poaceae (Poales): a developmental study reveals homologies and suggests insights into their functional role in young leaves” fornece dados sobre o desenvolvimento de 16 espécies de Poaceae distribuídas em seis subfamílias, além de uma espécie pertencente a cada um dos grupos externos (Flagellariaceae e Joinvilleaceae). Este trabalho visa fornecer principalmente dados sobre a origem das células fusoides em Poaceae comparando seu desenvolvimento com células similares observadas em Joinvilleaceae, bem como traz importante contribuição para o entendimento estrutural deste caráter anatômico amplamente utilizado na sistemática e filogenia da família. (iv) O quarto capítulo “An update on comparative leaf blade anatomy in the systematics of Poaceae (Poales): the past thirty years since Ellis” tem como referência o trabalho de Ellis (1987) e, a partir dele, fornece dados sobre o estado da arte e da importância da anatomia da lâmina foliar na sistemática de Poaceae. São apresentadas descrições anatômicas de cada subfamília e discussões sobre a aplicabilidade de caracteres anatômicos foliares na sistemática do grupo. 4. LITERATURA CITADA Angiosperm Phylogeny Group IV (APG) 2016 An update of the Angiosperm Phylogeny classification for the orders and families of flowering plants: APG IV. Botanical Journal of the Linnean Society 181: 1-20. Avdulov NP 1931 Kario-sistematicheskoe issledovanie Semeistva Zlakor. (A karyo-sistematic investigation of the grass family. Bulletin of Applied Botany, of Genetics and Plant- Breeding, suppl. 44. 7 Brown WV 1958 Leaf anatomy in grass systematics. Botanical Gazette 119: 170-178. Clark LG, Kobayashi M, Mathews S, Spangler RE, Kellogg EA 2000 The Puelioideae, a new subfamily of Poaceae. Systematic Botany 25: 181-187. Clayton WD, Renvoize SA 1986 Genera Graminum. Grasses of the world. Her Majesty's Stationery Office, London. Duval-Jouve MJ 1875 Histotaxie des feuilles de Graminées. Annales des Sciences Naturelles, Botanique 6(1): 227-346. Ellis RP 1976 A procedure for standardizing comparative leaf anatomy in the Poaceae. I. The leaf-blade as viewed in transverse section. Bothalia 12: 65-109. Ellis RP 1979 A procedure for standardizing comparative leaf anatomy in the Poaceae. II. The epidermis as seen in surface view. Bothalia 12: 641-671. Ellis RP 1987 A review of comparative leaf blade anatomy in the systematics of the Poaceae: the past twenty-five years. Pages 2-10 in Soderstrom TR, Hilu KW, Campbell CS, Barkworth ME (eds) Grass Systematics and Evolution. Smithsonian Institution Press, Washington, D.C. Filgueiras TS, Canto-Dorow TS, Carvalho MLS, Dórea MC, Ferreira FM, Mota AC, Oliveira RC de, Oliveira RP, Reis PA, Rodrigues RS, Longhi-Wagner, HM, Santos-Gonçalves AP, Shirasuna RT, Silva AS, Silva C,Valls JFM,Viana PL,Welker CAD,Zanin A. 2015. Poaceae in Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro. Disponivel em: . Acesso em: 13 outubro 2016. Fisher A, Triplett JK, Ho C-S, Schiller A, Oltrogge K, Schroder E, Kelchner SA, Clark LG 2009 Paraphyly in the bamboo subtribe Chusqueinae and a revised infrageneric classification for Chusquea. Systematic Botany 34: 673-683. http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB193 8 Fisher AE, Clark LG, Kelchner SA 2014 Molecular phylogeny estimation of the bamboo genus Chusquea (Poaceae: Bambusoideae: Bambuseae) and description of two new bamboo subgenera. Systematic Botany 39: 829-844. Grass Phylogeny Working Group (GPWG) 2001 Phylogeny and subfamilial classification of the grasses (Poaceae). Annals of the Missouri Botanical Garden 88: 373-456. Grass Phylogeny Working Group II (GPWG II) 2012 New grass phylogeny resolves deep evolutionary relationships and discovers C4 origins. New Phytologist 193: 304-312. Judziewicz EJ, Clark LG, Londoño X, Stern MJ 1999 American Bamboos. Smithsonian Institution Press, Washington, DC. 392 p. Kelchner SA, Bamboo Phylogeny Group (BPG) 2013 Higher level phylogenetic relationships within the bamboos (Poaceae: Bambusoideae) based on five plastid markers. Molecular Phylogenetics and Evolution 67: 404-413. Leandro TD, Shirasuna RT, Filgueiras TS, Scatena VL 2016a The utility of Bambusoideae (Poaceae, Poales) leaf blade anatomy for identification and systematics. Brazil Journal of Biology 76: 708-717. Leandro TD, Scremin-Dias E, Arruda RCO 2016b Micromorphology and anatomy of the leaf blade: a contribution to the taxonomy of Luziola (Poaceae, Oryzoideae) from the Pantanal, Brazil. Plant Systematics and Evolution 302: 265-273. Linder HP, Rudall PJ 2005 Evolutionary history of Poales. Annual Review of Ecology, Evolution and Systematics 36: 107-124. Longhi-Wagner HM 2012 Poaceae: an overview with references to Brazil. Rodriguésia 63: 89- 100. Metcalfe CR 1960 Anatomy of the Monocotyledons I: Gramineae. Oxford: Claredon Press. Prat H 1960 Vers une classification naturelle des Gramineés. Bulletin de la Societé Botanique de France 107: 32-79. 9 Prat H 1961 Emploi des characters epidermiques dans la classification des Gramineés in Recent Advances in Botany 1: 92-102, University of Toronto Press. Sánchez-Ken JG, Clark LG, Kellogg EA, Kay EE 2007 Reinstatement and emendation of subfamily Micrairoideae (Poaceae). Systematic Botany 32: 71-80. Soreng RJ, Peterson PM, Romaschenko K, Davidse G, Zuloaga FO, Judziewicz EJ, Filgueiras TS, Davis JI, Morrone O 2015. A worldwide phylogenetic classification of the Poaceae (Gramineae). Journal of Systematics and Evolution 53: 117-137. Stevens PF 2001 onwards Angiosperm Phylogeny Website. Version 12, July 2012 [and more or less continuously updated since]. Disponível em: . Acesso em: 12 outubro 2016. Schwendener S 1890 Die Mestomscheiden der Gramineen-blatter. Sitz.-ber. Akad. Berlin 1980: 405-426. Tateoka T 1963 Notes on some grasses. XII. Relationships between Oryzeae and Ehrharteae, with special reference to leaf anatomy and histology. Botanical Gazette 124: 264-270. Viana PL, Filgueiras TS, Paiva EAS 2011 A new combination in Aulonemia (Poaceae: Bambusoideae: Bambuseae) based on floral analysis, anatomical features, and distribution. Brittonia 63: 102-112. Viana PL, Filgueiras TS, Graciano-Ribeiro D 2013 A new woody (Poaceae, Bambusoideae) from Central Brazil, Aulonemia xerophylla. Novon 22: 371-376. Watson L, Dallwitz MJ 1992 The Grass Genera of the World. CAB International, Wallingford, U.K. 10 11 ILUSTRAÇÕES 12 Figuras 1-4. Relações filogenéticas de monocotiledôneas, Poales e Poaceae; e distribuição geográfica das famílias pertencentes ao clado graminídeo. 1. Relações filogenéticas das monocotiledôneas baseado em APG IV (2016). 2. Relações filogenéticas de Poales baseado em Bouchenak-Khelladi et al. (2014). 3. Relações filogenéticas de Poaceae baseado em Soreng et al. (2015). 4. Distribuição geográfica das famílias pertencentes ao clado graminídeo (Poaceae, Ecdeiocoleaceae, Joinvilleaceae e Flagellariaceae) retirado de Stevens (2001 onwards). 13 14 Figuras 5-19. Diversidade de espécies e aspecto geral lâmina foliar evidenciando caracteres úteis à sistemática de Poaceae. (16) secção transversal em microscopia ótica; (17-19) vista frontal em microscopia eletrônica de varredura. 5. Merostachys argyronema Lindm. 6. Merostachys pluriflora Munro ex E.G. Camus. 7. Imperata brasiliensis Trin. 8. Cenchrus echinatus L. 9. Oplismenus hirtellus (L.) P. Beauv. 10. Paspalum notatum Flüggé. 11. Merostachys neesii Rupr. 12. Merostachys riedeliana Rupr. ex Doell. 13. Merostachys scandens Send. 14. Chusquea capituliflora Trin. var. pubescens McClure & L.B. Sm. 15. Merostachys scandens. 16. Aulonemia pumila L.G. Clark & Lodoño (Clark 382). 17. Chusquea attenuata (Irwin et al. 29242). 18. Chusquea tenuiglumis (Hatschbach 42746). 19. Luziola bahiensis (Steud.) Hitchc. (Guglieri 1610). Imagens (5-6, 11-15) R T Shirasuna, (7-10) A. Guglieri-Caporal. es estômato; cs célula silicosa; ma macrohair; mi microhair; pr prickles. Barras de escala: 16 = 25 µm; 17 = 30 µm, 18 = 6 µm, 19 = 4 µm. 15 16 CAPÍTULO I The utility of Bambusoideae (Poaceae, Poales) leaf blade anatomy for identification and systematics T. D. Leandro, R. T. Shirasuna , T. S. Filgueiras e V. L. Scatena PUBLICADO NO PERIÓDICO BRAZILIAN JOURNAL OF BIOLOGY 76(3): 708-717 17 Abstract Bambusoideae is a diverse subfamily that includes herbaceous (Olyreae) and woody Arundinarieae and Bambuseae) bamboos. Species within Bambusae are particularly difficult to identify due to their monocarpic lifecycle and the often long durations between mass flowering events; whereas the herbaceous bamboos are pluricarpic, but often are found with no reproductive structures. The leaf blade anatomy of 16 sympatric species of native Brazilian bamboos (Olyreae and Bambuseae) from the Atlantic Rainforest was studied in order to detect useful features for their identification. All the studied species share the following features: epidermis with a single stratum of cells; adaxial bulliform cells; mesophyll with arm cells, rosette cells, and fusoid cells; and collateral vascular bundles. Herbaceous bamboos share two features: papillae scattered on the abaxial surface and parallel-sided arrays of bulliform cells; whereas woody bamboos share: centrally organized papillae and fan-shaped arrays of bulliform cells. Also within the woody bamboos, intercostal fibers and a midrib with only one vascular bundle (simple midrib) characterize the subtribe Arthrostylidiinae; whereas a midrib with more than one vascular bundle (complex midrib) and a stomatal apparatus with two pappilae per subsidiary cell characterize the subtribe Chusqueinae. There are also diagnostic features for the sampled species, such as: papillae shape, and the outline and structure of the midrib. An identification key for all the studied species is provided based on the anatomical features. Keywords: Arthrostylidiinae, Bambuseae, Chusqueinae, leaf blade, Olyreae. 18 Resumo Bambusoideae é uma subfamília que inclui diversas espécies de bambus herbáceos (Olyreae) e lignificados (Arundinarie e Bambuseae). Bambus lignificados geralmente apresentam dificuldades de delimitação e identificação, devido principalmente ao ciclo monocárpico e longa amplitude temporal entre florações; enquanto que bambus herbáceos possuem ciclo pluricárpico, porém frequentemente são encontrados em estágio vegetativo. Foi estudada a anatomia da lâmina foliar de 16 espécies de Bambusoideae (Olyreae e Bambuseae), simpátricas e nativas do Brasil, visando levantar caracteres úteis para sua identificação. Todos os táxons estudados compartilham: epiderme uniestratificada; células buliformes na face adaxial; mesofilo com células invaginantes, células em roseta e células fusoides; e feixes vasculares colaterais. Bambus herbáceos compartilham: papilas dispersas na face abaxial e grupos de células buliformes organizadas paralelamente; enquanto que bambos lignificados compartilham: papilas organizadas em colunas centrais e grupos de células buliformes em forma de leque. Ainda dentre os bambus lignificados, fibras intercostais e nervura central com apenas um feixe vascular (nervura central simples) caracterizam a subtribo Arthrostylidiinae; enquanto que nervura central com mais de um feixe vascular (nervura central complexa) e complexo estomático com duas papilas por célula subsidiária caracterizam a subtribo Chusqueinae. Há ainda caracteres anatômicos diagnósticos, tais como: forma da papila, e forma e estrutura da nervura central. Uma chave de identificação é fornecida baseada nos caracteres anatômicos relevantes à identificação das espécies estudadas. Palavras-chave: Arthrostylidiinae, Bambuseae, Chusqueinae, lâmina foliar, Olyreae. 19 1. Introduction The cosmopolitan family Poaceae comprise about 11,000 species found mainly in grasslands and forest formations (Watson and Dallwitz, 1992 onwards; GPWG II, 2012). Twelve subfamilies are recognized within Poaceae (GPWG II, 2012; Soreng et al., 2015), among them Bambusoideae, a monophyletic group that currently includes 1,482 described species (Clark et al., 2015). Three Bambusoideae tribes are recognized, two of which are found in the Neotropics: Bambuseae, which comprise the woody bamboos; and Olyreae, the herbaceous bamboos (Kelchner, 2013; Clark et al., 2015). The Atlantic Rainforest is considered an important center of bamboo diversity (Judziewicz et al., 1999), and Brazil occupies a leading position based on number of species (298) and high endemism (172) (Carvalho et al., 2016). Bambusoideae may be distinguished from other grass subfamilies by morphological, anatomical, and ecological characters. Monocarpic perennial lifecycle, lignified culms, branching nodes, pseudopetiolate leaves, and an outer ligule are characters worth mentioning for the woody bamboos (GPWG, 2001; BPG, 2012); whereas herbaceous bamboos are pluricarpic, usually unbranched, with quite weak culms and an inner ligule (Judziewicz et al., 1999). Together with, the strongly asymmetrically invaginated arm cells as seen in cross section are highly important for the recognition of Bambusoideae species (GPWG, 2001), and also represents one of the main synapomorphies for this group (Zhang and Clark, 2000; BPG, 2012). In general, the Poaceae taxonomy is mainly based on reproductive characters, such as the shape and structure of spikelets and inflorescence types (Longhi-Wagner, 2012). This is true more particularly for the herbaceous species, which generally bloom many times in their life cycle. In contrast, the woody bamboos bloom only once during a life cycle (Janzen, 1976; Filgueiras, 1988), and sometimes even herbaceous species are found with no reproductive 20 structures. For this reason, searching for vegetative characters in addition to the reproductive ones is highly important to aid in species identification, and anatomical features often have provided useful findings (e.g., Brandis, 1907; Prat, 1936; Brown, 1958; Metcalfe, 1960; Calderón and Soderstrom, 1973; Renvoize, 1987; Vieira et al., 2002; Guglieri et al., 2008; Oliveira et al., 2008; Pelegrin et al., 2009; Jesus Junior et al., 2012; Viana et al., 2013a, b; Leandro et al., 2016; Aliscioni et al., 2016). Considering that mostly bamboo plants have unique life cycles, but also the importance of the leaf blade anatomy for the taxonomy of grasses in general, we studied 16 sympatric species of native bamboos from the Atlantic Rainforest. We examined the leaf blade anatomy of three species of herbaceous bamboos and 13 species of woody bamboos in order to provide useful features for their identification. 2. Material and Methods 2.1. Sampling area The study was carried out with 16 native species sampled at Parque Estadual das Fontes do Ipiranga - PEFI (23° 38” 08” S and 23° 40’ 18” S - 46° 36’ 48” W and 46° 38’ 00” W) [Ipiranga State Park], a fragment of Atlantic Rainforest located in the State of São Paulo, Brazil. We have analysed three specimens per species, but only one voucher per specimens was included in the herbarium of the Instituto de Botânica (SP) (Table 1). The choice of taxa was based on a floristic study of the area that indicated the necessity of providing additional data in order to aid in species identification and conservation (Shirasuna and Filgueiras, 2013). Olyra loretensis Mez was not included in this study due to its uncertain occurrence in the PEFI [see Shirasuna and Filgueiras (2013) for details about each species]. 21 2.2. Anatomical analysis For the woody bamboos, mature leaf blades were taken from the branches at the mid- culm, whereas for the herbaceous bamboos mature leaves were taken from the third node from the base. Fresh plant material was fixed in FAA 50 (Johansen, 1940) and later stored in 70% ethanol. Found on leaves of Arthrostylidiinae species, the green stripe was excluded from this work due to its anatomical peculiarities in relation to the remainder of the leaf blade (Judziewicz et al., 1999). Samples from the middle portion of the leaf blade were embedded in polyethylene glycol 1500 solution (adapted from Richter, 1985) and cross-sectioned with a rotary microtome. Sections were cleared in sodium hypochlorite 50%, washed in distilled water, stained with Astra blue and Safranin (Bukatsh, 1972), and finally mounted on semi-permanent slides with glycerol. Also, a maceration technique was performed by the Jeffrey’s method (Johansen, 1940) in order to describe the epidermal features. Descriptions were primarily based on Ellis (1976, 1979), and optical images were obtained on a Leica DM4000B microscope using the software Leica Application Suite LASV4.0. 3. Results 3.1. Surface view All the studied taxa share an epidermis with long-short cell alternation (Figures 1A- M). Short cells occur as silica bodies (Figures 1D, F, K - arrow) or suberized cells (cork cells - arrowhead) (Figure 1A) – sometimes as silico-suberose couples in the intercostal zone (e.g., Figure 1A). The wall sinuosity of long cells may be deep (Figures 1B, E, K), moderate (Figures 1A, F) or slight (Figures 1G, J). Papillae commonly occur on the abaxial surface: less pronounced in the herbaceous species (e.g., Figure 1D) and more pronounced in the woody 22 species (e.g., Figures 1C, H). In Merostachys argyronema Lindm. papillae are very conspicuous (Figure 1H; Table 2), and in Merostachys neesii Rupr. they have a concave apex (Figure 1C; Table 2). A scattered distribution of papillae is observed in the herbaceous bamboos (e.g., Figure 1D; Table 2), whereas the organization in the woody bamboos is often in a single central row (e.g., Figures 1G, I), but may be variable in some intercostal cells (1-2 rows) (e.g., Figure 1H). Trichomes mainly occur on the abaxial surface and they may be of three types: (i) prickle hairs (short and silicified, microscopic unicellular) (Figures 1C, F); (ii) macrohairs (macroscopic unicellular) (Figures 1E); (iii) or microhairs (microscopic bicellular) (Figures 1E, L, M). The occurrence of these trichomes is variable among the studied species and only Chusquea capituliflora Trin var. pubescens McClure & L.B. Sm. has all the three types (Figures 1E, F). Prickle hairs of most of the species develop an enlarged base, usually as seen in Chusquea capituliflora var. pubescens (Figure 1F), but in M. neesii this base is more pronounced (Figure 1C). Macrohairs occur on the abaxial surface of C. capituliflora var. pubescens (Figure 1F) and Chusquea meyeriana Rupr. ex Doell (Figure 1I - scars). Bicellular microhairs often consist of cells of about the same size (e.g., Figure 1M), except for C. capituliflora var. pubescens, in which the apical cell is reduced (Figure 1L). Microhairs often occur on the abaxial surface in the woody species and Parodiolyra micrantha (Kunth) Davidse & Zuloaga. Stomata are paracytic and occur on the abaxial surface of all the studied species, but also on the adaxial surface in A. aristulata and Olyra humilis Nees (Figure 1B; Table 2). Stomatal apparatus comprise triangular subsidiary cells (Figures 1B, D, H, K) or semi-circular (cupuliform) cells (Figures 1A, E, G). In species of Chusquea the stomatal apparatus bears two papillae per subsidiary cell as seen in Chusquea capituliflora var. pubescens (Figure 1E detail inset; Table 2). 23 3.2. Cross section The epidermis consists of a single stratum of cells with slightly thickened outer walls (Figures 2A-Q). Epidermal cells are visually about the same size (Figures 2E, G, J), but may be larger on the adaxial side (Figures 2F, H, M) – excluding the bulliform cells. Bulliform cells occur as part of the adaxial epidermis (Figures 2D-M) and form a fan-shaped array in the woody bamboos, (e.g., Figures 2G, J, K; Table 2); whereas the herbaceous bamboos share a parallel-sided array of bulliform cells (e.g., Figures 2E, I; Table 2). The mesophyll comprise arm cells, fusoid cells, rosette cells, fibers, and vascular bundles. Asymmetrically invaginated arm cells are parallel to the epidermis (Figures 2E-M), including the midrib portion (Figures 2A-D). Herbaceous species (Figures 2E, I) and Chusquea bambusoides (Figure 2H) develop arm cells with invaginations only from the abaxial side, whereas the other species develop invaginations from both sides (Figures 2F, G, J-M). The number of rosette cells between each fusoid cell is often variable (one to four) within the same sample/specimen (Figures 2E-M). Fusoid cells occur adjacent to the vascular bundles and arm cells (Figures 2E-M); and their outline may be short and wide, as seen in A. aristulata (Figure 2G), or long and narrow, as in M. neesii (Figure 2F). Intercostal fibers located adjacent to the bulliform cells (sometimes also opposite) occur just among species of Arthrostylidiinae (e.g., Figures 2D, F, G; Table 2). Collateral vascular bundles are surrounded by a double sheath (Figures 2A-M): the outer one is parenchymatic and may be interrupted by fibers from both sides, as seen in Merostachys speciosa Spreng. (Figure 2M) or only from the abaxial side, as in Merostachys skvortzovii Send. (Figure 2L); and the inner one is pericyclic (mestome) with thick-walled cells (Figures 2A-M). First and third order vascular bundles are observed in all the studied species (Figures 2F-M). In most of the studied species the midrib is flat (e.g., Figure 2D), but it is abaxially projected in species of Chusquea (Figures 2A, B) and adaxially projected in Padoriolyra 24 micrantha (Figure 2C; Table 2). The midrib comprise one first order vascular bundle (Figures 2C, D), except among species of Chusquea, in which the midrib includes minor vascular bundles adjacent to the central one (Figures 2A, B; Table 2). With regard to the margin, the leaf blade may be acute (Figure 2N-P) or obtuse (Figures 2Q); always with thick-walled epidermal cells and fibers immediately subjacent to the epidermis (e.g., Figures 2N-Q). 3.3. Taxonomic treatment The main anatomical features are summarized in Table 2. These data in tabular form are available upon request from the first author. Identification key to the native Bambusoideae species from PEFI, SP, based on the leaf blade anatomical data (surface view and cross section) 1. Papillae scattered on the abaxial surface; parallel-sided arrays of bulliform cells (Tribe Olyreae) 2. Prickle hairs on the abaxial surface developed; midrib adaxially projected ……… ………………………………………………………………………… Parodiolyra micrantha 2’. Prickle hairs on the abaxial surface lacking; midrib slightly convex on both surfaces 3. Leaves amphistomatic; adaxial epidermal cells larger than abaxial epidermal cells (excluding the bulliform cells) .......................................................................... Olyra humilis 3’. Leaves hypostomatic; adaxial epidermal cells equal to sub-equal to the abaxial epidermal cells (excluding the bulliform cells) .......................................... Olyra glaberrima 1’ Papillae centrally organized in a single or double row on the abaxial surface; fan-shaped arrays of bulliform cells (Tribe Bambuseae) 25 4. Intercostal fibers developed; midrib with only one vascular bundle (simple midrib); stomata apparatus without papillae (Subtribe Arthrostylidiinae) 5. Stomata on both surfaces; papillae on the adaxial surface developed …….….... ……………………………………………………………………..….. Aulonemia aristulata 5’. Stomata only on the abaxial surface; papillae on the adaxial surface lacking 6. Central vascular bundle in the midrib (major one) with outer sheath interrupted by fibers from both sides 7. First order vascular bundle with outer sheath interrupted by fibers from the abaxial side …….……………………………………………...…..…… Merostachys scandens 7’. Fisrt order vascular bundle with outer sheath interrupted by fibers from both sides 8. Fusoid cells long and narrow ………………….……….. Merostachys magellanica 8’. Fusoid cells short and wide 9. Adaxial epidermal cells larger than abaxial epidermal cells (excluding the bulliform cells) ……………………………………………. Merostachys riedeliana 9’. Adaxial epidermal cells equal to sub-equal to the abaxial epidermal cells (excluding the bulliform cells) 10. Prickle hairs on the abaxial surface developed ………. Merostachys burmanii 10’. Prickle hairs on the abaxial surface lacking ……….... Merostachys pluriflora 6’. Central vascular bundle in the midrib (major one) with outer sheath interrupted by fibers only from the abaxial side 11. First order vascular vascular bundle with outer sheath interrupted by fibers from both sides 12. Bicellular microhairs on the adaxial surface developed; adaxial epidermal cells larger than abaxial epidermal cells (excluding the bulliform cells) ...……… 26 ……………………………………………………………………. Merostachys neesii 12’. Bicellular microhairs on the adaxial surface lacking; adaxial epidermal cells equal to sub-equal to the abaxial epidermal cells (excluding the bulliform cells) .………………………………………………….......……… Merostachys skvortzovii 11’. First order vascular vascular bundle with outer sheath interrupted by fibers only from the abaxial side 13. Fusoid cells short and wide; bicellular microhairs on the adaxial surface developed ………………………………………….......….. Merostachys argyronema 13. Fusoid cells long and narrow; bicellular microhairs on the adaxial surface lacking ……………………………………………….…......………….. Merostachys speciosa 4’. Intercostal fibers lacking; midrib with more than one vascular bundle (complex midrib); stomatal apparatus bearing two papillae per subsidiary cell (Subtribe Chusqueinae) 14. Midrib with two vascular bundles subjacent to the adaxial epidermis and opposite to the central one .................................................................................... Chusquea meyeriana 14’. Midrib with one vascular bundle subjacent to the adaxial epidermis and opposite to the central one 15. Prickle hairs on the adaxial surface developed; macrohairs on the abaxial surface developed; bicellular microhairs developed ...... Chusquea capituliflora var. pubescens 15’. Prickle hairs on the adaxial surface lacking, macrohairs on the abaxial surface lacking; bicellular microhairs lacking ……..…….…………….. Chusquea bambusoides 4. Discussion Our anatomical study demonstrates that papillae scattered on the abaxial surface and parallel-sided arrays of bulliform cells are exclusive features among the herbaceous bamboos sampled; whereas centrally organized papillae and fan-shaped arrays of bulliform cells are exclusive features among the woody bamboos sampled. 27 Within the herbaceous bamboos sampled, the midrib outline and amphistomatic leaves may distinguish Parodiolyra Soderstr. & Zuloaga from Olyra L. Although this may be true, it is not clear if these features are consistent among all Brazilian species of Olyra (20) and Parodiolyra (four) (Oliveira and Filgueiras, 2016a, b). Comparatively, within the woody bamboos sampled, intercostal fibers and a midrib with only one vascular bundle (simple midrib) characterize the subtribe Arthrostylidiinae; whereas a stomata apparatus bearing two papillae per subsidiary cell and a midrib with more than one vascular bundle (complex midrib) characterize the subtribe Chusqueinae. The presence of two papillae per subsidiary cell herein supports the assumption of this feature as a synapomorphy for Chusquea (Fisher et al., 2009, 2014), although there are not enough studies on micromorphology and anatomy to clarify its value. Currently, the set of features herein observed for Arthrostylidiinae and Chusqueinae is common among all species known within each subtribe and extremely applicable for recognizing these groups (BPG, 2012; Clark et al., 2015). The comparative anatomical analysis herein performed demonstrates that the variation in the distribution of papillae is useful for delimiting tribes. There are some reports showing the importance of this feature in bamboo systematics (e.g., Soderstrom and Ellis, 1987; Paisooksantivatana and Pohl, 1992; Yang et al., 2008; Gomes and Neves, 2009; Mota, 2013), but also for other closely related groups (e.g., Pelegrin et al., 2009). Our study is not able to define the value of this feature to the systematics of Olyreae and Bambuseae, therefore a detailed work to evaluate both distribution and type of papillae within different groups would be informative. Our study also indicates that some features may be considered diagnostic at the species level. Among them, the stomata on the adaxial surface in Aulonemia aristulata must be mentioned, since their occurrence is considered as rare for Aulonemia (Arthrostylidiinae) (Viana et al., 2013a), but usually typical for species within the subtribe Guaduineae 28 (Soderstrom and Ellis, 1987). Adaxial stomata were also recently observed in other species within Aulonemia (Viana, 2010; Viana et al., 2011), and thus it reinforces the anatomical affinity between the subtribes Arthrostylidiinae and Guaduineae (Bambuseae) (Soderstrom and Ellis, 1987; Zhang and Clark, 2000; Ruiz-Sanchéz et al., 2008), as well as the necessity of a broad anatomical study in order to elucidate the systematic value of this feature for Bambuseae. It is important to highlight that the size and shape of bulliform cells may be influenced by environmental factors (Shields, 1951), but the structural variation herein observed deserves more attention in order to verify its constancy among bamboo groups. Also, the fusoid cell is another feature that requires additional attention since its environmentally influenced morpho- anatomical variations (March and Clark, 2011; T. D. Leandro, unpubl. data). In the present study, we consider the structure of bulliform cells and the outline of fusoid cells as relevant features for delimiting species given that all specimens were sampled under the same environmental conditions. 5. Conclusion Although most of the information herein provided is not a novelty for Bambusoideae, our results reinforce the importance of leaf blade anatomy studies for grass systematics, specially when we consider the great number of questions that are still unclear. The inclusion of anatomical data as a routine on bamboo studies may be really useful for identifying diagnostic features and additional synapomorphies, in which certainly will aid in species circumscription. 29 Acknowledgments - This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (Ph.D. grant to the first author - proc. 163550/2012-3 and Productivity in Research grant to the last author - proc. 301692/2010-6). To Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP (proc. 2011/18275-0) and CNPq (proc. 471837/2011-3) also for the financial support. The authors are immensely grateful to Lynn G. Clark and Timothy J. Gallaher (EEOB, Iowa State University, U.S.A.) for their comments that greatly improved the manuscript. References ALISCIONI, S.S., OSPINA, J.C. and GOMIZ, N.E., 2016. Morphology and leaf anatomy of Setaria s.l. (Poaceae: Panicoideae: Paniceae) and its taxonomic significance. Plant Systematics and Evolution, vol 302, no. 2, pp. 173-185. doi: 10.1007/s00606-015-1251-9 BAMBOO PHYLOGENY GROUP (BPG), 2012. An updated tribal and subtribal classification of the bamboos (Poaceae: Bambusoideae). Bamboo Science and Culture: The Journal of the American Bamboo Society, vol. 24, no. 1, pp. 1-10. BRANDIS, D., 1907. V. Remarks on the structure of bamboo leaves. Transactions of the Linnean Society London Botany, vol 7, no. 5, pp. 69-89. doi: 10.1111/j.1095- 8339.1907.tb00152.x BROWN, W.V., 1958. Leaf anatomy in grass systematics. Botanical Gazette, vol. 119, p. 170-178. doi: 10.1086/335974 BUKATSH, F., 1972. Benerkemgem zeir Doppelfarbeing Astrablau-Safranina. Microkosmos, vol. 61, 255 p. CALDERÓN, C.E. and SODERSTROM, T.R., 1973. Morphological and anatomical considerations of the grass subfamily Bambusoideae based on the new genus Maclurolyra. Smithsonian Contribuitions to Botany, vol. 11, pp 1-55. doi: 10.5479/si.0081024X.11 http://dx.doi.org/10.1007/s00606-015-1251-9 30 CARVALHO, M.L.S., DÓREA, M.C., FERREIRA, F.M., MOTA, A.C., OLIVEIRA, R.C. DE, OLIVEIRA, R.P., FILGUEIRAS, T.S., REIS, P.A., SANTOS-GONÇALVES, A.P., SHIRASUNA, R.T., SILVA, A.S., VIANA, P.L., ZANIN, A. and LONGHI-WAGNER, H.M., 2016 [viewed 15 February 2016]. Bambusoideae In: Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro. Available from: http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB102232. CLARK, L.G., LONDOÑO, X. and RUIZ-SANCHEZ, E., 2015. Bamboo taxonomy and habitat. In: W. LIESE and M. KÖHL, (eds.). Bamboo: The plant and its uses, vol 10, pp. 1- 30. doi: 10.1007/978-3-319-14133-6_1 ELLIS, R.P., 1976. A procedure for standardizing comparative leaf anatomy in the Poaceae. I. The leaf-blade as viewed in transverse section. Bothalia, vol. 12, pp. 65-109. doi: 10.4102/abc.v12i1.1382 ELLIS, R.P., 1979. A procedure for standardizing comparative leaf anatomy in the Poaceae. II. The epidermis as seen in surface view. Bothalia, vol. 12, pp. 65-109. doi: 10.4102/abc.v12i4.1441 FILGUEIRAS, T.S., 1988. A floração dos bambus e seu impacto ecológico. Eugeniana, vol. 15, pp. 1-8. FISHER A.E., TRIPLETT J.K., HO C-S., SCHILLER A.D., OLTROGGE K.A., SCHRODER E.S., KELCHNER S.A., CLARK L.G., 2009. Paraphyly in the bamboo subtribe Chusqueinae and a revised infrageneric classification for Chusquea. Systematic Botany, vol. 34, no. 4, pp. 673-683. doi: 10.1600/036364409790139790 FISHER A.E., CLARK L.G. and KELCHNER S.A., 2014. Molecular phylogeny estimation of the bamboo genus Chusquea (Poaceae: Bambusoideae: Bambuseae) and description of two new bamboo subgenera. Systematic Botany, vol. 39, no. 3, pp. 829-844. doi: 10.1600/036364414X681554 http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB102232 http://dx.doi.org/10.1600/036364409790139790 31 GOMES, D.M.S. and NEVES, L.J., 2009. Scanning electron microscopy of the leaf epidermis of Merostachys Spreng. (Poaceae: Bambusoideae). Acta Botanica Brasilica, vol. 23, no. 2, pp. 516-525. doi: 10.1590/S0102-33062009000200023 GRASS PHYLOGENY WORKING GROUP (GPWG), 2001. Phylogeny and subfamilial classification of the grasses (Poaceae). Annals of the Missouri Botanical Garden, vol. 88, pp. 373-456. GRASS PHYLOGENY WORKING GROUP II (GPWG II), 2012. New grass phylogeny resolves deep evolutionary relationships and discovers C4 origins. New Phytologist, vol. 193, pp. 304-312. doi: 10.1111/j.1469-8137.2011.03972.x GUGLIERI, A., LONGHI-WAGNER, H.M. and ZULOAGA, F.O., 2008. Anatomia foliar das espécies de Panicum L. subg. Panicum (Poaceae: Panicoideae: Paniceae) no Brasil. Iheringia, Série Botânica, vol. 63, no. 2, pp. 279-293. JANZEN, D.H., 1976. Why bamboos wait so long to flower. Annual Review of Ecology and Systematics, vol. 7, no. 1, pp. 347-391. doi: 10.1146/annurev.es.07.110176.002023 JESUS-JUNIOR, L.A., OLIVEIRA, R.P., LEITE K.R.B. and SILVA, L.B., 2012. Comparative analysis of the leaf anatomy in two Parodiolyra species (Poaceae: Olyreae) occuring on forests in Earstern Brazil. Brazilian Journal of Biology, vol. 72, pp. 205-210. doi: 10.1590/S1519-69842012000100025 JOHANSEN, D., 1940. Plant microtechnique. McGrawn-Hill Book Co., New York. 523 p. JUDZIEWICZ, E.J., CLARK, L.G., LONDOÑO, X. and STERN, M.J., 1999. American Bamboos. Smithsonian Institution Press, Washington, DC. 392 p. KELCHNER, S.A. and BAMBOO PHYLOGENY GROUP (BPG), 2013. Higher level phylogenetic relationships within the bamboos (Poaceae: Bambusoideae) based on five plastid markers. Molecular Phylogenetics and Evolution, vol. 67, pp. 404-413. doi:10.1016/j.ympev.2013.02.005 http://dx.doi.org/10.1016/j.ympev.2013.02.005 32 LEANDRO, T.D., SCREMIN-DIAS, E. and ARRUDA, R.C.O., 2016. Micromorphology and anatomy of the leaf blade: a contribution to the taxonomy of Luziola (Poaceae, Oryzoideae) from the Pantanal, Brazil. Plant Systematics and Evolution, vol. 302, no. 3, pp.265-273. doi: 10.1007/s00606-015-1260-8 LONGHI-WAGNER, H.M., 2012. Poaceae: an overview with reference to Brazil. Rodriguésia, vol .63, pp. 89-100. doi: 10.1590/S2175-78602012000100008 METCALFE, R. 1960. Anatomy of the Monocotyledons I: Gramineae. Oxford, Claredon Press. 731 p. MOTA, A.C. 2013. Sistemática de Chusquea subgênero Rettbergia (Bambusoideae, Poaceae). Feira de Santana. Universidade Estadual de Feira de Santana, 229 p. Tese de Doutorado em Botânica. OLIVEIRA, R.P., LONGHI-WAGNER, HM. and LEITE, KRB., 2008. A contribuição da anatomia foliar para a taxonomia de Raddia Bertol. (Poaceae: Bambusoideae). Acta Botanica Brasilica, vol. 22, no. 1, pp. 1-19. doi: 10.1590/S0102-33062008000100002 OLIVEIRA, R.P. and FILGUEIRAS, T.S., 2016a [viewed 15 February 2016]. Olyra In: Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro [online]. Available from: http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB13360 OLIVEIRA, R.P. and FILGUEIRAS, T.S., 2016b [viewed 15 February 2016]. Parodiolyra In: Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro [online]. Available from: http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB13423 PAISOOKSANTIVATANA, Y. and POHL, RW., 1992. Morphology, anatomy and cytology of the genus Lithachne (Poaceae: Bambusoideae). Revista de Biologia Tropical, vol. 40, pp. 47-72. PELEGRIN, C.M.G. DE, LONGHI-WAGNER, H.M. and OLIVEIRA, P.L. DE, 2009. Anatomia foliar como subsídio à taxonomia de espécies do Complexo Briza L. (Poaceae: http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB13360 http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB13423 33 Pooideae: Poeae). Acta Botanica Brasilica, vol. 23, no. 3, pp. 666-680. doi: 10.1590/S0102- 33062009000300006 PRAT, H., 1936. La systemátique des Graminées. Annales des Sciences Naturelles Botanique, vol. 18, pp. 165-258. RENVOIZE, S.A., 1987. A survey of leaf blade anatomy in grasses X. Bambuseae. Kew Bulletin, vol. 42, pp. 201-207. doi: 10.2307/4109902 RICHTER, H.G., 1985. Wood and bark anatomy of Lauraceae II. Licaria Aublet. Iawa Bulletin, vol. 6, no. 3, pp. 187-199. doi: 10.1163/22941932-90000938 RUIZ-SANCHEZ, E., SOSA, V. and MEJÍA-SAULES, M.T., 2008. Phylogenetics of Otatea inferred from morphology and chloroplast DNA sequence data, and recircumscription of Guaduinae (Poaceae: Bambusoideae). Systematic Botany, vol. 33, no. 2, pp. 277-283. doi: 10.1600/036364408784571644. SHIELDS, L.M., 1951. The involution mechanism in leaves of certain xeric grasses. Phytomorphology, vol. 1, no. 3 and 4, pp. 225-241. SHIRASUNA, R.T. and FILGUEIRAS, T.S., 2013. Bambus nativos (Poaceae, Bambusoideae) no Parque Estadual das Fontes do Ipiranga, São Paulo, SP, Brasil. Hoehnea, vol. 40, no. 2, pp. 315-359. doi: 10.1590/S2236-89062013000200005 SODERSTROM, T.R. and ELLIS, R.P., 1987. The position of bamboo genera and allies in a system of grass classification. pp. 225-238. In: T.R. SOLDERSTROM, KW. HILU, C. S. CAMPBELL and M. E. BARKWORTH (eds.). Grass: Systematics and Evolution. Washington, D. C. Smithsonian Instituition Press. SORENG, R.J., PETERSON, P.M., KOMASCHENKO, K., DAVIDSE, G., ZULOAGA, F.O., JUDZIEWICZ, E.J., FILGUEIRAS, T.S., DAVIS, J.I., MORRONE, O., 2015. A worldwide phylogenetic classification of the Poaceae (Gramineae). Journal of Systematics and Evolution vol. 53, pp. 117-137. doi: 10.1111/jse.12150 34 VIANA, P.L., 2010. O gênero Aulonemia Goudot (Poaceae: Bambusoideae: Bambuseae) no Brasil. Belo Horizonte: Universidade Federal de Minas Gerais, 272 p. Tese de Doutorado em Biologia Vegetal. VIANA, P.L., FILGUEIRAS, T.S. and PAIVA, E.A.S., 2011. A new combination in Aulonemia (Poaceae: Bambusoideae: Bambuseae) based on floral analysis, anatomical features, and distribution. Brittonia, vol. 63, pp. 102-112. doi: 10.1007/s12228-010-9138-0 VIANA, P.L., FILGUEIRAS T.S. and GRACIANO-RIBEIRO, D., 2013a. A new woody bamboo (Poaceae, Bambusoideae) from Central Brazil, Aulonemia xerophylla. Novon, vol. 22, no. 3, pp. 371-376. doi: 10.3417/2010044 VIANA, P.L., FILGUEIRAS T.S. and CLARK, L.G., 2013b. Cambajuva (Poaceae: Bambusoideae: Bambuseae: Arthrostylidiinae), a new woody bamboo genus from Southern Brazil. Systematic Botany, vol. 38, no. 1, pp. 97-103. doi: 10.1600/036364413X662097 VIEIRA, R.C., GOMES, D.M.S., SARAHYBA, L.S. and ARRUDA, R.C.O., 2002. Leaf anatomy of three herbaceous bamboo species. Brazilian Journal of Biology, vol. 72, no. 1, pp. 205-210. doi: 10.1590/S1519-69842002000500021 WATSON, L. and DALLWITZ, M.J., 1992 (onwards) [viewed 25 February 2016]. The grass genera of the world: descriptions, illustrations, identification, and information retrieval Version: 8 December 2015. Available from: http://delta-intkey.com. YANG, H., WANG, H. and LI, D., 2008. Comparative morphology of the foliage leaf epidermis, with emphasis on papillae characters, in key taxa of woody bamboos of the Asian tropics (Poaceae: Bambusoideae). Botanical Journal of the Linnean Society, vol. 156, pp. 411-423. doi: 10.1111/j.1095-8339.2007.00736.x ZHANG, W. and CLARK, L.G., 2000. Phylogeny and classification of the Bambusoideae (Poaceae). pp. 35-42. In: S. W. L. JACOBS and J. E. EVERETT (eds.), Grasses: Systematics and Evolution. CSIRO Publishing, Collingwood, Victoria. http://dx.doi.org/10.3417/2010044 http://dx.doi.org/10.1600/036364413X662097 http://delta-intkey.com/ 35 TABLES 36 Table 1. Specimens used in this study, with classification and voucher information provided. Taxa and classification Voucher information TRIBE OLYREAE Subtribe Olyrineae Olyra glaberrima Raddi A. Custodio Filho 124 (SP 160961) O. humilis Nees R. T. Shirasuna 2617 (SP 415204) Parodiolyra micrantha (Kunth) Davidse & Zuloaga R. T. Shirasuna 2863 (SP 420323) TRIBE BAMBUSEAE Subtribe Chusqueinae Chusquea bambusoides (Raddi) Hack. R. T. Shirasuna & A. Costa 1809 (SP 409031) C. capituliflora Trin var. pubescens McClure & L.B. Sm. R. T. Shirasuna 2760 (SP 415735) C. meyeriana Rupr. ex Döll R. T. Shirasuna 2697 (SP 415247) Subtribe Arthrostylidiinae Aulonemia aristulata (Döll) McClure R. T. Shirasuna 2860 (SP 420320) Merostachys argyronema Lindm. R. T. Shirasuna 2868 (SP 420326) M. burmanii Send. J. F. Toledo s/nº (SP 238492) M. magellanica Send. M. T. Grombone s/nº (SP 412132) M. neesii Rupr. R. T. Shirasuna 2864 (SP 430324) M. pluriflora Munro ex. E.G. Camus R. T. Shirasuna 1811 (SP 409023) M. riedeliana Rupr. ex Döll R. T. Shirasuna 2872 (SP 426233) M. scandens Send. R. T. Shirasuna 2993 (SP 441817) M. skvortzovii Send. T. Sendulsky 1318 (SP 166796) M. speciosa Spreng. R. T. Shirasuna 2798 (SP 417980) 37 Table 2. Summary of leaf blade features useful for delimiting the tribes and subtribes, and also for recognizing the species. 1. Olyra humilis; 2. O. glaberrima; 3. Parodiolyra micrantha; 4. Chusquea bambusoides; 5. C. capituliflora var. pubescens; 6. C. meyeriana; 7. Aulonemia aristulata; 8. Merostachys argyronema; 9. M. burmanii; 10. M. magellanica; 11. M. neesii; 12. M. pluriflora; 13. M. riedeliana; 14. M. scandens; 15. M. skvortzovii; 16. M. speciosa. (+) presence, (-) absence. SPECIES Tribe Olyreae herbaceous bamboos Bambuseae woody bamboos Subtribe Olyrinae Chusqueinae Arthrostylidiinae FEATURES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Epidermis Papillae scattered on the abaxial surface + + + - - - - - - - - - - - - - Papillae centrally organized in a single or double row - - - + + + + + + + + + + + + + Papillae with concave apex - - - - - - - - - - + - - - - - Papillae (two) per subsidiary cell - - - + + + - - - - - - - - - - Microhairs with reduced apical cell - - - - + - - - - - - - - - - - Amphistomatic leaves + - - - - - + - - - - - - - - - Mesophyll Parallel-sided arrays of bulliform cells + + + - - - - - - - - - - - - - Fan-shaped arrays of bulliform cells - - - + + + + + + + + + + + + + Intercostal fibers - - - - - - + + + + + + + + + + Midrib Adaxially projected - - + + + + - - - - - - - - - - 38 Complex midrib - - - + + + - - - - - - - - - - Simple midrib + + + - - - + + + + + + + + + + With one vascular bundle opposite to the central one - - - - + - - - - - - - - - - - 39 FIGURES 40 Figure 1. Surface view of leaf blades of Bambusoideae species. Adaxial surface (B, F, K) and abaxial surface (A, C-E, G-J). (A) Chusquea bambusoides with cupuliform subsidiary cells (*) and silico-suberose couples; (B) Olyra humilis with triangular subsidiary cells (*); (C) Merostachys neesii showing papillae with concave apex (white circle); (D) Olyra glaberrima with silica body (black arrow) and papillae scattered; (E) Chusquea capituliflora var. pubescens with macrohairs (Ma), microhairs (Mi), and a detail showing the stomata apparatus bearing two papillae per subsidiary cell (white arrows); (F) Chusquea capituliflora var. pubescens with prickle (Pr); (G) Aulonemia aristulata with microhair (Mi); (H) Merostachys argyronema with papillae organized in a central row (or double row in some long cells); (I) Chusquea meyeriana showing the epidermis with macrohair scars (Ma); (J) Merostachys riedeliana with silica body (black arrow); (K) Parodiolyra micrantha showing silica body (black arrow); (L) Chusquea capituliflora var. pubescens detail of a bicellular trichome (microhair) with reduced apical cell; (M) Aulonemia aristulata detail of a bicellular trichome (microhair) with cells about the same size. (Ma) macrohair; (Mi) microhair; (Pr) prickles. Black arrows: silica bodies; white arrows: papillae; arrowhead: suberized cells; asterisks: stomata; white circle: papillae with concave apex. 41 FIG 1. 42 Figure 2. Cross section of leaf blades of Bambusoideae species. Midrib (A-D), mesophyll (E- M) and margin (N-Q). (A) Chusquea capituliflora var. pubescens showing complex midrib with one vascular bundle opposite to the central one; (B) Chusquea meyeriana showing complex midrib with two vascular bundles opposite to the central one; (C) Parodiolyra micrantha showing midrib with only one vascular bundle (simple midrib); (D) Aulonemia aristulata with intercostal fibers (If); (E) Olyra humilis showing arm cells with invaginations only from the abaxial side; (F) Merostachys neesii with intercostal fibers (If), bulliform cells (Bc) and arm cells (Ac); (G) Aulonemia aristulata showing arm cells with invaginations from both sides; (H) Chusquea bambusoides showing rosette cells (Rc); (I) Parodiolyra micrantha with papillae (black circle); (J) Chusquea capituliflora var. pubescens showing prickle hair (Pr); (K) Merostachys burmanii with silica body (arrowhead); (L) Merostachys skvortzovii showing vascular bundles with outer sheath interrupted by fibers from the abaxial side; (M) Merostachys speciosa showing vascular bundles surrounded by a double sheath; (N) Aulonemia aristulata with acute margin and thick-walled epidermal cells; (O) Chusquea capituliflora var. pubescens with acute margin and a few fibers; (P) Chusquea bambusoides with thick-walled epidermal cells; (Q) Chusquea meyeriana with obtuse margin and many fibers. (Ac) arm cells; (Bc) bulliform cells; (Fi) fibers; (If) intercostal fibers; (Pr) prickle hair; (Rc) rosette cells. Arrows: fusoid cells; arrowheads: silica bodies; black circles: papillae. 43 FIG 2. 44 CAPÍTULO II The contribution of foliar micromorphology and anatomy to the circumscription of species within the Chusquea ramosissima Clade (Poaceae, Bambusoideae, Chusqueinae) T. D. Leandro, V. L. Scatena e L. G. Clark ENVIADO PARA O PERIÓDICO PLANT SYSTEMATICS AND EVOLUTION 45 Abstract Chusquea is a diverse but monophyletic genus of Neotropical woody bamboos from primarily montane forests that comprises four well-supported lineages: subg. Magnifoliae, subg. Platonia, subg. Rettbergia and the Euchusquea clade (comprising subg. Swallenochloa and subg. Chusquea). The Euchusquea clade encompasses about 75% of the described species diversity within Chusquea and includes five minor clades with at least moderate support from molecular data. However, the relationships among and within the minor clades inferred from molecular data are mostly not congruent with those inferred from morphological evidence, consequently limiting our ability to understand species relationships. In this study we generated foliar micromorphological and anatomical data for one of the minor clades (referred to as the Chusquea ramosissima Clade) including three sampled species Chusquea ramosissima, C. tenella, and C. longispiculata, and for the morphologically closely related species C. attenuata, C. tenuiglumis and Chusquea sp. nov., in order to test the value of these types of characters for defining species and to seek potential synapomorphies for this clade. Our results demonstrate that epidermal features, mainly with regard to the stomatal apparatus, proved to be more valuable in distinguishing species than anatomical characters. The type of trichomes, type and arrangement of silica bodies, type of arm cells and the number of their layers in the mesophyll, and midrib structure may be useful to lesser degree. The inclusion of C. tenuiglumis within the Chusquea ramosissima Clade and the C. ramosissima informal group is supported based on morpho-anatomical similarities. Support for the recognition of a new species in this group from Bolivia is also provided by micromorphological characters. An identification key based on leaf blade features is provided for the six studied species. Keywords Bambuseae, Monocotyledons, Poales, woody bamboos 46 Introduction Bambuseae comprise one of three lineages within Bambusoideae and include at least 812 species in 66 genera (Clark et al. 2015). This tribe is mostly diversified in tropical forests, both lowland and montane, and humid habitats (Judziewicz et al. 1999; Clark et al. 2015). Two major lineages are currently recognized for Bambuseae based on molecular data (Sungkaew et al. 2009; Kelchner and BPG 2013): Paleotropical woody bamboos (four subtribes, 407 species), and Neotropical woody bamboos (three subtribes, 405 species) (BPG 2012; Clark et al. 2015). The phylogenetic relationship of the three subtribes within the Neotropical woody bamboos [(Arthrostylidiinae + Guaduinae) + Chusqueinae] has good support (Kelchner and BPG 2013; Wysocki et al. 2015), although no defining characters have been identified (Clark et al. 2015). Sister to each other, Arthrostylidiinae and Guaduinae are each well supported as monophyletic also based on morphological and molecular data (Ruiz-Sanchez 2011; Tyrrell et al. 2012; Kelchner and BPG 2013), while the monophyly of Chusqueinae, which includes only the genus Chusquea Kunth, is currently based primarily on molecular evidence although all members share the presence of two papillae per subsidiary cell and one-flowered spikelets with four glumes and no rachilla extension (Fisher et al. 2009; Kelchner and BPG 2013; Fisher et al. 2014). Bamboos grow vegetatively for long periods and then their life cycle usually ends after a single gregarious flowering episode (Seifriz 1950; Janzen 1976; Judziewicz et al. 1999). For this reason, vegetative morphological data from their rhizomes, culms and leaves remains extremely important for bamboo studies. Anatomical and micromorphological surveys in addition to morphological ones have established the importance of these types of features in providing the basis for a more natural classification within the grass family (e.g., Guglieri et al. 2008; Oliveira et al. 2008; Gomes and Neves 2009; Pelegrin et al. 2009; Aliscioni, et al. 2016; Leandro et al. 2016a,b). Although diagnosable macro-morphological characters of Chusquea 47 and its subgenera are well defined (Fisher et al. 2009; 2014), there is a lack of knowledge with respect to the value of leaf blade anatomical and micromorphological features across the genus, even considering the amount of data available in the literature (Clark 1986; Clark et al. 1989; Clark 1990; Guerreiro et al. 2013). Chusquea is the most diverse genus of Neotropical woody bamboos, comprising more or less 175 described species primarily from montane forests (Judziewicz et al. 1999; Fisher et al. 2014). Considering the classification previously provided by taxonomic work [see Clark (1989) and Fisher et al. (2009; 2014) for a detailed taxonomic history of the genus], plastid sequence data supports the existence of four clades within Chusquea (Fisher et al. 2014) (Fig. 1): (i) subg. Magnifoliae and (ii) subg. Platonia; (iii) subg. Rettbergia; and (iv) the major Euchusquea clade, comprising subg. Swallenochloa and subg. Chusquea and including about 75% of the species diversity of the genus. Fisher et al. (2014) also identified five minor clades (I-V) within the Euchusquea clade, but inferring their relationships with confidence remains a challenge because the morphologically-based infrageneric classification is largely incongruent with the molecular topology. In this study, our focus was on the minor but relatively well supported clade referred to as the Chusquea ramosissima Clade (Clade II in Fisher et al. 2014; Fig. 1), a group that includes several species distributed in Paraguay, Uruguay, Argentina and Brazil. Well supported as monophyletic, the Chusquea ramosissima Clade includes three sampled species (Fisher et al. 2014): Chusquea ramosissima Lindman, C. tenella Nees, and C. longispiculata L.G. Clark. Morphologically, all three species are classified within subg. Chusquea based on their extravaginal or infravaginal branching and the more or less constellate arrangement of subsidiary buds with respect to the central bud (Clark 2004; Fisher et al. 2009, 2014). [Note that the statement in the caption of Fig. 3 in Fisher et al. 2014 that C. longispiculata and several other species were not placed in a subgenus is erroneous.] In Fisher et al. (2009), C. ramosissima and C. tenella were included within the C. ramosissima informal group, within 48 subg. Chusquea, along with C. tenuiglumis Döll, based on culm leaves with pseudopetiolate, deciduous blades that usually remain green and racemose or weakly paniculate synflorescences (Fisher et al. 2014). Also, C. longispiculata, along with C. anelythra Nees, C. anelythroides Döll, C. attenuata (Döll) L.G. Clark and C. meyeriana Rupr. ex Döll, was classified as a member of the C. meyeriana informal group within subg. Chusquea based on the presence of spatheate bracts subtending the synflorescences, reflexed lower synflorescence branches and reduced glumes I and II (Clark 2004; Fisher et al. 2009). Previous work identified C. ramosissima, C. tenella, C. tenuiglumis, C. anelythra, C. anelythroides and others as included within subg. Rettbergia (Judziewicz et al. 1999), but Fisher et al. (2009, 2014) and Mota (unpublished data) based on molecular and morphological evidence excluded these species from subg. Rettbergia and placed them within subg. Chusquea. Considering its complex taxonomic history and the incongruence between the chloroplast phylogeny versus the morphology-based classification of Chusquea, the main objective of the current work was to test the value of leaf blade micromorphology and anatomy across species within the Chusquea ramosissima Clade of Fisher et al. (2014), and some of the species assigned to the two informal morphological groups that comprise this clade. To that end, Chusquea ramosissima, C. tenella, C. longispiculata, C. attenuata and C. tenuiglumis were studied. We also included a putatively undescribed species of Chusquea from Bolivia due to its strong vegetative similarity to C. ramosissima. Materials and Methods Taxon sampling The following three species comprising the Chusquea ramosissima Clade (Fisher et al. 2014) were studied: C. ramosissima and C. tenella of the C. ramosissima informal group and C. 49 longispiculata of the C. meyeriana informal group. For comparison, we also included two morphologically related species assigned to these informal groups (Fisher et al. 2009): Chusquea attenuata of the C. meyeriana informal group and C. tenuiglumis of the C. ramosissima informal group; and a putative new species similar to C. ramosissima referred to as Chusquea sp. nov. The species were studied from herbarium material deposited as vouchers at the Ada Hayden Herbarium of Iowa State University (ISC) and the Missouri Botanical Garden (MO) for those specimens of Chusquea sp. nov. (Table 1). While we were able to sample all species assigned to the C. ramosissima informal group, we sampled only the two species in the C. meyeriana informal group that have been well characterized morphologically. Circumscriptions for C. meyeriana, C. anelythra and C. anelythroides are currently being worked out but we did not sample these species for this study in order to avoid creating additional confusion. Micromorphology (scanning electron microscopy) Two pieces of approximately 0.5 cm2 of the middle portion of the leaf blade were removed from herbarium specimens. Pieces representing both surfaces were attached to a cylindrical sample holder (stub) and coated with a thin sample of gold (Denton vacuum Desk III). At least two specimens per species were treated with xylene to eliminate the epicuticular wax in order to provide a better view of micromorphological features (Dávila and Clark 1990). Observation and images of the surface view were obtained with the aid of a scanning electron microscope (JEOL JSM-5800LV) in the Microscopy and Nano-Imaging Facility (MNIF) at Iowa State University (ISU). 50 Anatomy (light microscopy) Fully expanded leaf blades were taken from herbarium specimens and the middle portion was excised and then immersed in polyethylene glycol 1.500 solution and kept in an incubator at 60ºC for fifteen days (adapted from Richter 1985). Cross sections were made using a Spencer 820 rotary microtome, and then they were cleared in 50% sodium hypochlorite, rinsed in distilled water, and then stained in Epoxy Tissue Stain (Spurlock et al. 1966). Semi-permanent slides were mounted in 50% glycerin and analyzed under a light microscope (Olympus BX- 40). Leaf clearings were performed in order to describe the structures in surface view. For this purpose, pieces of approximately 0.5 cm2 from the middle portion of the leaf blade were removed. These pieces were hydrated through a graded series of ethyl alcohol (50, 25 and 10%) and then soaked in 1:1/dH20 until the samples were translucent. Samples were rinsed with dH20, dehydrated through a graded series of ethyl alcohol (25, 50, and 70%), and then stained with Safranin and Fast Green (Johansen 1940). Stained samples were treated with ethyl alcohol (95 and 100%), xylene, and then xylene and Permount (1:1). Permanent slides were mounted with Permount. Images and terminology For both sections and clearings, photomicrographs were obtained with the aid of a Zeiss AXIO Observer microscope in the MNIF at ISU using ZEN 2.0 blue software, and applying different light contrast regimes. The terminology for the leaf blade in cross-section and in surface view primarily followed Ellis (1976; 1979). 51 Results The following data in tabular form are available upon request from the first author. A summary of the most important micromorphological and anatomical features for each species is provided in Table 2. Epidermal surfaces (SEM and clearings) Data for epidermal cells as seen with scanning electron microscopy (Figs. 2a-i, 3a-i) and with light microscopy of clearings (Fig. 4a-j, l, n) are merged in order to provide a better understanding. The epidermis consists of alternating long and short cells (Figs. 2a-i, 3a-i) with epicuticular wax (e.g., Fig. 2g). In general, long cells are tabular-shaped with thin, undulating anticlinal walls (Figs. 2a-i, 3a-i), except in C. attenuata, which exhibits highly thick anticlinal walls (Fig. 4f). Intercostal long cells on both leaf blade surfaces are usually densely covered by dome-shaped papillae that are often organized in one central row (Figs. 2a-c, g, 3a, g, 4i), but scattered papillae may be observed on the abaxial long cells of C. longispiculata (Fig. 2e-f), C. ramosissima (Fig. 2i) and C. tenella (Fig. 3b-c). Papillae are usually absent or poorly developed in the interstomatal band of the intercostal zone in C. longispiculata, C. ramosissima, and C. tenuiglumis (Figs. 2e, h, 3e), but are well developed in C. attenuata, C. tenella, and Chusquea sp. nov. (Fig. 2b, 3b, h). Intraspecific variation in the density of papillae is observed, in which papillae may be well-developed or poorly developed. Short cells may be developed as silica bodies or cork cells (Fig. 4d, i). Silica bodies occur in the costal zone on both surfaces and they are associated with tabular short cells (e.g., Fig. 4g, h), but also occur scattered on the adaxial surface in the intercostal zone (e.g., Figs. 3g, 4c). Cork cells are tall and narrow and occur as silico-suberose pairs mostly in the intercostal zone (Fig. 4d, i). In the costal zone, silica bodies are vertically oriented (e.g., Fig. 4a) or 52 horizontally oriented (e.g., Fig. 4b), but in the interstomatal band of the intercostal zone, silica bodies are always vertically oriented (e.g., Fig. 3b, 4i). With regard to shape, silica bodies are mainly saddle-shaped (e.g., Fig. 4a, g), but dumbbell-shaped (Fig. 4b, h) and equidimensional- shaped (not shown) bodies can be observed in the same specimen/sample. The vertically oriented pattern in the costal zone is observed just in C. attenuata and C. longispiculata (Fig. 4a), and in these two species, the silica bodies are exclusively saddle-shaped. Stomata are always observed on the abaxial epidermis (Figs. 2b-c, e-f, h-i, 3b-c, e-f, h- i), but they occur scattered on the adaxial epidermis in C. longispiculata, C. tenuiglumis (Figs. 3d, 4l), and Chusquea sp. nov. (Fig. 4n). On the abaxial epidermis, the number of rows of stomata can be variable (three to six) among species (e.g., Figs. 2h, 3e), and usually there is only one interstomatal long cell separating the stomata (e.g., Fig. 3f). The stomatal apparatus is paracytic (e.g. Fig. 4j) with subsidiary cells that are low dome-shaped (Fig. 4c) or low triangular-shaped (Fig. 4j) and bearing two papillae, each one branched (Figs. 3c, i, 4f) or not (Fig. 2f, 3c, i). Branched papillae completely overarch the guard cells and form a chamber above the pore in C. ramosissima (Fig. 2i) and C. tenuiglumis (Fig. 3f), whereas in C. attenuata the branches do not overarch the pore (Fig. 2c). In C. tenella and Chusquea sp. nov., the simple papillae arch over and meet across the guard cells, but do not form a chamber (Fig. 3c, i). Papillae are not branched in C. longispiculata, but the stomatal apparatus is often overarched by papillae from adjacent intercostal long cells (Fig. 2f). Trichomes can be of five types: (i) silicified and pointed unicellular with the base forming an integral part of the epidermis (prickle hair); (ii) silicified unicellular with a cell that emerges straight out of the epidermis (microhair); (iii) non-silicified unicellular (macrohair); (iv). non-silicified bicellular with a basal and a distal cell (microhair); (v). non-silicified bicellular with many specialized epidermal cells adjacent to the hair base (microhair). Prickle hairs occur on both surfaces in C. attenuata (Fig. 2a-b), C. tenella (Fig. 3a-b), and Chusquea sp. nov. (Fig. 3h). Non-silicified macrohairs occur on both surfaces of Chusquea sp. nov. (Fig. 53 3g-h) and may be observed on the abaxial surface of C. ramosissima, C. attenuata (Fig. 2b), and C. tenella (Fig. 3b). Silicified unicellular microhairs occur often on the abaxial surface in C. tenella, C. attenuata (Fig. 2c), Chusquea sp. nov. (Fig. 3g), and C. longispiculata (Fig. 4e), whereas bicellular microhairs occur on the adaxial surface of all the studied species (e.g., Figs. 2h, 3b, e, h). Cross section The outline of the leaf blade as seen in cross section is gently undulated (Fig. 4k-n). The epidermis consists of a single layer of cells with slightly thickened outer periclinal walls in most of the species (Fig 4l-n), but strongly thickened ones in C. longispiculata (Fig. 4k, q). Epidermal cells are all about the same size except for the presence of fan-shaped arrays of bulliform cells in between the bundles as part of the adaxial epidermis (Fig. 4k-n). The number of bulliform cells in each array is variable among samples within the same species. The mesophyll comprises asymmetrically invaginated arm cells, fusoid cells, rosette cells, and vascular tissue (Fig. 4k-n). Arm cells are organized in one or two layers in C. longispiculata (e.g., Fig. 4k, q), and in two layers in the remainder of species (e.g., Fig. 4l-n). Invaginations of the arm cells are mostly from the abaxial side (Fig. 4l-n, r), but invaginations from both sides are observed in C. longispiculata (Fig. 4k, q), C. attenuata, and C. ramosissima. Just one layer of arm cells is observed adjacent to the abaxial surface, in which the invaginations often occur just from the adaxial side (Fig. 4k, r). Surrounded by arm cells and between vascular bundles, the fusoid cells are highly variable in shape and size among and within species/specimens (Fig. 4k-n). These cells occur along the entire lamina, except at the margins. Rosette cells occur between fusoid cells (Fig. 4k-n), with the number of cells extremely variable in each group along the leaf blade. 54 With regard to the vascular tissue, xylem and phloem are collateral and surrounded by a double sheath, the inner mestome sheath and the outer parenchymatic sheath (Fig. 4k-n). Vascular bundles comprise two types: (i) first order with distinguishable metaxylem and phloem (Fig. 4k-n); and (ii) third order, small vascular bundles with usually a few lignified tracheary elements and a small patch of phloem (Fig. 4k-n). Girders occur in both types of vascular bundles, but only the parenchymatic sheath from the first order vascular bundles is abaxially interrupted by this arrangement of fibers (Fig. 4k-n). The midrib can be of two types: (i) composed of two opposing vascular bundles sharing the same inner sheath (Fig. 4o); or (ii) composed of three vascular bundles, two opposite to the central one, but all three sharing the same inner sheath (Fig. 4p). Leaf blade margins are mainly acute, but one acute and one obtuse margin (dimorphic) may be observed within the same leaf blade of C. attenuata and C. ramosissima. Also, the amount of sclerenchyma cells in this region is variable among species and within the same sample. Discussion Micromorphological and anatomical data Long cells with papillae; fan-shaped arrays of bulliform cells; a mesophyll with arm cells, fusoid cells, rosette cells, collateral vascular bundles surrounded by a double sheath; and a C3 photosynthetic pathway are features shared by all the studied species, and also are general for Bambusoideae (Metcalfe 1960; Hattersley 1987; Judziewicz et al. 1999; Sanchez-Ken et al. 2001; Oliveira et al. 2008; Gomes and Neves 2009; Viana 2010; Mota 2013; Leandro et al. 2016b). This study reveals many anatomical similarities among the sampled species while also demonstrating that micromorphological features prove to be more systematically informative 55 even considering notable intraspecific variation observed in papillae development. Presence or absence of trichomes and their type, type and arrangement of silica bodies, type of arm cells and the number of their layers in the mesophyll, and the midrib structure may be useful at the species level. Implications for systematics Anatomically, the occurrence of intercostal fibers, presence or absence of papillae on the subsidiary cells and midrib structure seem to be the primary differences between the subtribes Chusqueinae and Arthrostylidiinae (Soderstrom and Ellis 1987; Leandro et al. 2016b). Intercostal fibers and a simple midrib are diagnostic features for Arthrostylidiinae, whereas species within Chusqueinae may be recognized by the presence of stomatal apparatus bearing two papillae per subsidiary cell, lack of intercostal fibers, and a complex midrib (Fisher et al. 2009; 2014; Clark et al. 2015). Comparatively, Guaduinae may be distinguished from these two subtribes by the upper epidermis having abundant stomata and often well-developed papillae (Judziewicz et al. 1999; Ruiz-Sanchez et al. 2008; BPG 2012; Clark et al. 2015). Guaduinae also lack intercostal fibers and possess a complex midrib, and both the Arthrostylidiinae and Guaduinae lack any papillae on the subsidiary cells (Clark et al. 2015). The stomatal apparatus in Bambusoideae (and Poaceae) is composed of a pair of guard cells surrounded by two subsidiary cells (Metcalfe 1960; Ellis 1979; Judziewicz et al. 1999). Subsidiary cells bearing two papillae is a feature shared by all the studied species, and is putatively a synapomorphic character for Chusquea (Fisher et al. 2009, 2014; Clark et al. 2015). Although the presence of branched papillae has been previously reported for other species of Chusquea (Clark 1986, Clark 1989), the presence of two branched papillae that completely overarch the guard cells herein observed in Chusquea ramosissima and C. tenuiglumis is highly informative to identify these two species. 56 Another interesting feature is the midrib. Species herein studied share a complex midrib vasculature as expected for Chusquea (BPG 2012; Clark et al. 2015), although not composed solely of true vascular bundles. The complex midrib in species placed within subg. Rettbergia (currently 15 described species) often comprise at least two vascular bundles, each one usually with their own bundle sheaths (except for C. pulchella L.G. Clark and C. sellowii Rupr.) (Mota 2013); whereas a midrib composed of one central vascular bundle that shares its own bundle sheaths with adjacent vascular bundles is a common feature among the species herein studied, which are all placed within subg. Chusquea (Fisher et al. 2009). In the latter case, a midrib with a few elements of xylem and phloem embedded in fibers is observed adjacent to the central vascular bundle in the midrib—and sometimes these are difficult to distinguish. Hence, considering the differences in midrib development observed thus far between these subgenera, midrib vasculature in Chusquea deserves a broad study in order to verify its value for systematics across the genus. With regard to morphology, the sympatric C. ramosissima and C. tenella, and also C. tenuiglumis exhibit culm leaves with pseudopetiolate, deciduous blades that usually remain green and racemose or weakly paniculate synflorescences (Fisher et al. 2014). Micromorphologically, these three species exhibit branched papillae, but they may be easily distinguished by a set of features including type of papillae on the stomatal apparatus (simple or branched), type of invagination of the arm cells (abaxial or from both sides), and midrib structure (comprising two or three vascular bundles). The stomatal apparatus is also useful to distinguish C. ramosissima and Chusquea sp. nov., species closely related by sharing a similar culm leaf structure, branch complement, foliage leaf blade size, and horizontally oriented silica bodies—although the much wider leaf blades of Chusquea sp. nov. suggested that it might deserve recognition as a distinct species. The results of this study support this, and we are in the process of describing it as a new species. 57 The Chusquea meyeriana informal group (Fisher et al. 2009), herein represented by C. longispiculata and C. attenuata, can be recognized morphologically by the spatheate bracts often subtending the synflorescences, reflexed lower inflorescence branches, and reduced glumes I and II (Fisher et al. 2014). The species exhibit many micromorphological and anatomical similarities, such as silica bodies vertically oriented, subsidiary cells low-dome shaped, and arm cells with invaginations from both sides. The latter is also one of the features observed in C. meyeriana (Leandro et al. 2016b), and C. ramosissima, within the Chusquea meyeriana and C. ramosissima informal groups, respectively (Fisher et al. 2009). The orientation of the silica bodies was the only consistent difference herein observed between these informal groups, in which species in the Chusquea ramosissima informal group exhibit horizontally oriented silica bodies and in the Chusquea meyeriana informal group vertically oriented. As a final conclusion, considering our results and in parallel with the anatomical data presented by Mota (2013) for subg. Rettbergia, the inclusion of C. ramosissima and similar species within the Euchusquea clade as proposed by Fisher et al. (2014) is confirmed. The presence of branched papillae on the subsidiary cells and horizontally oriented silica bodies support the inclusion of C. tenuiglumis as member of the Chusquea ramosissima Clade (Fisher et al. 2014), whereas vertically oriented silica bodies support the recognition of the Chusquea meyeriana informal group (Fisher et al. 2009). We are not yet able to define an additional feature or a set of features that support the monophyly of the Chusquea ramosissima Clade. Lastly, we corroborate the molecular evidence that supported C. ramosissima as sister to C. tenella (Fisher et al. 2014)—although C. tenuiglumis (not sampled in their work) exhibits more morpho-anatomical similarities to C. ramosissima. 58 Taxonomic treatment Identification key based on micromorphological and anatomical data of species of Chusquea herein studied 1a. Each subsidiary cell bearing two branched papillae (e.g., Fig. 2c, i) …...……...…...…….... 2 1b. Each subsidiary cell bearing two simple papillae (e.g., Fig. 3c) ……………...………..…... 4 2a. Papillae with branches that completely overarch the guard cells forming a chamber above the pore (e.g., Fig. 2i) …………………...….……………………………...…………………… 3 2b. Papillae with branches that do not overarch the guard cells (Fig. 2c) ..…...… 4. C. attenuata 3a. Arm cells with invaginations from the abaxial side only (e.g., Fig. 4r); stomata on both surfaces of the leaf blade (Fig. 4l) ..……………………...……………….…... 5. C. tenuiglumis 3b. Arm cells with invaginations from both sides (e.g., Fig. 4q); stomata on only the abaxial surface of the leaf blade …………….....…...................................................… 3. C. ramosissima 4a. Midrib composed of two opposing vascular bundles (e.g., Fig. 4o) …………....…….…..... 5 4b. Midrib composed of three vascular bundles, two smaller ones opposing the major one (e.g., Fig. 4p) ….. ………………………………………………………………………… 1. C. tenella 5a. Costal silica bodies horizontally oriented (e.g., Fig. 4b); prickles and macrohairs developed; arm cells with invaginations from the abaxial side only (e.g., Fig. 4r) ...… 6. Chusquea sp. nov. 5b. Costal silica bodies vertically oriented (Fig. 4a); prickles and macrohairs lacking; arm cells with invaginations from both sides (Fig. 4q) …………………………......... 2. C. longispiculata General features for all species of the Chusquea ramosissima Clade herein studied ADAXIAL SURFACE: LONG CELLS tabular with undulate anticlinal walls. PAPILLAE often present on the long cells (usually absent or poorly developed in C. longispiculata and C. 59 tenuiglumis); often present on the bulliform cells (except in C. longispiculata and C. tenuiglumis). SHORT CELLS over the veins and usually scattered between the veins: SILICA BODIES saddle-shaped, dumbbell-shaped or equidimensional; vertically oriented or horizontally oriented. CORK CELLS usually present; occurring in silico-suberose pairs. PRICKLES often present (absent in C. longispiculata and C. tenuiglumis). MICROHAIRS often present; unicellular and silicified or bicellular and non-silicified. MACROHAIRS rarely present (observed in Chusquea sp. nov.); unicellular. STOMATAL APPARATUS usually absent (but often observed in C. tenuiglumis and rarely in C. longispiculata); with subsidiary cells low dome-shaped or low triangular-shaped; two papillae per subsidiary cell, branched or simple. ABAXIAL SURFACE: LONG CELLS tabular with undulate anticlinal walls. PAPILLAE often present on the long cells (except over the veins in C. longispiculata); often present on the bulliform cells (except in C. longispiculata and C. tenuiglumis). SHORT CELLS mainly over the veins: SILICA BODIES saddle-shaped, dumbbell-shaped or equidimensional; vertically oriented or horizontally oriented. CORK CELLS usually present; occurring in silico-suberose pairs. PRICKLES often present (absent in C. longispiculata and C. tenuiglumis). MICROHAIRS often present; unicellular and silicified or bicellular and non-silicified. MACROHAIRS usually observed in C. attenuata, C. tenella, and