Universidade Estadual De Campinas Instituto De Biologia

UNIVERSIDADE ESTADUAL DE CAMPINAS INSTITUTO DE BIOLOGIA

ARINAWA LIZ DEL PRADO FILARTIGA

MORPHOANATOMICAL AND ESSENTIAL OIL ANALYSIS OF FOUR SPECIES OF ALDAMA LA LLAVE (ASTERACEAE: HELIANTHEAE)

ANÁLISES MORFOANATÔMICAS E DO ÓLEO ESSENCIAL DE QUATRO ESPÉCIES DE ALDAMA LA LLAVE (ASTERACEAE: HELIANTHEAE)

CAMPINAS

2016

ARINAWA LIZ DEL PRADO FILARTIGA

Morphoanatomical and essential oil analysis of four species of Aldama La Llave (Asteraceae: Heliantheae)

Análises morfoanatômicas e do óleo essencial de quatro espécies de Aldama La Llave (Asteraceae: Heliantheae)

Thesis presented to the Institute of Biology of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Plant Biology.

Tese apresentada ao Instituto de Biologia da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do Título de Doutora em Biologia Vegetal.

ESTE ARQUIVO DIGITAL CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELA ALUNA ARINAWA LIZ DEL PRADO FILARTIGA E ORIENTADA PELA PROFª. DRª. BEATRIZ APPEZZATO DA GLÓRIA.

Orientadora: PROFª. DRª. BEATRIZ APPEZZATO DA GLÓRIA

Co-Orientadora: DRª. VERA LÚCIA GARCIA

CAMPINAS

2016 Agência(s) de fomento e nº(s) de processo(s): FAPESP, 2012/02476-0; FAPESP, 2010/51454-3

Ficha catalográfica Universidade Estadual de Campinas Biblioteca do Instituto de Biologia Mara Janaina de Oliveira - CRB 8/6972

Filartiga, Arinawa Liz, 1986- F472m FilMorphoanatomical and essential oil analysis of four species of Aldama La Llave (Asteraceae : Heliantheae) / Arinawa Liz del Prado Filartiga. – Campinas, SP : [s.n.], 2016.

FilOrientador: Beatriz Appezzato da Glória. FilCoorientador: Vera Lúcia Garcia. FilTese (doutorado) – Universidade Estadual de Campinas, Instituto de Biologia.

Fil1. Asteraceae. 2. Pampas (Rio Grande do Sul). 3. Estruturas secretoras. 4. Óleo essencial. 5. Órgãos subterrâneos. I. Appezzato-da-Glória, Beatriz. II. Rehder, Vera Lúcia Garcia,1959-. III. Universidade Estadual de Campinas. Instituto de Biologia. IV. Título.

Informações para Biblioteca Digital

Título em outro idioma: Análises morfoanatômicas e do óleo essencial de quatro espécies de Aldama La Llave (Asteraceae : Heliantheae) Palavras-chave em inglês: Asteraceae Pampas (Rio Grande do Sul) Secretory structures Essential oil Underground parts Área de concentração: Biologia Vegetal Titulação: Doutora em Biologia Vegetal Banca examinadora: Beatriz Appezzato da Glória [Orientador] Ricardo Cardoso Vieira Marcia Ortiz Mayo Marques Juliana Lischka Sampaio Mayer André Olmos Simões Data de defesa: 08-08-2016 Programa de Pós-Graduação: Biologia Vegetal

Campinas, 08 Agosto de 2016

COMISSÃO EXAMINADORA

Profª. Drª. Beatriz Appezzato da Glória

Prof. Dr. Ricardo Cardoso Vieira

Drª. Marcia Ortiz Mayo Marques

Profª. Drª. Juliana Lischka Sampaio Mayer

Prof. Dr. André Olmos Simões

Os membros da Comissão Examinadora acima assinaram a Ata de defesa, que se encontra no processo de vida acadêmica da aluna.

AGRADECIMENTOS

Agradeço a todos que permitiram que essa tese fosse realizada e àqueles que tornaram este caminho mais prazeroso.

Primeiramente, agradeço à minha orientadora, Profa. Dra. Beatriz Appezzato-da- Glória, da Escola Superior de Agricultura ‘Luiz de Queiroz’, pela possibilidade de desenvolver este trabalho e especialmente pela confiança e amizade. Levarei comigo todos os aprendizados e experiências que tive durante este período.

À minha co-orientadora Dra. Vera Lúcia Garcia, do Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas, por possibilitar que meu desejo de conhecer um pouco mais sobre a química das plantas fosse concretizado. Agradeço por todo auxílio técnico, pelo direcionamento e pelo carinho.

À Dra. Jitka Klimešová, chefe da Seção de Ecologia Vegetal, da Academy of Science of the Czech Republic, por me mostrar a beleza das plantas clonais e da simplicidade da vida. Agradeço também às famílias Klimeš e Bartušk pelo acolhimento e carinho durante a estadia na República Tcheca.

À Profa. Dra. Mara Magenta, da Universidade Santa Cecília, pelas recomendações nas coletas das Aldama, pela identificação dos exemplares e pela simpatia.

Aos alunos Vanessa Bassinello e Gustavo Filippi pela cooperação, dedicação e também por me mostrarem os diversos aprendizados de uma orientação.

Aos amigos e colegas do Laboratório de Anatomia Vegetal (LanVeg), da Escola Superior de Agricultura ‘Luiz de Queiroz’, pelo apoio, amizade e paciência. Em especial agradeço à Ms. Marli Kasue, técnica do LanVeg, pelo suporte técnico, pela simpatia e por dividir conhecimentos fantásticos sobre a cultura Japonesa.

Agradeço à Fundação de Amparo à Pesquisa do Estado de São Paulo pela concessão das bolsas de estudo nacional (n° 2012/02476-0) e internacional (2014/09401-0), e pelo financiamento proveniente do projeto temático (nº 2010/51454- 3), os quais permitiram a realização deste trabalho.

Também deixo meus agradecimentos aos meus pais Sarita e Carlos, às minhas irmãs Jana e Luna, e ao meu sobrinho Omam, seu incentivo e carinho foram muito importantes durante este período.

Por fim, agradeço ao William Motta de Abreu por seu companheirismo, pela paciência, e pelo apoio ao longo de toda essa trajetória. O seu amor foi fundamental. RESUMO

Asteraceae é considerada uma das maiores famílias dentre as Eudicotiledôneas, representando cerca de 10% da flora mundial. O sucesso desta família nos diferentes ecossistemas está relacionado à diversidade de formas de vida, aos métodos de propagação e à produção de metabólitos bioativos. Dentre os 1.600-2.000 gêneros que compõem a família, Aldama La Llave é constituído por 112 espécies sul-americanas, das quais 35 ocorrem em território brasileiro. A análise filogenética mais recente feita para o grupo foi realizada em 2011, mas foi considerada inconsistente devido ao baixo suporte estatístico dos ramos das espécies brasileiras. Desde então, muitos esforços têm sido aplicados para a obtenção de novos dados que possam auxiliar na filogenia e taxonomia do grupo. Por esse motivo, neste trabalho são investigadas espécies do gênero Aldama, com ênfase em A. anchusifolia, A. megapotamica, A. nudibasilaris e A. pilosa, as quais apresentam uma estreita relação. As análises apresentadas focam na morfoanatomia, fitoquímica e perfil dos óleos essenciais dos órgãos vegetativos aéreos e subterrâneos, buscando (i) novos caracteres com valor diagnóstico; (ii) evidenciar possíveis marcadores químicos; (iii) caracterizar as particularidades dos órgãos subterrâneos e sua capacidade de rebrotamento. Para as análises estruturais e histoquímicas, amostras dos órgãos aéreos (folha e caule) e subterrâneos (caule subterrâneo e raiz) foram processadas de acordo com as técnicas usuais em anatomia vegetal. Para as análises fitoquímicas, os óleos essenciais foram analisados por cromatografia a gás e espectrometria. No decorrer das análises morfoanatômicas foi possível identificar para folhas e caules aéreos um conjunto de características anatômicas único para cada espécie. Além disso, foi constatado que o número e o posicionamento de canais secretores na nervura central são bastante variáveis e devem ser utilizados com cuidado para a distinção de espécies. Para os caules subterrâneos foram observadas duas novidades relacionadas ao desenvolvimento de extensões e ao armazenamento de frutanos do tipo inulina, os quais diferem das características já relatadas para o gênero Aldama. A análise detalhada dos órgãos subterrâneos também aprimorou o conhecimento sobre o banco de gemas dessas estruturas. A avaliação do perfil químico dos óleos essenciais revelou a presença de terpenos exclusivos para cada espécie. Os dados gerados nesse estudo representam uma significativa contribuição para o conhecimento sobre as espécies de Aldama, uma vez que forneceram informações com potencial para auxiliar a distinção das espécies e novidades para o grupo, as quais serão úteis para futuras pesquisas em diversas áreas da botânica.

Palavras-chave Banco de gemas, canais secretores, frutanos, órgãos vegetativos, órgãos subterrâneos, padrão de venação, terpenos

ABSTRACT

Asteraceae is considered one of the largest families among the eudicots, representing about 10% of the world's flora. The success of this family in different ecosystems is related to the diversity of life forms, methods of propagation and production of bioactive metabolites. Among the 1600-2000 genera within the family, Aldama La Llave consists of 112 South American species, of which 35 occur in Brazil. The most recent phylogenetic analysis for the group was held in 2011, however was considered to be inconsistent due to the low statistical support of the branches of the Brazilian species. Thereafter, many efforts have been applied to obtain new data that may assist in the phylogeny and taxonomy of the group. For this reason, this work investigates Aldama species, with an emphasis on A. anchusifolia, megapotamica A., nudibasilaris and A. pilosa, of which have a close relationship. The analyzes focus on morphoanatomy, and phytochemical profile of essential oils of vegetative above and belowground organs, seeking (i) to find new characters with diagnostic value; (ii) to highlight potential chemical markers; (iii) to characterize the peculiarities of belowground organs and its regrowth capacity. For structural and histochemical analyzes, samples of aerial organs (leaf and stem) and underground organs (stems and roots) were processed according to the usual techniques for plant anatomy. For phytochemical analysis, essential oils were analyzed by gas chromatography and spectrometry. Morphoanatomical analysis allowed the identification of a unique set of anatomical features to leaves and aerial stems of each species. Furthermore, it was observed that the number and positioning of the secretory ducts in the midrib are variable and must be used carefully for distinguishing between species. For the underground stems were identified two new features related to the development of extensions and to the storing of fructans, of which differ from the features already reported to Aldama. The detailed analysis of the belowground organs also improved the knowledge of its bud bank. The chemical profile of essential oils showed the presence of a unique set of terpenes to each species. The data obtained in this study represent a significant contribution to the knowledge of Aldama species, as provided information with the potential to assist species distinction, as well as new aspects of the group, of which will be useful for future research in different botany areas.

Key words Belowground organs, bud bank, secretory ducts, fructans, terpenes, vegetative organs, venation pattern SUMÁRIO

Agradecimentos ...... 5 Resumo...... 7 Abstract...... 8

Introdução ...... 12 Objetivos...... 14 Referências...... 16

Capítulo 1...... 21 Leaf and stem anatomy and essential oil composition of four Brazilian Aldama species (Asteraceae) and their taxonomic significance Abstract ...... 22 Resumo...... 22 Introduction ...... 23 Material and methods ...... 23 Plant species and study area ...... 23 Structural analyses ...... 24 Histochemical analysis ...... 24 Essential oil extraction ...... 25 Essential oil analysis - Gas Chromatography...... 25 Statistical analyses ...... 25 Results ...... 26 Leaf anatomy ...... 26 Stem anatomy ...... 27 Essential oils...... 28 Discussion...... 29 Conclusions...... 32 Acknowledgments ...... 32 References ...... 33

Capítulo 2...... 47 Secretory ducts distribution and leaf venation pattern of Aldama species (Asteraceae) and their application in taxonomy Abstract ...... 48 Resumo...... 48

Introduction ...... 49 Material and methods ...... 49 Plant species...... 49 Secretory duct analysis ...... 50 Venation analysis...... 50 Statistical analyses ...... 50 Results ...... 51 Secretory ducts traits in Aldama ...... 51 Secretory duct arrangements ...... 51 Variation of secretory duct between leaves with different sizes ...... 52 Venation analysis...... 52 Discussion...... 53 Conclusions...... 55 Acknowledgments ...... 55 References ...... 55

Capítulo 3...... 65 Belowground organs of four Brazilian Aldama (Asteraceae): morphoanatomical traits and essential oil profile Abstract ...... 66 Resumo...... 66 Introduction ...... 67 Material and methods ...... 68 Plant species...... 68 Anatomical analysis ...... 68 Essential oil extraction and analysis...... 69 Results ...... 69 Morphoanatomy ...... 69 Essential oils ...... 72 Discussion...... 73 Conclusions...... 75 Acknowledgments ...... 75 References ...... 75 Capítulo 4...... 90 Underground organs of Brazilian Asteraceae: testing CLO-PLA database traits Abstract ...... 91 Resumo...... 91 Introduction ...... 92 Material and methods ...... 93 Asteraceae species – clonal growth organ...... 93 Aldama genus – clonal and bud bank traits ...... 94 Results ...... 94 Asteraceae species – clonal growth organ ...... 94 Aldama genus – clonal and bud bank traits ...... 95 Discussion...... 95 Translation of morphological categories of belowground organs ...... 96 Assessment of clonal and bud bank traits...... 98 Conclusions...... 98 Acknowledgments ...... 98 References ...... 98

Considerações finais ...... 116 Anexo 1 ...... 118 Anexo 2 ...... 119

INTRODUÇÃO

Asteraceae é a maior família dentre as Eudicotiledôneas, sendo representada por cerca de 23.000 espécies distribuídas em 1.600 a 2.000 gêneros (Panero & Funk 2008) constitui cerca de 10% da flora mundial (Funk et al. 2005). Apresenta distribuição cosmopolita (Souza & Lorenzi 2008), e é considerada uma das famílias botânicas mais bem sucedidas, não só pela diversidade de formas de vida, as quais incluem ervas, subarbustos, árvores e epífitas (Magenta 2006), mas também pelos diferentes métodos de propagação (Cronquist 1981) e ocorrência de metabólitos secundários bioativos (Funk et al. 2005). Asteraceae também apresenta grande importância econômica, sendo utilizada popularmente como plantas ornamentais (dálias, margaridas, crisântemo), na culinária (girassol, alcachofra, almeirão, escarola e a alface) e na medicina (carqueja, camomila, calêndula e guaco) (Souza & Lorenzi 2008). Quimicamente, é caracterizada por terpenoides, poliacetilenos e compostos fenólicos (Judd et al. 2007). A família é aceita como um grupo monofilético, pertence ao clado Magnoliophyta e à ordem Asterales, também composta por Campanulaceae e Menyanthaceae. Asteraceae é dividida em 20 tribos, das quais se destaca Heliantheae que apresenta 189 gêneros e aproximadamente 2.500 espécies (Bremer 1994). Dentre os maiores gêneros de Heliantheae estão Bidens L. com 240 espécies, Wedelia Jacq. com 130 (Bremer 1994) e Aldama La Llave com 112 (Magenta & Pirani 2014), das quais 35 ocorrem em áreas de Cerrado e Campos Sulinos (Magenta 2006). Em 2011, as espécies sul-americanas do gênero Viguiera foram transferidas para o gênero Aldama através de análises moleculares (Schilling & Panero 2011), pois formavam um grupo coeso e separado das demais espécies do gênero. Porém, ainda existe baixa consistência dos dados e questionamentos sobre o tema, uma vez que na análise filogenética de Schilling & Panero (2011) os ramos das espécies brasileiras apresentaram baixo suporte estatístico, evidenciando uma lacuna no conhecimento sobre estas espécies. Nos últimos anos, diversos estudos tem sido desenvolvidos com o objetivo de fornecer dados para auxiliar a taxonomia do grupo e de ampliar o conhecimento sobre a morfologia (Almeida et al. 2005; Magenta 2006; Magenta 2010a, b; Magenta & Pirani 2014), anatomia (Oliveira et al 2012; Silva et al. 2014; Bombo et al. 2012, 2014), fitoquímica (Da Costa et al. 1996, 2001; Ambrosio et al. 2002, 2004; Bombo et al. 13

2016) e bioquímica de Aldama (Tirapelli et al. 2002; Arakawa et al. 2008; Carvalho et al. 2011; Chagas-Paula et al. 2015). Dentre os estudos anatômicos, destacam-se àqueles que analisam as estruturas secretoras responsáveis pela produção, estocagem e secreção de substâncias resultantes do metabolismo secundário (Oliveira et al. 2012; Bombo et al. 2012, 2014, 2016; Silva et al. 2014). As secreções produzidas têm despertado grande interesse, especialmente os óleos essenciais, uma vez que já foram demonstradas atividade antimicrobiana (Canales et al. 2008), ação anti-inflamatória (Valério et al. 2007; Chagas-Paula et al. 2015) e combate ao protozoário Trypanosoma cruzi (Ambrosio et al. 2008). A maior parte dos trabalhos desenvolvidos neste tema se concentra na análise dos órgãos aéreos destas espécies, mesmo após a comprovação de atividade biológica para compostos desenvolvidos nos órgãos subterrâneos (Ambrosio et al. 2004; Arakawa et al. 2008; Nicolete et al. 2009; Porto et al. 2009; Carvalho et al. 2011). Além da importância fitoquímica, a presença de estruturas secretoras abaixo do solo também é uma das estratégias adaptativas da planta na defesa contra a herbivoria (Rasmann et al. 2005; Appezzato-da-Glória et al. 2008). Dados sobre a morfoanatomia de sistemas subterrâneos na família Asteraceae têm demonstrado uma diversidade de órgãos espessados, como xilopódios, raízes tuberosas, raízes gemíferas e rizóforos (Hayashi & Appezzato-da-Glória 2007; Vilhalva & Appezzato-da-Glória 2006; Appezzato-da-Glória et al. 2008; Appezzato-da-Glória & Cury 2011; Oliveira et al 2012; Silva et al. 2014; Bombo et al. 2014). A presença desses órgãos pode influenciar na dinâmica populacional das espécies, visto que são capazes de recuperar os órgãos aéreos através do desenvolvimento de novos ramos, originados do banco de gemas subterrâneo (Soares et al. 2006; Medeiros & Miranda 2008; Fidelis 2008). Verdaguer e Ojeda (2005) associam a importância das gemas subterrâneas à reserva de carboidratos. Dessa forma, o investimento no acúmulo subterrâneo destes compostos possibilita a rápida recomposição da biomassa aérea perdida, e consequentemente a sobrevivência das plantas submetidas a condições severas periódicas (Hoffmann, 1999). Os frutanos têm sido considerados, após o amido, como uma importante reserva de carboidratos (Soja et al., 1989), e estão envolvidos em inúmeras estratégias adaptativas relacionadas à seca e ao frio (Figueiredo-Ribeiro 1993; Alberdi et al. 2002), uma vez que apresentam rápidas reações de polimerização e

despolimerização envolvidas nos processos de osmoregulação (Figueiredo-Ribeiro 1993). A presença de frutanos foi registrada para várias espécies de Asteraceae (Figueiredo-Ribeiro et al. 1986; Dias-Tagliacozzo et al. 1999; De Moraes et al. 2016), incluindo espécies de Aldama (Isejima et al. 1991; Tertuliano & Figueiredo-Ribeiro 1993; Oliveira et al. 2012; Silva et al 2014; Bombo et al. 2014). Mediante o exposto, neste estudo são apresentadas análises mais detalhadas sobre a distribuição de estruturas secretoras, e sobre a morfoanatomia dos sistemas subterrâneos de quatro espécies de Aldama, visando buscar novos caracteres morfoanatômicos e químicos que possam contribuir para a elucidação dos problemas taxonômicos do gênero, e aprimorar o conhecimento sobre os órgãos subterrâneos para entender as estratégias de sobrevivência destas plantas. A escolha das espécies baseou-se na alta similaridade em estágio vegetativo entre Aldama nudibasilaris (S.F.Blake) E.E.Schill. & Panero (Figura 1A-1B) e A. pilosa (Baker) E.E.Schill. & Panero (Figura 1C-1D), assim como na possibilidade de formação de híbridos entre A. anchusifolia (DC) E.E.Schill. & Panero (Figura 1E-1F) e A. megapotamica (Malme) Magenta & Pirani (Figura 1G-1H), o que evidencia a estreita relação estabelecida entre elas. Além disso, tais espécies ocorrem em regiões campestres do sul do Brasil e em áreas de Cerrado da região Sudeste, as quais têm sido submetidas a uma acelerada degradação e fragmentação (Klink & Machado 2005; Almeida et al. 2005; Overbeck et al. 2005, 2006; Fidelis 2008).

OBJETIVOS Os objetivos principais dessa tese foram: 1) Analisar a morfoanatomia dos órgãos vegetativos aéreos e subterrâneos de espécies de Aldama, visando levantar caracteres com valor diagnóstico que auxiliem na circunscrição das mesmas; 2) Avaliar o rendimento e a composição química dos óleos essenciais dos diferentes órgãos vegetativos buscando apontar o potencial dos mesmos para futuros estudos farmacológicos, além de identificar potenciais marcadores químicos; 3) Identificar as variações morfológicas dos sistemas subterrâneos das quatro espécies de Aldama, procurando caracterizar as particularidades destes órgãos e a sua capacidade de rebrotamento.

Figura 1. Aldama nudibasilaris (A-B) coletada na região Sul de Minas Gerais; A. pilosa (C-D), A. anchusifolia (E-F) e A. megapotamica (G-H) provenientes do Rio Grande do Sul. Aspecto geral da planta no ambiente (A, C, E, G) e detalhe da inflorescência (B, D, F, H).

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______Capítulo 1

Leaf and stem anatomy and essential oil composition of four Brazilian Aldama

species (Asteraceae) and their taxonomic significance

“Anatomia de folha e caule e composição do óleo essencial de quatro espécies

brasileiras de Aldama e sua significância taxonômica”

Filartiga AL1, 2, Bombo AB1, 2, Garcia VL3, Appezzato-da-Glória B1, 2

(submitted to Brazilian Journal of Botany)

1 Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brasil. 2 Departamento de Ciências Biológicas, Escola Superior de Agricultura ‘Luiz de Queiroz’, Piracicaba, SP, Brasil. 3 Divisão de Química Orgânica e Farmacêutica, Centro de Pesquisas Químicas, Biológicas e Agrícolas, CPQBA, UNICAMP, Campinas, SP, Brasil. 22

Abstract: Aldama La Llave is one of several Asteraceae genera that pose phylogenetic problems. The close similarity between species, as well as the inconsistencies found in the most recent phylogenetic analysis, show that new data are needed to help delimit group species. Aldama anchusifolia, A. megapotamica, A. nudibasilaris and A. pilosa are difficult to identify because they are very closely related. Therefore, the aim of this study was to detect anatomical and phytochemical characteristics to help elucidate phylogenetic issues raised by Aldama. Aerial vegetative organs were prepared using the standard histological techniques. Essential oils were obtained by hydrodistillation and their components identified using a gas chromatograph coupled to a mass spectrometer and flame ionization detector. Each species presented a set of unique leaf and stem anatomical features. The front view of the epidermal cell walls in the leaves, presence of secretory ducts in the phloem and medulla sclerification in the stems proved useful in delimiting these species. The essential oils were characterized by the predominance of sesquiterpenes such as t-Caryophyllene, Germacrene D and Bicyclogermacrene. Some unique constituents in each species were also identified as potential chemical markers.

Keywords Compositae, fructan, secretory duct, socket cells, terpenes

Resumo: Aldama La Llave é um dos diversos gêneros de Asteraceae que apresenta problemas filogenéticos. A estreita similaridade entre as espécies, assim como as inconsistências encontradas na mais recente análise filogenética, mostram que novos dados são necessários para auxiliar a delimitação deste grupo de espécies. Aldama anchusifolia, A. megapotamica, A. nudibasilaris e A. pilosa são plantas de difícil identificação porque são estreitamente relacionadas. Sendo assim, o objetivo deste trabalho foi detectar características anatômicas e fitoquímicas para ajudar na elucidação de questões filogenéticas de Aldama. Órgãos vegetativos aéreos foram preparados com o uso de técnicas histológicas usuais. Os óleos essenciais foram obtidos mediante hidrodestilação e seus componentes foram identificados através de cromatografia gasosa acoplada ao espectrômetro de massas e ao detector de ionização de chamas. Cada espécie apresentou um conjunto único de características anatômicas foliares e caulinares. A visão frontal da parede das células epidérmicas nas folhas, assim como a presença de canais secretores no floema e a esclerificação da medula nos caules provaram ser úteis na delimitação destas espécies. Os óleos essenciais foram caracterizados pela predominância de sesquiterpenos, como t-Cariofileno, Germacreno D e Biciclogermacreno. Alguns constituintes exclusivos de cada espécie também foram identificados como potenciais marcadores químicos.

Palavras-chave Canal secretor, Compositae, frutanos, socket cells, terpenos

Introduction The South American species of Viguiera Kunth genus were transferred to Aldama La Llave due to results of molecular analyses (Schilling and Panero 2011) since these species have formed a cohesive group apart from the other species in this genus (Schilling and Jansen 1989; Schilling and Panero 1996, 2011). However, inconsistencies found in the phylogenetic analysis performed by Schilling and Panero (2011) highlighted the need for more data to circumscribe Aldama genus, whose species share many morphological similarities (Magenta and Pirani 2014). Anatomical and chemical features in Aldama are important tools used to help identifying the species (Bombo et al. 2012, 2014; Oliveira et al. 2013; Silva et al. 2014). The secretory structures represent an important taxonomic character in among the anatomical features in Asteraceae due to their position and variety (Castro et al. 1997; Appezzato-da- Glória et al. 2008). Phytochemical analyses also emphasize the use of secondary metabolites in the phylogeny and chemotaxonomy of Aldama (Schilling et al. 2000; Da Costa et al. 2001) as well as their pharmacological potential (Tirapelli et al. 2002; Valério et al. 2007; Canales et al. 2008). It is worth highlighting the essential oils among such metabolites, which are a complex mixture of lipophilic substances mostly composed of volatile low molecular weight terpenes (Fahn 2000). Essential oils have been already reported in several Asteraceae genera (Heinrich et al. 2002; Alvarenga et al. 2005; Agostini et al. 2005; Maia et al. 2010; Chagas et al. 2012); however only three species have been reported in genus Aldama (Bombo et al. 2012, 2014). The strong vegetative similarities between Aldama nudibasilaris (S.F.Blake) E.E.Schill. & Panero and A. pilosa (Baker) E.E.Schill. & Panero, as well as the possible formation of hybrids between A. anchusifolia (DC) E.E.Schill. & Panero and A. megapotamica (Malme) Magenta & Pirani highlighted the difficulty in identifying them. Therefore, the aim of the current study is to point out some anatomical and phytochemical features that can help circumscribing these Aldama species and elucidating the phylogenetic issues of the genus.

Materials and methods Plant species and study area The aerial organs of Aldama anchusifolia, A. megapotamica, A. nudibasilaris and A. pilosa which have derived from plants in the flowering stage were collected in grasslands and 24

in the borders of highways in the Brazilian Southern and Southeastern regions between 2012 and 2013. A total of nine specimens from three distinct populations, spaced at least 30 km from each other, were sampled for each species. The species were identified by a specialist and the vouchers were deposited at the ESA Herbarium (Luiz de Queiroz College of Agriculture).

Structural analyses The middle region and the petiole of fully expanded leaves and stems were analyzed for each specimen collected. The thinner and the median size diameters, as well as the internode near to the ground, were sampled in the stem. The samples were fixed in FAA 50 (formaldehyde, acetic acid and 50 % ethanol) (Johansen 1940) or in Karnovsky solution (Karnovsky 1965). Subsequently, they were subjected to vacuum to remove the air from the tissues and dehydrated in an ethanol series up to 70% ethanol, wherein they were stored until the time to be processed. A fraction of each material was embedded in Leica Historesin® plastic resin (Heraeus Kulzer, Hanau, Germany). The blocks were sectioned by means of Leica RM 2245 rotary microtome at 6 µm. The sections were stained with 0.05 % toluidine blue O in a citrate–phosphate buffer, pH 4.5 (Sakai 1973) and mounted on glass slides in Entellan® synthetic resin (Merck, Darmstadt, Germany). Thicker sections (20-60 µm) of fixed samples were also prepared in Leica SN 2000 R sliding microtome. The sections were clarified in 20% sodium hypochlorite, washed in distilled water, stained in safranin and astra blue (Bukatsch 1972), and mounted in 50% glycerin. The classification of the glandular trichomes was based on Castro et al. (1997). Leaf and stem surfaces were also analyzed through the epidermal dissociation technique using the Jeffrey’s solution (Johansen 1940). The epidermis was also analyzed through scanning electron microscopy (SEM). The samples were dehydrated in ethanol series up to absolute ethanol, dried according to the CO2 critical-point method (Horridge and Tamm 1969), mounted on aluminum stubs and coated with a gold layer (30-40 nm). The observations and photomicrographs were obtained in a LEO 435 VP SEM (Zeiss, Oberkochen, Germany) operated at 20 kV. . Histochemical analysis The histochemical analyses were performed in sections obtained from the material embedded in historesin and from the fixed material that had not been embedded in historesin. The following reagents and dyes were used: NADI reagent, to identify the essential and resin

oils (David and Carde 1964); zinc-chloride iodide, to detect the starch grains (Strasburger 1913); phloroglucinol in acid medium, to detect lignin; ferric chloride, for the phenolic compounds (Johansen 1940); Sudan IV for the lipophilic substances (Jensen 1962); Sudan black B for the total lipids (Pearse 1968); and ruthenium red for the pectic substances (Johansen 1940). Inulin-type fructans were observed under polarized light. The digital photomicrographs were obtained in Leica DM LB microscope equipped with Leica DC 300F camera.

Essential oil extraction The fresh material was subjected to hydrodistillation for three hours, in Clevenger- type apparatus. The aqueous phase was collected after cooling and the set-up was washed with dichloromethane (50 ml) to obtain the essential oils. Each solution was dried over anhydrous sodium sulfate, weighed on an analytical scale to set the yield and stored at -5°C in sealed amber glass flasks.

Essential oil analysis - Gas Chromatography The essential oil constituents were set in a gas chromatography coupled to a mass spectrometer (GC-MS) using HP 5890 chromatograph Series II (Palo Alto, CA, USA) equipped with the Hewlett-Packard 5971 mass selective detector and the HP-5 capillary column (25 m × 0.20 mm × 0.33 µm). The GC-MS was performed through split/splitless injection by using the injector at 220 °C; the detector at 280 °C; the column, at 60 °C, with increments of 3 °C.min-1 up to the final temperature of 240 °C. The constituents were also set in a flame ionization detector (FID/DIC/ULTRA FAST) coupled to the Thermo Scientific TRACE GC Ultra gas chromatograph with AS 3000 autosampler, split/splitless injection, HP- 5 capillary column (30 m × 0.25 mm × 0.25 μm), temperatures equal to the aforementioned ones, and final temperature of 250 °C. Helium at 1 mL.min-1 was used as carrier gas. The samples were dissolved in ethyl acetate at 20 mg/mL concentration. The constituents of the essential oils were identified through the comparison of their mass spectra and the NIST-05 library data, by co-injection of hydrocarbons patterns in order to calculate the Arithmetic index and through data described in the literature (Adams 2007).

Statistical analyses The values found for essential oil yield were submitted to variance analysis (ANOVA) and the means were compared through Tukey’s test (p <0.05). 26

Results Leaf anatomy The epidermal cells have sinuous walls on both leaf sides of Aldama anchusifolia, A. nudibasilaris and A. pilosa from the front side view (Table 1; Fig. 1A). A. megapotamica has straight cell walls (Fig. 1B). The stomata were anomocytic and occurred on both leaf sides, except for A. nudibasilaris, which has hypostomatic leaves. The indumentum of the four species comprised two glandular trichomes (Types II and IV) and a non-glandular trichome. The non-glandular type (Fig. 1A, 1C) occurred on both leaf sides and consisted of three cells: two cone-shaped basal cells and a terminal cell with an acute apex. The walls were thickened in pectin; the verrucous ornamentations (humps) were more commonly found in the basal cells and they were absent in the apical cells. The basis of each non-glandular trichome was delimited by concentric series of epidermal cells (Fig. 1A). The amount of series may vary from one species to another (Table 1). These cells had mucilaginous content and their walls were thickened in pectin. The type II glandular trichome occurred on both leaf sides and accumulated phenolic substances, it was linear and uniseriate; its terminal cell was spatulate (Fig. 1C). The type IV trichome just occurred on the abaxial surface of the epidermis (Fig. 1D); its exudate had lipophilic nature; it was capitate and biseriate and comprised of two basal cells and 3-8 pairs of secreting cells, which corresponded to the trichome head. Both sides of the leaf presented uniseriate epidermis with thickened outer periclinal cell walls covered with a thin cuticle (Fig. 1E-1G). The mesophyll was dorsiventral in all species (Fig. 1E-1F). Aldama megapotamica was the only one to present tiny prismatic crystals in this tissue. Lipid droplets were found in the palisade cells of A. anchusifolia and A. megapotamica (Fig. 1F, inset). Revolute leaf margins were found in the four species. Their tissue organization was similar to that visualized in the rest of the leaf blade. The ornamentation regions in A. nudibasilaris and A. pilosa leaves may have hydathodes (Fig. 1G), which are constituted by water pores, incomplete parenchymatous sheaths that surround the thin-walled cells of the epithem, and the terminal tracheids of the vascular bundle. The mid-rib had a conspicuous projection filled with collenchyma cells in adaxial side of Aldama nudibasilaris and A. pilosa leaves (Fig. 1H). This projection was less prominent in A. anchusifolia, and it was missing in A. megapotamica (Fig. 1I). The fundamental parenchyma surrounded the vascular bundles (Fig. 1H), but the palisade parenchyma cells extend to the lateral side of the mid-rib in A. anchusifolia and in A. megapotamica (Fig. 1I).

The number of ducts in the fundamental parenchyma can vary from one leaf to another in the same individual and also between individuals in different populations in A. megapotamica, A. nudibasilaris and A. pilosa (Table 1). Such variation was not observed in A. anchusifolia, which only had two secretory ducts facing the abaxial surface. The ducts had different sizes, secreted lipophilic substances and occurred in the adaxial and abaxial regions of the fundamental parenchyma (Fig. 1H-1I). Some parenchyma cells around the vascular bundles exhibited starch grains in all the species analyzed. The vascular system was collateral, arranged in a larger central bundle and in two smaller lateral bundles in A. anchusifolia, A. nudibasilaris and A. pilosa (Fig. 1I, inset). A. megapotamica had one large central bundle, which was associated with fiber caps (Fig. 1I). The secretory ducts were found in the primary phloem just in A. nudibasilaris and A. pilosa. The lateral veins may present parenchymatous sheaths that can reach the epidermis on both sides of the leaf; however, the secretory ducts just occurred in the adaxial bundle extension (Fig. 1E). The description of petiole was held herein for the first time to the genus selected. Only Aldama anchusifolia, A. nudibasilaris and A. pilosa presented petiole, which had an epidermis structure similar to that one observed in the leaf blade (Fig. 1J). The indumentum was formed by the non-glandular trichome and by the Type II glandular trichome. The collenchyma and the subjacent fundamental parenchyma are arranged in layers immediately below the epidermis. The layers may vary in number, depending on the sample analyzed (Fig. 1K). Secretory ducts of different sizes are immersed in this parenchyma (Fig. 1J). Ducts in A. anchusifolia only developed towards the abaxial region, whereas those in A. nudibasilaris and A. pilosa also occurred in the adaxial region. The vascular system consisted of three major bundles and of a varying number of smaller lateral bundles, which may be surrounded by cells containing starch grains. The secretory ducts only occurred in the phloem of A. nudibasilaris and A. pilosa (Fig. 1L).

Stem anatomy In an incipient secondary structure, the epidermis of all studied species was uniseriate (Fig. 2A) and exhibited non-glandular trichomes and glandular trichomes Type II, similarly to that described in the leaves (Fig. 2B). There were secretory ducts in the cortical parenchyma (Fig. 2A, 2C) and the endodermal cells presented Casparian strips and starch grains (Fig. 2A, inset). The pericycle region opposite to the primary phloem formed fiber cap, and it was be interrupted by phloematic cells in Aldama pilosa (Table 1; Fig. 2A). The vascular bundles were collateral, and only A. nudibasilaris formed secretory ducts in the primary phloem. The 28

medulla was parenchymatic and its ducts were distributed in the perimedullary zone (Fig. 2A), which rarely occurred in A. pilosa. In stems with established secondary structure, the epidermis was replaced by suberized thick-walled cells, which were originated by periclinal divisions of subepidermal cells (Fig. 2D). Sclereids emerged in the cortical region, either alone or in small groups (Fig. 2D). In the fascicular and interfascicular regions of the vascular cylinder, the cambium produced secondary xylem and phloem with all elements of the axial and radial systems (Fig. 2E-2G). Secretory ducts occurred in the secondary phloem in all studied species (Fig. 2E). The stem thickening in A. anchusifolia, A. nudibasilaris and A. pilosa mainly resulted from the expansion of the medulla. In the first two species, the vascular ray cells elongated in periclinal direction, whereas the cells belonging to the perimedullary zone divided and elongated in anticlinal direction (Fig. 2F). On the other hand, the expansion of the medulla in A. pilosa resulted from cellular hyperplasia (Fig. 2H) and hypertrophy (Fig. 2I). The stem thickening was not very pronounced in A. megapotamica and the medulla became sclerified at the end of the development (Fig. 2G). There were inulin-type fructans in the cortical and medullary parenchyma cells (Fig. 2J), in the cambium and in other vascular tissues, often inside the tracheary elements, only in thicker stems of Aldama pilosa.

Essential oils The yield of essential oils extracted from the aerial organs was different among the populations analyzed (Table 2). However, there was no significant difference between the mean values of the leaves and stems in each species (Fig. 3). In Aldama anchusifolia the mean yield of leaf EO was 0.26±0.07 for leaves and 0.26±0.03 for stems, and it was almost four times higher than the mean value for A. megapotamica leaves (0.07±0.07) and five times higher for stems (0.05±0.06). Furthermore, the mean values of A. pilosa (0.26±0.03 and 0.19±0.06) were also higher than those of A. megapotamica, and similar to those of A. nudibasilaris (0.18±0.06 for leaves and 0.05±0.12 for stems). The essential oils from the aerial organs were featured by the presence of monoterpenes, sesquiterpenes and diterpenes, and by the prevalence of sesquiterpenes in all four species (Supplementary Table 1). Sixty-seven constituents were identified, totaling approximately 85% of the total amount. Thirty-five constituents were common to both aerial organs of the species analyzed. Carotol was the major compound in Aldama anchusifolia, although α- and -Pinene (monoterpenes) and 5-neo-Cedranol (sesquiterpene) also stood out in more than one

population due to the high relative percentage of them both organs (Supplementary Table 1). Germacrene D (sesquiterpene) presented the highest relative percentage of essential oil in A. megapotamica leaves in the three populations, whereas t-Caryophyllene showed the highest value in the EO’s from stems (24.91%; Table 3). As for A. nudibasilaris, the constituents showing significant relative percentage were Caryophyllene oxide and Bicyclogermacrene, respectively, on the leaves and stems of the three populations. Bicyclogermacrene (sesquiterpene) was detected as the major compound in the leaves and stems of A. pilosa (Table 3). The following constituents were identified in all four species: t-Caryophyllene, Germacrene D and Bicyclogermacrene (in both organs), Caryophyllene oxide (only in the leaves) and Spathulenol (only in the stems; Table 3). When the constituents were found in leaf and stem EOs at least in two populations of a single species, they were considered unique. Five constituents were unique in Aldama anchusifolia (Camphene, Bornyl acetate, δ-Amorphene, Carotol and 5-neo-Cedranol), four in A. megapotamica (δ-Elemene, 1,7-di-epi--Cedrene, -Gurjunene and Isodaucene) and six constituents in A. nudibasilaris (Copaene, -Atlantol, Caryophilla-4(14),8(15)-dien-5--ol, allo-Aromadendrene epoxide, (Z)-14-hydroxy Caryophyllene and Eudesma-4 (15),7-dien-1- -ol). No constituent was considered unique in A. pilosa (Table 3).

Discussion The vegetative anatomy of Aldama has helped in morphologically differentiating of similar species that pose taxonomic problems (Bombo et al. 2012, Oliveira et al. 2013, Silva et al. 2014). The available data suggest that certain leaf and stem characteristics, such as the shape of epidermal cells, positioning of the stomata and presence of secretory structures, play a decisive role in differentiating species. Indeed, the evaluation herein provided a set of useful leaf and stem characteristics for distinguishing four closely related Aldama species. Epidermal features such as the distribution of stomata on the leaf surface (to distinguish Aldama nudibasilaris) and the outline of the anticlinal walls of epidermal cells (to distinguish A. megapotamica) have been of important taxonomic value. Both characteristics have also been used to contrast other Aldama species (Bombo et al. 2012, Oliveira et al. 2013, Silva et al. 2014). The non-glandular trichome described herein is common among Asteraceae (Cornara et al. 2001, Adedeijo and Jewoola 2008) and has already been reported in Aldama species (Bombo et al. 2012, Oliveira et al. 2013, Silva et al. 2014). However, the presence of epidermal cells with pectin-thickened walls surrounding the non-glandular trichome was 30

reported only by Silva et al. (2014) in the four Aldama species analyzed herein. The number of rings in the arrangement of these mucilage-containing cells proved to be an important distinguishing feature for the species. Apart from the role these cells play as supports (socket cells, Evert 2006), they are also important for storing carbohydrate (Clifford et al. 2002), moisture uptake (Westhoff et al. 2009), reducing transpiration (Zimmermann et al. 2007) and protection against herbivory (Thompson et al. 2014). The existence of secretory structures of various types and in varying positions in plant body is useful in identifying Asteraceae species (Metcalfe and Chalk 1979, Kelsey 1984, Castro et al. 1997, Appezzato-da-Glória et al. 2008). The presence of glandular trichomes, hydathodes, ducts and cavities has already been reported in Aldama (Bombo et al. 2012, Oliveira et al. 2013, Silva et al. 2014) and was confirmed for the species analyzed in this study. Hydathodes were identified in the leaf margin of A. nudibasilaris and A. pilosa, with structures similar to those reported for other Aldama (Oliveira et al. 2013, Silva et al. 2014). These hydathodes are responsible for guttation, which occurs under conditions of low transpiration and high soil moisture content (Fahn 1979), but this phenomenon has, to date, not been reported for Aldama species. Secretory ducts have already been used for differentiating Aldama species (Bombo et al. 2012, Oliveira et al. 2013, Silva et al. 2014). In the leaves, these ducts occur on the mid-rib parenchyma, primary phloem and also in the sheath extensions of lateral bundles. Nevertheless, those authors did not examine whether the number and distribution of these structures are constant from one leaf or specimen to another. Variations in secretory ducts have already been reported in Burseraceae leaves of different sizes (Kakrani et al. 1991) and in Pinus taiwanensis Havata needles from distinct populations (Sheue et al. 2003). For the Aldama species examined herein, these characteristics are constant only in A. anchusifolia. In the other three species, the number of secretory ducts can vary from one leaf to another in the same individual. Therefore, we recommend that this characteristic should be used carefully, or combined with other characteristics, for differentiating Aldama species. This is the first study to describe the anatomy of the Aldama petiole, which helped in differentiating three of the species selected. According to Magenta (2006), the presence or absence of the petiole in Aldama leaves may also play a part in differentiating species. Among the 35 Brazilian Aldama, A. megapotamica is one of 11 species that develop sessile leaves. In contrast, A. nudibasilaris and A. pilosa, together with another 9 species, have leaves with petioles. The leaves of the remaining 13 taxa can be either sessile or petiolate. In this case, the

petioles are usually tiny, ranging from 1 to 3 mm (Magenta 2006), as in A. anchusifolia. The presence of secretory ducts in the petiole is useful for identifying the species mentioned. In terms of stem anatomy, despite close similarities, some features were important in distinguishing species: phloem cells interrupting the pericycle and the presence of inulin-type fructans (only in A. pilosa), inconspicuous hypertrophy and hyperplasia of medulla cells and sclerified medulla (only in A. megapotamica), and the presence of secretory ducts in the primary phloem (A. nudibasilaris and A. pilosa). The last two features have already been reported for Aldama and proved useful in delimiting species with very similar morphologies (Bombo et al. 2012, Oliveira et al. 2013, Silva et al. 2014). Inulin crystals (fructans) were detected only in the stem of A. pilosa. This carbohydrate is typical in the underground organs of Asteraceae species (Figueiredo-Ribeiro 1993, Silva et al. 2015, Abdalla et al. 2016, Moraes et al. 2016) and has already been reported, but only in tuberous roots of Aldama (Oliveira et al. 2013, Silva et al. 2014, Bombo et al. 2014). It is well known that fructan storage enables plants to tolerate stress conditions, such as drought (Valluru and Van den Ende 2008, Vilhalva et al. 2011, Oliveira et al. 2013) and low temperatures (Hendry 1987, Pontis 1989, Vijn and Smeekens, 1999, Portes et al. 2008), both common environmental conditions in the regions in which A. pilosa is usually found. Secretory ducts proved to be the main essential oil secretion sites in all the species studied. The yield of essential oil, as well as its constituents, varied from species to species and from one organ to another in the same species. These variations from one organ to another may be related to the number of secretory structures (Fahn 1979), plant genotype (Gershenzon et al. 2000) and the environmental conditions they are subjected to (Erdtman 1963). The highest yield values were observed for leaves and stems of A. anchusifolia, with A. pilosa in second place. In contrast, low yield values were found for stems of A. nudibasilaris and for the aerial organs of one population of A. megapotamica. In terms of chemical composition, the predominance of sesquiterpenes was noteworthy in oils from leaves and stems of the selected species, in contrast to the predominance of monoterpenes reported for another three species of Aldama (Bombo et al. 2012). Diterpenes were also detected in the four Aldama species studied. However, they were not useful in differentiating the species, since they occurred in only one population of A. anchusifolia, A. megapotamica and A. pilosa. The presence of these high molecular weight terpenes has already been reported in essential oils from other Asteraceae (Heinrich et al. 2002; Meragelman et al. 2003; Chagas-Paula et al. 2012). Furthermore, the medicinal 32

importance of some diterpenes, such as the Kaurene and Pimarane, has already been reported for Aldama (=Viguiera) (Ambrosio et al. 2002; 2006, Carvalho et al. 2011). However, the authors identified these constituents by analyzing root extracts rather than essential oils. Several components identified herein occur widely in Asteraceae (Agostini et al. 2005; Godinho et al. 2014) and in other Aldama species (Canales et al. 2008; Bombo et al. 2012). Some of them are highlighted in phytochemical studies as being active biological agents. The antibacterial agents reported in these studies are Spathulenol, Bornyl acetate, trans- Caryophyllene, Myrcene, -Myrcene, Germacrene D, Bicyclogermacrene, and α- and - Pinene (Constantin et al. 2001; Canales et al. 2008; Souza et al. 2007; Carvalho et al. 2011). Bicyclogermacrene and α- and -Pinene also have antifungal effects (Constantin et al. 2001; Silva et al. 2007), whereas α- and -Thujene have anthelmintic effects (Godinho et al. 2014). As well as being of considerable phytochemical importance, the identification of potential chemical markers to lend support to taxonomic studies is also widely recognized in the Aldama genus (Da Costa et al. 1996, 2001; Spring et al. 2003; Ambrosio et al. 2004; Carvalho et al. 2011; Bombo et al. 2012, 2014). In the present study, determining unique constituents in three of the four selected species provides very useful information for chemical differentiation.

Conclusions This research determined that despite the strong morphological similarities shared by the Aldama studied herein, some anatomical features of leaves and stems can be decisive to distinguish these species. Furthermore, we also succeeded in identifying the unique chemical constituents for each Aldama analyzed. In conclusion, this study serves as an indication that the anatomical and chemical features have potential to elucidate the taxonomical issues raised by this genus.

Acknowledgments We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the Grant (Proc. nº 303715/2014-6) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for providing financial support (Thematic project Proc. n° 2010/51454-3) and for the Grants to the first (2012/02476-0) and second (2012/01586-6) authors. We would also like to thank Professor Mara Angelina Galvão Magenta for species identification.

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Table 1. The main anatomical features distinguishing Aldama anchusifolia (Aa), A. megapotamica (Am), A. nudibasilaris (An) and A. pilosa (Ap). Amph = amphistomatic, Hyp = hypostomatic, Ab = abaxial region, Ad = adaxial region, no = not observed, conspic = conspicuous, inconspic = inconspicuous. Trait Aa Am An Ap

Stomata position Amph Amph Hyp Amph

Anticlinal walls of epidermis Sinuous Straight Sinuous Sinuous

Number of cells surrounding the non- 1-2 Ad, 1-3 Ad, 0 Ad, 1 Ad, glandular trichome 0-1 Ab no 1-3 Ab 1-3 Ab

Hydathode on leaf margin Absent Absent Present Present

Palisade parenchyma extended to the Present Present Absent Absent abaxial side of the mid-rib 0 Ad, 1 Ad, 2 Ad, 0-2 Ad,

Leaf Leaf Number of secretory ducts on the mid-rib 2 Ab 0-2 Ab 2-3 Ab 2 Ab

Fiber caps on the mid-rib bundle Absent Present Absent Absent

Phloem secretory duct on the mid-rib Absent Absent Present Present

Secretory ducts on abaxial region of the Absent - Present Present petiole Phloem secretory duct on the petiole Absent - Present Present

Secretory duct on the primary phloem Absent Absent Present Present

Phloem cells interrupting the pericycle Absent Absent Absent Present

Hypertrophy and hyperplasia of medulla

Stem Stem cells Conspic Inconspic Conspic Conspic

Sclerified medulla Absent Present Absent Absent

Innulin Crystal Absent Absent Absent Present

Figure 1. Surface view and cross-sections of the leaf blade and petiole in Aldama anchusifolia (D, E, J), A. megapotamica (B, F, I), A. nudibasilaris (C, G, H, K, L) and A. pilosa (A). (A and B) Sinuous (A) and straight (B) epidermal cell walls. See the thickening of cells surrounding the basis of the non-glandular trichomes (A, arrow) and of the anomocytic stomata (A-B). (C and D) Scanning electron micrograph of the non-glandular (C, arrows), glandular type II (C, arrowheads) and glandular type IV (D) trichomes. (E and F) Uniseriate epidermis (Ep), palisade parenchyma (Pp), spongy parenchyma (Sp), lateral bundles and secretory ducts (arrow). Detail of the lipid droplets in chlorophyllous parenchyma stained in NADI reagent (inset in F). (G) Hydathode consisting of tracheids, epitema (Ept) and incomplete sheath (arrows). (H and I) Mid-rib with secretory ducts (arrows) and fiber caps (I, arrowheads). Detail of the secretory duct (inset in I). (J, K and L) Petiole with secretory ducts (J, arrows). Detail of the epidermis (Ep), chollenchyma (Cl), parenchyma (Pr) and ducts in the primary phloem (L, arrows). Scale bars = 10 µm (J inset), 25 µm (B, D, F inset, G, H, I, L), 50 µm (A, C, E, K), 100 µm (F), 200 µm (10).

Figure 2. Cross-sections and longitudinal-section of stems of Aldama anchusifolia (B, C), A. megapotamica (G), A. nudibasilaris (E, F) and A. pilosa (A, D, H, I, J). (A) Internode in an incipient secondary structure. Epiderm (Ep), collenchyma (Cl), phloem cells interrupting the pericycle (arrowhead) and the secretory ducts in the medulla (arrow). Detail of the Casparian strips (inset in A). (B) Non-glandular (arrow) and Type II glandular (arrowhead) trichomes. (C) Longitudinal section of the secretory duct (arrow). (D) Periclinal divisions (*) of the outmost cortical region that originates the suberized tissue (St) in the secondary structure and in the sclereids in the cortex (arrow). (E) Secreting ducts (arrows) in the secondary phloem. (F) Secondary structure of the internode. Periclinal elongation of the ray cells (arrows) and anticlinal elongation and division of the cells in the perimedullary zone (arrowheads). (G) Secondary structure of the thickened stem, with sclerified medulla (Md). (H and I) Hypertrophy (H) and hyperplasia (I) of the medullary cells. (J) Inulin-type fructans observed under polarized light. Scale bars = 50 µm (A inset, B, C, D), 100 µm (A, G, J), 200 µm (E, F, H, I). 42

Table 2. Fresh matter (M) and essential oil yield (Y) of leaves and stems of Aldama populations (Pop).

Species Organ Pop M (g) Y (% w/w) 1 194.8 0.24 Leaf 2 152.35 0.34 3 240.83 0.21 1 549.61 0.48 Stem 2 122.48 0.19

A. anchusifolia A. anchusifolia

3 308.11 0.12 1 9.60 0.0025 Leaf 2 40.83 0.12 3 19.76 0.11

1 21.46 0.0025 Stem 2 70.95 0.12 A. megapotamica A. megapotamica 3 78.57 0.05 1 99.90 0.15 Leaf 2 156.56 0.25 3 71.04 0.16

1 210.45 0.03 Stem 2 341.7 0.05

A. nudibasilaris A. nudibasilaris 3 102.7 0.07 1 124.11 0.28

Leaf 2 126.25 0.23 3 139.44 0.28 1 117.02 0.12

A. A. pilosa Stem 2 131.55 0.21

3 153.83 0.24

Figure 3. Essential oil yield of leaves and stems of Aldama anchusifolia (Aa), A. megapotamica (Am), A. nudibasilaris (An) and A. pilosa (Ap). Bars represent mean ± standard deviation (n = 3). Values are not significantly different (p > 0.05) under the same organ. 44

Table 3. Chemical profile and relative percentage area (%) of the essential oils extracted from leaves and stems of Aldama anchusifolia, A. megapotamica, A. nudibasilaris and A. pilosa. P = population, AIlit= arithmetic index from literature, AIcal= Calculated arithmetic index.

Aldama anchusifolia Aldama megapotamica Aldama nudibasilaris Aldama pilosa Constituent AI lit AI cal Leaf Stem Leaf Stem Leaf Stem Leaf Stem P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 Monoterpene hydrocarbons Camphene 946 948 0.51 0.52 - 0.71 0.48 ------Oxigenated monoterpenes Bornyl acetate 1287 1285 0.84 0.65 ------Sesquiterpene hydrocarbons δ-Elemene 1335 1337 ------0.88 1.80 ------Copaene 1374 1375 ------0.87 0.32 5.11 ------1,7-di-epi--Cedrene 1411 1413 ------1.38 - 3.31 12.66 ------2.39 t-Caryophyllene 1417 1418 1.34 0.93 1.42 - 0.60 0.52 5.38 2.72 4.16 9.21 - 24.91 6.10 1.39 12.15 1.68 1.39 - 13.72 7.98 7.20 16.77 1.25 - -Gurjunene 1431 1431 ------0.64 - - 1.43 3.24 ------Germacrene D 1484 1480 6.72 3.40 7.88 4.80 2.94 2.43 27.94 32.84 24.65 9.46 1.21 - 4.79 4.10 5.53 12.49 8.58 11.40 17.46 14.68 17.44 16.77 7.19 9.72 Biciclogermacrene 1500 1495 6.51 12.11 17.42 6,10 13.20 6.66 16.74 41.10 22.38 9.12 3.94 16.81 7.29 12.45 7.91 13.35 14.17 16.70 25.19 23.27 15.58 33.76 16.70 - Isodaucene 1500 1504 ------1.59 3.20 4.65 ------δ-Amorphene 1511 1514 0.83 0.50 1.18 - 0.67 0.86 ------Oxigenated sesquiterpenes Espatulenol 1577 1576 6.73 4.1 3.41 7.67 5.64 6.71 - - - 3.51 - 9.24 13.21 9.53 6.29 19.29 15. 6 2.70 5.43 3.15 11.70 16.17 6.52 - Caryophyllene oxide 1582 1582 1.64 1.32 1.50 - - - 1.14 - - 4.75 - 13.78 26.30 12.38 38.60 4.16 4.16 18.00 2.53 1.19 2.25 3.85 - 1.16 Carotol 1594 1594 15.93 21.13 18.70 12.11 2.86 17.26 -------Atlantol 1608 1607 ------2.10 1.26 - - 2.20 ------Caryophyla-4(12),8(13)- 1639 1634 ------6.22 1.10 ------dien-5α-ol - - - Alloaromadendrene 1639 1636 ------1.20 - 2.37 1.80 - - - - epoxide - - - - (Z)-14-hidroxi 1666 1668 ------2.69 0.83 ------Caryophylene - Eudesma-4(15),7-dien-1 1687 1683 ------3.18 2.21 - 1.31 2.47 -------ol 5-neo-Cedranol 1684 1689 4.24 10.53 7.84 0.95 4.31 2.90 ------

Supplementary Table 1. Chemical composition and relative percentage area (%) of the essential oils extracted from leaves and stems of Aldama anchusifolia, A. megapotamica, A. nudibasilaris and A. pilosa. P = population; AIlit = arithmetic index from literature; AIcal = calculated arithmetic index; ni = not identified

Aldama anchusifolia Aldama megapotamica Aldama nudibasilaris Aldama pilosa

Constituent AI lit AI cal Leaf Stem Leaf Stem Leaf Stem Leaf Stem P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 Monoterpene hydrocarbons 30.71 30.61 30.54 33.08 29.64 49.48 - 1.39 4.64 - - 13.45 13.61 22.94 - 6.55 35.26 - 8.00 29.19 29.01 - 49.96 36.59 α-Thujene 924 926 - - 1.20 0.62 0.28 1.99 ------0.94 - - 0.71 - - 1.81 0.62 - 3.68 4.19 α-Pinene 932 933 6.13 2.68 5.67 24.40 8.90 8.78 ------1.25 0.94 - - - - 0.38 7.59 3.60 2.20 1.25 - Camphene 946 948 0.51 0.52 - 0.71 0.48 ------Sabinene 969 973 0.64 7.75 3.82 1.14 6.20 6.19 ------0.67 1.99 - - - - 0.38 3.92 0.73 - 5.81 1.81 -Pinene 974 976 8.52 7.01 8.28 16.74 9.29 12.60 - - - - - 5.35 5.24 0.75 - 2.29 1.56 - 2.30 1.45 3.11 - 14.57 3.86 Mircene 988 990 6.55 4.12 1.26 10.05 5.41 3.70 ------1.88 0.27 - 2.32 0.61 - 0.49 1.75 1.75 - 5.45 - 3-Carene 1008 1010 ------1.00 0.30 - - - - - 0.59 0.94 - - - Ocimene 1022 1023 0.67 ------0.88 - - 26.73 D-Limonene 1024 1027 2.65 3.80 8.20 2.89 4.17 14.5 - - - - - 4.33 2.67 16.39 - 1.94 32.38 - 4.56 7.93 11.64 - 19.20 - (Z)--Ocimene 1032 1035 ------0.90 1.36 - - - - - 2.58 4.12 - - - (E)--Ocimene 1044 1046 3.75 3.83 2.67 0.93 0.69 1.19 - 1.39 4.64 - - 3.77 ------1.72 1.57 4.00 - - - -Terpinene 1054 1057 0.88 0.36 0.64 - 0.42 0.53 ------0.47 - 1.22 - - - Oxigenated monoterpenes 0.84 1.42 - - 1.42 1.38 1.28 - - - - - 3.56 - - 4.22 4.94 18.57 - - - 5.43 1.64 3.15 (E)-pinocarveol 1135 1138 ------1.16 ------3.15 4-Terpineol 1174 1176 - 0.77 - - 1.42 1.38 ------4.22 4.94 18.57 - - - 5.43 1.64 - α-Terpineol 1186 1189 ------2.40 ------Bornyl acetate 1287 1285 0.84 0.65 ------4-Vinilguaiacol 1309 1312 ------1.28 ------Sesquiterpene hydrocarbons 17.58 17.73 28.53 12.39 18.39 11.23 60.75 38.67 55.84 38.29 21.05 52.99 20.20 19.34 43.28 34.85 26.65 31.90 57.36 48.62 41.52 67.30 25.14 20.87 δ-Elemene 1335 1337 ------0.88 1.80 -------Elemene 1389 1391 ------3.85 1.73 ------2.15 0.90 ------Copaene 1374 1375 ------0.87 0.32 5.11 ------1,7-di-epi--Cedrene 1411 1413 ------1.38 - 3.31 12.66 ------2.39 t-Caryophyllene 1417 1418 1.34 0.93 1.42 - 0.60 0.52 5.38 2.72 4.16 9.21 - 24.91 6.10 1.39 12.15 1.68 1.39 - 13.72 7.98 7.20 16.77 1.25 - -Gurjunene 1431 1431 ------0.64 - - 1.43 3.24 ------α-(E)-Bergamotene 1432 1435 ------1.30 ------α-Humulene 1452 1452 0.39 - 1.30 - - - 1.33 - - 1.67 - 11.27 - 1.15 - - - - 1. 20 1.00 0.62 - - - -Chamigrene 1476 1474 ------1.78 - - - - - 1. 90 - - 4.40 - - Germacrene D 1484 1480 6.72 3.40 7.88 4.80 2.94 2.43 27.94 32.84 24.65 9.46 1.21 - 4.79 4.10 5.53 12.49 8.58 11.40 17.46 14.68 17.44 16.77 7.19 9.72 -Selinene 1485 1482 ------1.28 1.99 12.58 2.93 1.61 15.20 1.30 0.34 - - - 8.76 Biciclogermacrene 1500 1495 6.51 12.11 17.42 6.10 13.2 6.66 16.74 41.10 22.38 9.12 3.94 16.81 7.29 12.45 7.91 13.35 14.17 16.70 25.19 23.27 15.58 33.76 16.70 - Isodaucene 1500 1504 ------1.59 3.20 4.65 -------Bisabolene 1505 1507 ------1.40 ------δ-Amorphene 1511 1514 0.83 0.50 1.18 - 0.67 0.86 -------Cadinene 1513 1514 ------1.79 0.64 - - - - - 0.97 0.68 - - - δ-Cadinene 1522 1522 1.57 0.67 0.63 1.49 0.98 0.76 0.96 - - 3.24 - - - - - 2.25 - - 0.99 0.38 - - - - (E)-iso--Bisabolene 1528 1526 ------0.90 ------Germacrene B 1559 1556 ------1.44 - - 0.85 ------Oxigenated sesquiterpenes 47.37 48.89 34.04 25.37 19.75 29.87 8.98 4.97 - 9.61 - 23.02 57.52 30.54 52.92 30.26 12.95 20.70 25.53 13.29 12.64 20.02 10.13 16.35 Germacren D-4-ol 1574 1575 13.85 7.42 2.19 - 2.15 - 3.29 3.27 ------11.52 3.62 - - - 16.35 Espatulenol 1577 1576 6.73 4.10 3.41 7.67 5.64 6.71 - - - 3.51 - 9.24 13.21 9.53 6.29 19.29 15.60 2.70 5.43 3.15 11.70 16.17 6.52 - Caryophyllene oxide 1582 1582 1.64 1.32 1. 50 - - - 1.14 - - 4.75 - 13.78 26.30 12.38 38.6 4.16 4.16 18.00 2.53 1.19 2.25 3.85 - 1.16 Globulol 1590 1590 ------4.20 - - - 1.81 2.81 6.97 - 2.15 - Carotol 1594 1594 15.93 21.13 18.70 12.11 2.86 17.26 -------Atlantol 1608 1607 ------2.10 1.26 - - 2.20 ------Junenol 1618 1618 ------1.94 - - - - - Isolongifolene-7-α-ol 1618 1617 ------0.81 ------0.60 1.45 - - - 46

Caryophylla-4(12).8(13)- 1639 1634 ------6.22 1.10 ------dien-5α-ol - - - Alloaromadendrene 1639 1636 ------1.20 - 2.37 1.80 - - - - epoxide - - - - ent-Espatulenol n.i. 1636 ------1.50 ------Epi-α-Cadinol 1638 1639 2.80 1.75 1.90 2.15 2.91 2.10 0.78 1.70 ------0.51 1.29 - - - Epi-α-Muurolol 1640 1640 ------2.21 ------α-Muurolol 1644 1644 ------2.30 - - - - - -Eudesmol 1649 1648 ------1.41 - - 1.46 - α-Cadinol 1652 1652 3.41 1.50 - 2.49 1.88 0.90 0.84 - - 1.35 - - 1.70 3.13 3.83 3.13 2.32 ------(Z)-14-hidroxi 1666 1668 ------2.69 0.83 ------Caryophyllene - Khusinol 1679 1682 ------0.68 - - - Germacra-4(15),5,10- 1685 1683 ------0.62 ------trien-1-α-ol - Eudesma-4(15),7-dien-1 1687 1683 ------3.18 2.21 - 1.31 2.47 -------ol 5-neo-Cedranol 1684 1689 4.24 10.53 7.84 0.95 4.31 2.90 ------Diterpene hydrocarbons - - 1.40 - - 0.85 2.53 - - 2.66 ------Kaurene n.i. n.i. - - 1.40 - - 0.85 2.53 - - 2.66 ------Oxigenated diterpens - - 3.14 - - 3.41 - - - 4.37 ------4.77 - - - Manool 2056 2054 ------4.77 - - - Pimaral n.c. 2154 - - 1.74 - - 2.56 - - - 4.37 ------Ent-8(14),15pimaradien- n.c. 2195 - - 1.40 - - 0.85 ------γ-ol - - - - - Total identified (%) 97.10 96.85 96.25 70.84 69.20 79.07 73.54 45.03 60.48 54.93 21.05 89.46 93.08 72.82 96.20 75.88 79.80 71.17 90.89 89.65 87.94 92.75 86.87 76.96

______Capítulo 2

Secretory ducts distribution and leaf venation pattern of Aldama species

(Asteraceae) and their application in taxonomy

“Distribuição de canais secretores e padrão de venação em espécies de Aldama

(Asteraceae) e sua aplicação na taxonomia”

Filartiga AL1,2, Bassinello V3, Filippi GM4, Bombo AB1,2, Appezzato-da-Glória B1,2

(manuscript accepted by Botany)

1 Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brasil. 2 Departamento de Ciências Biológicas, Escola Superior de Agricultura ‘Luiz de Queiroz’, Piracicaba, SP, Brasil. 3 Graduanda em Ciências Biológicas, Escola Superior de Agricultura ‘Luiz de Queiroz’, Piracicaba, SP, Brasil. 4 Graduando em Agronomia, Escola Superior de Agricultura ‘Luiz de Queiroz’, Piracicaba, SP, Brasil. 48

Abstract: Morphological leaf features such as secretory duct distribution and venation patterns are important taxonomical tools. However, they might vary in different plants of the same species. This study aimed to evaluate whether the secretory duct distribution in the midrib and venation pattern are similar across different leaves of 17 Aldama species. Six fully expanded leaves (three each of the largest and smallest size) from five distinct plants were selected to analyze the duct distribution. The samples were histologically examined, and the quantitative data were statistically analyzed. The venation pattern was analyzed by subjecting five fully expanded leaves of different plants to diaphanization. In all, 23 secretory duct distribution patterns were identified; they showed intra- and inter-species variations except in Aldama anchusifolia and A. trichophylla. The largest number of ducts was not correlated with leaf and midrib dimensions. Further, Aldama venation could be divided into two groups: (1) pinnate camptodromous brochidodromous type (four species), and (2) acrodromous venation type and its basal and suprabasal variations (thirteen species). Thus, distinct secretory duct arrangements of the midrib might assist in the discrimination of Aldama species. The venation patterns were also important for distinguishing the majority of species selected.

Keywords Compositae, leaf anatomy, leaf architecture, micromorphology, midrib

Resumo: Características anatômicas foliares, como a distribuição de canais secretores e o padrão de venação, têm sido utilizadas como uma ferramenta importante para a resolução de problemas taxonômicos de diversos gêneros. Porém, já foi reportado que estes parâmetros podem variar em diferentes plantas da mesma espécie. O objetivo deste trabalho foi avaliar se a distribuição dos canais na nervura central e o padrão de venação são parâmetros constantes entre folhas distintas de 17 espécies de Aldama. As técnicas histológicas usuais foram usadas para a preparação das lâminas para as análises quantitativas, cujos dados foram analisados estatisticamente. Para analisar o padrão de venação, cinco folhas completamente expandidas de plantas diferentes foram submetidos à diafanização. Foram identificados 23 padrões diferentes de distribuição de canais secretores, os quais apresentaram variação intra e interespecífica, exceto em A. anchusifolia e A. trichophylla. Análises estatísticas mostraram que, em Aldama, o maior número dos canais não está correlacionado com as dimensões das folhas e da nervura central. Também foi identificado que a venação das Aldama puderam ser divididas em dois grupos: o primeiro deles com quatro espécies que apresentaram um tipo pinado, acródromo, boquidódromo, e o segundo com treze espécies que mostraram um tipo acródromo com suas variações basal e suprabasal. Foi confirmado que os diferentes arranjos dos ductos secretores da nervura central podem auxiliar na separação de espécies de Aldama quando combinado a outros dados estruturais foliares. Além disso, os padrões de venação forneceram importantes características para distinguir a maioria das espécies aqui selecionadas.

Palavras-chave Compositae, anatomia foliar, arquitetura foliar, micromorfologia, nervura central

Introduction Several studies have shown that leaf traits such as the type and position of secretory structures (Castro et al. 1997; Fahn 2000; Bombo et al. 2012; Oliveira et al. 2013; Silva et al. 2014) are useful to identify Asteraceae species (Solereder 1908; Metcalfe and Chalk 1950; Wagenitz 1976; Castro et al. 1997; Adedeji and Jewoola 2008; Fritz and Saukel 2011). However, the distribution of secretory ducts as a criterion for distinguishing species should be applied with care since their positioning and number are known to vary, as has been reported for other families. For example, in Burseraceae, the number of ducts in the midrib varied in relation to leaf size (Kakrani et al. 1991) whereas, in Pinaceae, seven duct distribution patterns were described for the needles of different populations of Pinus taiwanensis Havata (Sheue et al. 2003). In Aldama, in addition to the occurrence of secretory ducts on leaves, other features have been used in distinguishing species (Bombo et al. 2012; Oliveira et al. 2013; Silva et al. 2014); however, whether the occurrence and distribution of secretory structures are constant in the leaves of different sizes from distinct individuals of species cannot be confirmed. Venation patterns are taxonomically useful features in many groups of plants (Fonseca et al. 2007; Pacheco-Trejo et al. 2009; Gomes-Bezerra et al. 2011; Andrés-Hernández et al. 2012; Mashile and Tshisikhawe 2015). However, only few studies, including representative data for Asteraceae (Felippe and Alencastro 1966; Ravindranath and Inamdar 1982; Milan et al. 2006), are available in this regard. The genus Aldama currently includes 112 species that are distributed from the southwest of North America to South America; of these, 35 representative species occur in Brazil (Magenta and Pirani 2014). Morphologically, pollen grain features can be used to distinguish Aldama species (Magenta et al. 2010); however, a high similarity in their morphology is noted. Thus, obtaining more useful data in order to contribute to their circumscription is necessary. This study aimed to evaluate whether the number and position of secretory ducts in the midrib and the patterns of leaf venation are useful traits to assist in the taxonomical classification of Aldama genera.

Material and methods Plant species Seventeen species of Aldama were collected during expeditions between 2010 and 2015 from the South, Southeast, and Midwest regions of Brazil. The species were identified by a specialist, and the vouchers were deposited at distinct herbaria (Table 1). Aldama

filifolia, A. linearifolia, A. megapotamica, and A. trichophylla have filiform leaves with a narrow leaf blade, but all other species have a wide leaf blade.

Secretory duct analysis Samples were collected from five different individuals of each species; they were fixed in FAA 50 (formalin, acetic acid, and 50% ethyl alcohol in a ratio of 1:1:18; Johansen 1940). The distribution of secretory ducts in the midrib was determined by selecting six fully expanded leaves (three each of the largest and smallest size) of each individual depending on the amplitude of the leaf blade, as described by Magenta (2006). The width and length of each leaf were measured before histological analysis. The middle region of the leaf blade was dehydrated in a graded ethanol series and embedded in plastic resin—Leica Historesin® (Heraeus Kulzer, Hanau, Germany). Cross-sections were obtained using a Leica RM 2045 rotary microtome, and sections (5–8 µm thick) were stained with 0.05% toluidine blue O in citrate–phosphate buffer, pH 4.5 (Sakai 1973). From each block, five slides were prepared: two of them at the beginning, one at the middle, and two at the end of the block. For each character of the midrib, 10 measurements and counting were obtained: width of the midrib; number of vascular bundles; number of secretory ducts in the parenchyma, phloem, and between bundles; number of duct epithelium layers; and diameter of the lumen duct. The images were digitally captured using a Leica DMLB microscope (Leica, Wetzlar, Germany) equipped with a video camera connected to a computer. IM50 software (Leica) was used for image analysis. The measurements and counting were obtained using the measuring software Image-Pro Plus (version 4.5.0.29).

Venation analysis The venation pattern was analyzed by subjecting five fully expanded leaves of different plants to diaphanization technique, as described by Daudi and O’Brien (2012). The leaves were then washed in distilled water, stained with 1% safranin, and mounted in glycerin. The venation patterns were analyzed under a Leica M205C stereomicroscope and Leica DFC295, and the terminology used was based on Hickey (1973) and Ash et al. (1999).

Statistical analysis The average and standard deviation of each parameter were obtained separately for both leaf sizes of each species; the normality was confirmed using Kolmogorov–Smirnov & Lilliefors and Shapiro–Wilk’s tests. The values were then submitted to analysis of similarity

(ANOVA) and Tukey test to determine the existence of potential differences between leaf sizes for each species. When necessary, the data were transformed by log 10 (n + 1). Statistical significance was set at p < 0.05. The correlations between the number of secretory ducts and width of their lumen with the leaf dimensions (width and length) and width of the midrib were also analyzed. All analyses were performed using STATISTICA 10 software (StatSoft Inc, Tulsa, USA).

Results Secretory duct traits in Aldama In the Aldama species selected in this study, the secretory ducts located in the midrib consisted of a lumen surrounded by one or two epithelial cell layers (Figure 1A–D, F). In all the species, the ducts in the adaxial (DDP) and abaxial (DBP) parenchyma were evaluated according to their number and position (Table 2). Moreover, we identified secretory ducts associated with the phloem in several species, except in Aldama anchusifolia, A. bakeriana, A. filifolia, A. linearifolia, A. megapotamica, A. squalida, and A. tenuifolia. Ducts were rarely found between vascular bundles and only observed in few samples of A. arenaria, A. nudibasilaris, A. pilosa, and A. squalida. The width of the secretory duct lumen did not vary among small and large leaves of almost all species (Table 3). While the lowest average value was recorded for small leaves of Aldama megapotamica, the highest average value was noted for large leaves of A. filifolia. Some trends were observed for the width of the duct lumen. For example, in A. arenaria, the lumen of DDP tended to be narrower when the number of ducts increased (R = -0.887 for small leaves and R = -0.873 for large leaves). Similar trend was observed for DBP of A. robusta (R = -0.958 for small leaves and R = -0.995 for large leaves). In contrast, the DDP lumen tended to be wider when the DDP number increased in A. filifolia (R = 0.984 for small leaves and R = 0.978 for large leaves) and A. tenuifolia (R = 0.96 for small leaves and R = 0.995 for large leaves), as well as when the DBP number increased in A. bakeriana (R = 0.875 for small leaves and R = 0.968 for large leaves).

Secretory duct arrangements In the leaves of Aldama, secretory ducts in the midrib showed remarkable diversity in terms of arrangements. In all, 23 different patterns based on the number and position of leaves were found (Table 2). The number of DDP varied from 1 to 4 and that of DBP from 1 to 5. While pattern I had no secretory duct, a total of 9 ducts were found in pattern XXIV. The

pattern with two ducts in the adaxial region of the midrib had more variations (six) based on the number of ducts in the abaxial region than in the other arrangements, since the pattern with only one duct in the adaxial region showed only five variations, and patterns with three, four, and no secretory ducts in the adaxial region showed four variations (Table 2).

Variation of secretory ducts between leaves with different sizes In the majority of Aldama species, the leaf dimensions (width and length) showed significant differences among small and large leaves, except in Aldama bracteata, A. megapotamica, and A. tenuifolia (Table 3). Such differences were less frequent for width values of the midrib, since only five species showed different average values (Table 3). The variation in the number of secretory ducts in the midrib (Figure 1A-F) of each species might vary within a leaf; the range of these variations is shown in Table 3. Only one pattern each was observed in A. anchusifolia (pattern III, Figure 1C) and A. trichophylla (pattern VIII, Figure 1A). The secretory ducts were absent on the midrib of only the smallest leaves of A. megapotamica (pattern I, Figure 1E). A. corumbensis (eleven) and A. arenaria (ten) showed the highest number of patterns (Table 3). The most frequent patterns were VIII and XIII (Table 3, Figures 1A–1B). In the majority of Aldama species, the duct arrangements differed between two leaf sizes; only in four species (A. gardneri, A. pilosa, A. robusta, and A. squalida), the patterns were the same in both the leaf sizes (Table 3). No trend was noted for Aldama species with regard to the increase in number of ducts; only isolated cases of such trends were identified. In the large leaves of A. arenaria, for example, the DDP and DBP numbers decreased with increasing leaf width (R = -0.821 for DDP and R = -0.831 for DBP); in A. corumbensis, leaves with the same size and a wider midrib had more DDP (R = 0.914) and DBP (R = 0.841).

Venation analysis Three groups of species were formed based on two types of venation patterns. The first type was pinnate (group I; Figures 2A–2B, 2D), which was identified in filiform leaves with a single midvein. The second type was acrodromous (Figures 2F, 2J, 2M, 2P, 2S), which was found in leaves with a wide leaf blade and three or more primary veins. Acrodromous venation was distinguished according to its position at the leaf base: basal (group II), when it originated at the base of the leaf (Figures 2F, 2J); suprabasal (group III), when the veins

originated at some distance from the leaf base (Figures 2M, 2P, 2S). No variation was noted in the venation patterns among individuals analyzed for each species. Group I included four species with a narrow leaf blade. Aldama linearifolia (Figures 2A–2C) and A. megapotamica had pinnate camptodromous brochidodromous (PCB) venation, with simple and curved veinlets, whereas A. filifolia and A. trichophylla (Figure 2E) had a venation pattern that was a mixture between PCB and the pinnate camptodromous cladodromous (Table 4). Branched veinlets were found only for species with PCB venation type (Table 4). In group II, five species had the perfect basal acrodromous venation type, which included three to five primary veins running to the leaf apex. Only A. tenuifolia (Figures 2F– I) showed an imperfect development, with primary veins that did not reach the leaf apex (Figure 2F). The number of secondary veins was higher in A. bracteata and A. tenuifolia than in other species of this group (Table 4). The areolation shape was quadrangular and pentagonal (Figures 2H–2I, 2L), except in A. robusta and A. squalida, which showed hexagonal areolas. Veinlet branching was observed in four species, except in A. robusta, which had only simple and curved veinlets. The species of group III had a suprabasal acrodromous venation type, which was imperfect in A. anchusifolia (Figures 2M–2O), A. corumbensis, and A. rubra, and was perfect in A. bakeriana (Figures 2P–2R), A. gardneri, A. pilosa (Figures 2S–2U), and A. nudibasilaris. The areolation shape and veinlets had diverse features among species of this group; however, only A. corumbensis had simple and linear veinlets (Table 4). Intramarginal veins, parallel to leaf margins, and orthogonal tertiary veins were reported for all Aldama species with an expanded lamina (Figures 2G, 2K, 2N, 2Q, 2T; Table 4). The majority of these species could be distinguished only on the basis of venation features, except for A. linearifolia and A. megapotamica from group I, which had very similar characteristics (Table 4).

Discussion The diversity of leaf features such as the type and distribution of secretory structures has been frequently used to characterize Asteraceae taxa (Anderson and Creech 1975; Breitwieser 1993; Castro et al. 1997; Gregio and Moscheta 2006; Milan et al. 2006; Delbón et al. 2012). The number of ducts, along with other leaf characters, permitted differentiation among Aldama species (Bombo et al. 2012; Oliveira et al. 2013; Silva et al. 2014). However,

we found that, in 88% of species, the number and position of secretory ducts in the midrib varied in the same individual and within the same leaf, except in A. anchusifolia and A. trichophylla. Despite the variability of such traits has been considered to be significant in Pinaceae taxonomy (Wu and Hu 1997; Ghimire et al. 2015), the position and number of ducts should be used with care for distinguishing Aldama species. The variation in duct number has been reported to depend on the midrib size in Commiphora mukul Hook Ex Stocks (Burceraceae; Kakrani et al. 1991), altitude in Pinus species (Hengxiao et al. 1999; Sheue et al. 2003; Tiwari et al. 2013), nitrogen soil concentration in Scots pine seedlings (Jokela et al. 1998), low temperature (4°C) in Pinus halepensis Mill. (Fahn and Benayoun 1976), and genetic factors in Pinus roxburghii Sarg (Tiwari et al. 2013). Among these studies, only those about the influence of altitude (Hengxiao et al. 1999; Sheue et al. 2003; Tiwari et al. 2013) analyzed plants from different populations and reported that not only the number of ducts but also their position might change with altitude. Although we did not evaluate the influence of elevation in this study, we speculated that, in Aldama, altitude might influence differently, since two species with the highest number of ducts and patterns occurred in areas at an altitude of 7 606 m and 1 512 m, whereas A. trichophylla had only 3 secretory ducts and occurred at an altitude of 8 604 m. Moreover, in some species, the width of the duct lumen might be influenced by the number of secretory ducts. High nitrogen soil concentration and mid elevation (1 100 m altitude) have also been reported to be associated with the enlargement of the duct lumen (Jokela et al. 1998; Sheue et al. 2003). Further, we found that venation patterns of Aldama could be used to distinguish the majority of species selected, including those without capitula. These findings are consistent with those reported by Magenta (2006). Although some of the venation features (presence of intramarginal and orthogonal tertiary veins) were common in almost all species with a wide leaf blade, other traits (secondary veins, areolation shape, and veinlets) exhibited remarkable variation among Aldama species, including those with filiform leaves, and were useful to distinguish them. Such variability of areolation shape and veinlets have already been reported in Asteraceae (Ravindranath and Inamdar 1982) and other families such as Calycanthaceae (Nicely 1965), Convolvulaceae (Inamdar and Shenoy 1981) and Lauraceae (Moraes and Paoli 1999). However, these traits were not useful to distinguish the taxonomic features of our selected species.

The acrodromous pattern and its variations (basal and suprabasal, perfect and imperfect) were consistent with those reported for Heliantheae tribe (Asteraceae; Felippe and Alencastro 1966). The venation of several species of Asteraceae from Cerrado, including two Aldama (= Viguiera), were analyzed by Felippe and Alencastro (1966), who classified them according to the terminology defined by Ettinghausen (1861). Our classification for A. arenaria venation was similar to their observations; however, they also described a brochidodromous pattern in A. robusta with thick median primary vein and a pair of thick basal secondary vein, which was not similar to our description. The differences in the classification might be attributed to the use of different terminologies and not the variation in venation patterns of this species.

Conclusions The differences of secretory duct distribution in the midrib, reported in the present work, might occur not only in different individuals of Aldama species, but also in a single leaf. Furthermore, our data showed that the number and position of secretory ducts have no potential to be used alone as a tool for distinguishing species of this genus, but might assist taxonomical problems when combined with other features. In terms of taxonomical significance of the venation patterns, emphasizing that, although four species could not be distinguished from the others, the number of primary veins, intersecondary veins, areolation shape, and veinlets are important taxonomic features of Aldama.

Acknowledgments We thank The National Council for Scientific and Technological Development (CNPq) for grant (Proc. No. 303715/2014-6) and the São Paulo Research Foundation (FAPESP) for providing financial support (Thematic project number 2010/51454-3) and for grants to the first (2012/02476-0) and second (2012/01586-6) authors. We would also like to thank Professor Mara Angelina Galvão Magenta for species identification.

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Table 1. Brazilian Aldama species analyzed, and their respective locality, altitude and collection information.

Species Locality Altitude (m) Collection information (Aan) Aldama anchusifolia (DC) E.E.Schill. & Panero RS 65,6 ESA 135106; ESA 135107; ESA 135108 (Aar) Aldama arenaria (Baker) E.E.Schill. & Panero SP 760,6 ESA111847 (Aba) Aldama bakeriana (S.F.Blake) E.E.Schill. & Panero MS 927,7 ESA 123041; ESA 123042; ESA 123043 (Abr) Aldama bracteata (Gardner) E.E.Schill. & Panero GO 803,1 ESA 135113; ESA 135114; ESA 135115 (Ac) Aldama corumbensis (Malme) Magenta & Pirani MS 151,2 SPF 00221339 (Ad) Aldama discolor (Baker) E.E.Schill. & Panero GO 844,8 ESA 123044; ESA 123045; ESA 123046 (Af) Aldama filifolia (Sch.Bip. ex Baker) E.E.Schill. & Panero GO 803,1 ESA111850 (Ag) Aldama gardneri (Baker) E.E.Schill. & Panero GO 778,3 ESA 135116; ESA 135117 (Al) Aldama linearifolia (Chodat) E.E.Schill. & Panero MG 658,3 ESA113164 (Am) Aldama megapotamica (Malme) Magenta & Pirani RS 51,9 ESA 135103; ESA 135104; ESA 135105 (An) Aldama nudibasilaris (S.F.Blake) E.E.Schill. & Panero MG 814,3 ESA 118188; ESA 135101; ESA 135102 (Ap) Aldama pilosa (Baker) E.E.Schill. & Panero RS 785,1 ESA 135110; ESA 135111; ESA 135112 (Aro) Aldama robusta (Gardner) E.E.Schill. & Panero SP 598,3 ESA114255 (Aru) Aldama rubra (Magenta & Pirani) E.E.Schill. & Panero SP 631,1 SPF 151642 (As) Aldama squalida (S.Moore) E.E.Schill. & Panero MS 381,3 SPF 215969; SPF 215970 (Ate) Aldama tenuifolia (Gardner) E.E.Schill. & Panero MG 832,3 ESA 122869; ESA 122870; ESA 122871 (Atr) Aldama trichophylla (Dusén) Magenta PR 860,4 ESA111848

Figure 1. Cross-sections of the midrib of A. pilosa (B). A. anchusifolia (C), A. trichophylla (A), A. arenaria (D) and A. megapotamica (E-F), and the distinct arrangements of secretory ducts (circled). Detail of the patterns I (E), III (C), VI (F), VIII (A), XIII (B) and XXIII (D). Scale bars = 25 µm (D); 50 µm (A-C, E-F)

Table 2. Number of secretory ducts in the parenchyma of the midrib of Aldama species. The shape and the number of bundles in the midrib is only a general illustration; the rightward slanting stripes area represent the xylem and the small dotted sector the phloem, large black dots represent the ducts. DDP = duct in the adaxial parenchyma; DBP = duct in abaxial parenchyma.

Pattern Arrangement Number of Pattern Arrangement Number of ducts ducts

2 DDP; I None XIII 2 DBP

2 DDP; II 1 DBP XIV 3 DBP

2 DDP; III 2 DBP XV 4 DBP

2 DDP; IV 3 DBP XVI 5 DBP;

3 DDP; V 4 DBP XVII 1 DBP;

3 DDP; VI 1 DDP XVIII 2 DBP;

1 DDP; 3 DDP; VII 1 DBP; XIX 3 DBP;

1 DDP; 3 DDP; VIII 2 DBP; XX 4 DBP;

1 DDP; 4 DDP; IX 3 DBP; XXI 2 DBP;

1 DDP; 4 DDP; X 5 DBP; XXII 3 DBP;

2 DDP; 4 DDP; XI XXIII 4 DBP;

2 DDP; 4 DDP; XII 1 DBP; XXIV 5 DBP;

62 Table 3. Leaf dimensions, number of midrib bundles and number of secretory ducts in the midrib of two leaf sizes of Aldama. from DDP = duct in the adaxial parenchyma; DBP = duct in the abaxial parenchyma; - = parameter absent. For species name abbreviations see Table 1.

Leaf Width of leaf Length of leaf Width of the Number Number Number of Width of Species size blade (cm) blade (cm) midrib (mm) of bundles of DDP DBP duct lumen Duct distribution pattern (µm) Aa Small 0.96 ± 0.28 b 4.47 ± 0.78 b 0.48 ± 0.007 a 1 to 4 0 2 24.55 ± 6.85 a III Large 1.19 ± 0.51 a 6.73 ± 1.37 a 0.56 ± 0.011 a 1 to 4 0 2 27.27 ± 5.94 a III Aar Small 1.79 ± 0.73 b 3.05 ± 1.03 b 0.82 ± 0.013 a 1 to 3 1-4 1 to 4 25.93 ± 7.04 a VIII; XIII; XVIII; XXI; XXII; XIV; XV; XXIII Large 2.45 ± 0.532 a 4.75 ± 0.92 a 7.03 ± 0.012 a 2-3 1-4 1 to 5 29.89 ± 8.18 a VII; VIII; XIII; XVIII; XIV; XV; XXIII; XXIV Aba Small 1.76 ± 0.52 b 4.69 ± 0.90 b 0.6 ± 0.010 b 1 to 2 0 to 2 0 to 2 6.31 ± 2.39 a II; III; VIII; XIII Large 2.19 ± 0.49 a 6.51 ± 1.23 a 0.7 ± 0.011 a 1 to 2 0 to 2 0 to 2 6.19 ± 1.62 a III; VIII; XIII Abr Small 0.23 ± 0.05 a 15.80 ± 3.90 a 0.75 ±0.0049a 1 to 3 0 to 2 2 45.69 ± 14.8 a III; VIII; XIII Large 0.30 ± 0.11 b 16.36 ± 3.50 a 0.74± 0.0096a 2 to 3 0 to to 2 2 to 3 36.39 ± 9.25 b III; VIII; XIII; XVIII; XIV Ac Small 2.62 ± 0.50 b 8.96 ± 1.2 b 0.79 ± 0.013 a 1 to 5 0 to 2 1 to 3 15.01 ± 4.78 a VIII; XIII; XVIII; IX; XIV; XIX; XV; XX Large 3.78 ± 0.72 a 11.89 ± 1.8 a 0.85 ± 0.015 a 1 to 5 0 to 2 1 to 4 15.01 ± 5.06 a XIII; XVI; XVII; XVIII; XIX; XV; XX; XXIII Ad Small 2.40 ± 0.93 b 3.58 ± 0.67 b 0.92 ± 0.0123 a 3 to 7 0 to 2 2 21.96 ± 7.24a III; VIII; XIII Large 4.20 ± 1.70 a 5.41 ± 1.35 a 0.95 ± 0.0114 a 2 to 4, 8 0 to 2 2 to 3 21.12 ± 6.67 a III; IV; VIII; XIII Af Small 0.064 ± 14.14 b 5.67 ± 1.09 b 0.38 ± 0.0049 b 1 to 3 0 to 2 2 58.07± 21.92b III; XIII Large 0.084 ± 8.66 a 13.49 ± 1.8 a 0.42 ± 0.0048 a 1 to 3 0 to 2 2 99.83± 21.93a III; VIII; XIII Ag Small 1.57 ± 0.30 b 3.73 ± 0.60 b 0.8 ± 0.0099 a 1 to 3 1 to 3 1 to 2 9.80 ± 3.58 a VIII; XIII a a a b Large 2.59 ± 0.81 5.31 ± 0.54 0.8 ± 0.0010 1 to 3 1 to 2 2 6.35 ± 1.83 VIII; XIII a Al Small 0.13 ± 0.01 b 9.31 ± 2.78 b 0.6 ± 0.0104 a 3 to 4 1 to 2 1 to 2 7.68 ± 3.63 VII; VIII; XIII a Large 0.23 ± 0.01 a 13.17 ± 1.89 a 0.7 ± 0.0104 a 3 to 4 1 2 8.81 ± 3.40 VIII

Am Small 0.011 ± 0.04 a 2.86 ± 0.61 b 0.67 ± 0.0048 a 1 to 4 0 to 1 0 5.18 ± 1.68 a I; VIII

Large a a b 1 to 4 1 2 to 3 5.70 ± 1.91 a VI; VII; VIII 0.14 ± 0.05 5.59 ± 1.22 0.56 ± 0.0122 An Small b b b 1 to 4 0 to 2 2 to 3 12.62 ± 4.23 b III; VIII; XIII; IX; XIV 1.61 ± 0.49 6.13 ± 1.28 0.50 ± 0.0057 Large 2.80 ± 0.62 a 10.9 ± 1.16 a 0.70 ±0.0097 a 1 to 6 0 to 2 2 to 4 16.59 ± 5.48 a III; IV; V; VIII; XIII; XV b b b b Ap Small 0.77 ± 1.18 6.87 ± 1.94 0.48 ± 0.0048 1 to 4 0 to 2 0 to 2 9.97 ± 3.45 III; VIII; XIII Large 1.40 ± 0.375 a 11.88 ± 1.22 a 0.62 ± 0.0065 a 1 to 4 0 to 2 2 14.85 ±3.02 a III; VIII; XIII b b a a Aro Small 1.57 ± 0.76 3.42 ± 0.96 0.78 ± 0.022 1 to 4 1 to 3 2 to 4 28.54 ± 7.06 VIII; XIII; XIV; XV a a a a Large 2.82 ± 0.79 5.48 ± 1.25 0.93 ± 0.016 1 to 4 1 to 2 2 to 4 26.06± 6.34 VIII; XIII; XIV; XV b b a a Aru Small 1.68 ± 1.59 3.52 ± 0.85 0.71 ± 0.0271 1 to 5 1 to 2 2 to 4 56.52 ± 7.21 VIII; XIII; XIV; XV a a a a Large 3.57 ± 1.30 7.41 ± 0.90 0.81 ± 0.0271 1 to 6 0 to 2 1 to 5 61.50±25.40 III; VII; VIII; XIII; XV; X a As Small 3.44 ± 1.45 b 7.67 ± 1.37 b 0.93 ± 0.025 a 2 to 6 0 to 2 2 19.24± 5.85 III; VIII; XIII a Large 5.01 ± 1.37 a 10.06 ± 1.52 a 0.99 ± 0.025 a 1 to 7 0 to 2 2 21.53 ± 9.57 III; VIII; XIII a Ate Small 5.39 ± 0.66 a 50.46 ± 1.26 a 0.70 ± 0.0021 a 3 to 4 1 2 8.11 ± 2.39 II; III; VIII Large 5.99 ± 0.005 a 57.15 ± 0.002 a 0.50 ± 0.0021 a 3 to 4 1 2 8.34 ± 2.66 a II; III; VI Atr Small 0.05 ± 0.01 b 4.63 ± 0.66 b 0.50 ± 0.0020 a 1 to 3 1 2 66.75±15.32 a VIII Large 0.07 ± 24.51 a 8.20 ± 1.26 a 0.61 ± 0.0021 a 1 to 3 1 2 73.93 ± 8.96 a VIII Values represent mean ± standard deviation. Values followed by the same letter under the same row for each species, are not significantly different (p > 0.05).

Figure 2. Leaf diagrams of Aldama linearifolia (A-C), A. trichophylla (D-E), A. tenuifolia (F-I), A. discolor (J-L), A. anchusifolia (M-O), A. bakeriana (P-R) and A. pilosa (S-U). Overview of the venation types: pinnate camptodromous brochidodromous (PCB) (B), PCB/pinnate camptodromous cladodromous (E), acrodromous basal imperfect (F), acrodromous basal perfect (J), acrodromous suprabasal imperfect (M) and acrodromous suprabasal perfect (S). The details of intersecondary (is.v.), intramarginal (im.v.) and terciary veins, as well as the areolation shape and veinlets (circled) are illustrated in B-C, E, G-I, K-L, N-O, Q-R and T-U. Scale bars = 250 µm (E); 500 µm (R, U); 1 mm (C, H, I, L, O); 3 mm (B, G, K, N, Q, T); 5 mm (D, J, M, P, S); 1 cm (A, F).

Table 4. Summary of venation features of Aldama species. ABI = acrodromous basal imperfect; ABP = acrodromous basal perfect; ASI = acrodromous suprabasal imperfect; ASP = acrodromous suprabasal perfect; PCB = pinnate camptodromous brochidodromous; PCC = pinnate camptodromous cladodromous; - = not observed. For species name abbreviations see Table 1.

Group Species Venation type Primary Secondary Intramarginal Intersecondary Tertiary Areolation Veinlets veins veins; distance vein vein veins shape (sides) Al PCB (Fig. 2B) 1 Absent Absent (Fig. 2C) Absent Absent (Fig. 2B) 3-5 (Fig. 2C) Simple and curved or branched once Am PCB 1 Absent Absent Absent Absent 4-5 Branched once I Af PCB/PCC 1 Absent Absent Absent Absent - Simple and curved

Atr PCB/PCC (Fig. 2E) 1 Absent Absent (Fig. 2E) Absent Absent 3-4 (Fig. 2E) Simple and curved (Fig. 2E) Ate ABI (Fig. 2F) 3 10-12; irregular Present Composite (Fig. 2G) Orthogonal 4-5 (Fig. 2H-I) Simple and linear or branched once Aar ABP 3 6-8; irregular Present Composite Orthogonal 4-5 Simple and curved or branched once II Ad ABP (Fig. 2J) 3 to 5 6-10; irregular Present (Fig. 2K) - Orthogonal 4-5 (Fig. 2L) Simple and curve d or branched once Abr ABP 3 10-14; irregular Present Simple - - Simple and curved or branched once/ twice Aro ABP 3 6-8; irregular Present Composite Orthogonal 4-6 Simple and curved As ABP 3 6-8; irregular Present - Orthogonal 5-6 Simple and curved or branched once Simple and curved or branched twice/3 times Aa ASI (Fig. 2M) 3 6-4; decrescent Present (Fig. 2N) Composite Orthogonal 4-5 (Fig. O) (Fig. 2M) Ac ASI 3 8; irregular Present Simple Orthogonal 4 Simple and linear or branched once Aru ASI 3 6-8; irregular Present Composite Orthogonal 4 Simple and curved or branched once

III Aba ABP (Fig. 2P) 3 6-8; irregular Present (Fig. 2Q) Composite Orthogonal 4-5 (Fig. 2R) Simple and curved or branched once Ag ASP 3 4,6 or 8 Present Composite Orthogonal 4-5 Branched twice Ap ASP (Fig. 2S) 3 4-12; irregular Present (Fig. 2T) Composite Orthogonal 4-6 (Fig. 2U) Branched once (Fig. 2U) An ASP 3 4-8; irregular Present Composite Orthogonal 4-6 Simple and curved or branched once/twice

______Capítulo 3

Belowground organs of four Brazilian Aldama (Asteraceae): morphoanatomical traits

and essential oil profile

“Órgãos subterrâneos de quarto Aldama (Asteraceae) brasileiras: características

morfoanatômicas e perfil do óleo essencial”

Filartiga AL1, 2, Bombo AB1, 2, Garcia VL3, Appezzato-da-Glória B1, 2

(submitted to South African Journal of Botany)

Abstract: Asteraceae are one of the most abundant families in Brazilian Campos (grasslands) and Cerrado (savanna). Several Asteraceae species are able to resprout owing to the development of bud-banks in the rhizophores, xylopodium, soboles, and gemmiferous roots. The bud-bearing and thickened underground organs of some Aldama species tend to become odoriferous and produce compounds having biological activities. This study aimed to evaluate the morphoanatomy of the underground organs and to describe the chemical profile of the essential oils produced by them. The belowground organs of four Aldama species were prepared using standard histological techniques. Essential oils were obtained by hydrodistillation, and their components were identified using gas chromatography-mass spectrometry. Some morphological aspects of the underground stems were peculiar for the species investigated, such as the development of extensions and storage of inulin-type fructans, whereas some differed from the traits reported for the genus. The chemical profile of the essential oils differed among the four species analyzed, and only Limonene was the common constituent. Sixteen compounds had the potential to be considered as chemical markers. The underground organs are related to the adaptation of these plants to withstand unfavorable climate conditions characteristic of their habitats.

Keywords Bud-bearing organs, fructans, secretory duct, terpenes, thickened organ

Resumo: Asteraceae é uma das famílias mais abundantes nos Campos e Cerrado brasileiros. Vários representantes são capazes de rebrotar devido ao desenvolvimento de um banco de gemas localizado em rizóforos, xilopódios, sóboles e raízes gemíferas. Os órgãos gemíferos e espessados de algumas espécies de Aldama tendem a ser odoríferos e apresentar compostos químicos com atividades biológicas. A fim de melhorar a compreensão sobre os órgãos subterrâneos das espécies de Aldama, os objetivos deste estudo foram avaliar a morfoanatomia dos sistemas subterrâneos e caracterizar o perfil químico de seus óleos essenciais. Órgãos subterrâneos de quatro Aldama foram preparados utilizando técnicas histológicas usuais. Os óleos essenciais foram obtidos por hidrodestilação e seus componentes identificados utilizando cromatógrafo a gás acoplado ao espectrômetro de massa e ao detector de ionização de chamas. Os caules subterrâneos analisados apresentaram alguns aspectos distintos, tais como o desenvolvimento de extensões e o armazenamento de frutanos do tipo inulina, que diferem das características já relatadas para o gênero. O perfil químico dos óleos essenciais revelou algumas diferenças entre as Aldama analisadas, e apenas o Limoneno foi comum no óleo essencial das quatro espécies. Dezesseis compostos apresentaram potencial para serem considerados marcadores químicos. Os órgãos subterrâneos estão relacionados à adaptação de tais plantas aos períodos desfavoráveis em seus habitats. Palavras-chave Canais secretores, frutanos, órgãos espessados, órgãos gemíferos, terpenos

Introduction

Brazilian grasslands (Campos) and savannas (Cerrado), which have been subjected to accelerated degradation and fragmentation (Fidelis 2008; Overbeck and Pfadenhauer 2007; Parr et al. 2014), are ecosystems characterized by several species developing thickened belowground organs and a protected bud-bank that assist them to survive after abiotic and biotic disturbances (Almeida et al. 2005; Appezzato-da-Glória et al. 2008; Fidelis et al. 2009; Hayashi & Appezzato-da-Glória 2005; Medeiros & Miranda 2008; Vilhalva & Appezzato-da- Glória 2006a, 2006b). The most common bud-bearing organs found in Campos and Cerrado are rhizophores, xylopodium, soboles, and gemmiferous roots (Appezzato-da-Glória et al. 2015; Fidelis et al. 2009). These underground systems differ morphologically across species and might develop a complex anatomical structure. Asteraceae has several resprouter species with thickened underground organs, including herbs, subshrubs, and shrubs, and has been considered as one of the most abundant families in both vegetations (Nakajima et al. 2012). The Aldama La Llave genus comprises 112 species (Magenta and Pirani 2014), of which 35 are found in Campos and Cerrado (Magenta et al. 2010; Overbeck et al. 2006). Many Aldama species from Cerrado develop a xylopodium associated with tuberous roots as the belowground system (Bombo et al. 2014; Magenta 2006; Magenta and Pirani 2014; Silva et al. 2014), and their main storage compound is fructan, a type of carbohydrate that has an important role in osmoregulatory processes and is essential for aerial biomass recovery of species under harsh environmental conditions such as drought, cold, and fire events (Figueiredo-Ribeiro et al. 1991; Figueiredo-Ribeiro 1993; Itaya et al. 2007; Melo-de-Pina and Menezes 2003). The only species that occurs in Campos ecosystem and has already been evaluated is Aldama trichophylla, which also develops a woody xylopodium and storage inulin-type fructans (Bombo et al. 2014). The belowground organs of Aldama might also be odoriferous owing to the production of essential oils (Oliveira et al. 2013; Silva et al. 2014), which are characterized by a mixture of volatile and lipophilic substances rich in low-weight terpene molecules (Moraes 2009). In addition to their importance in attracting pollinators and protecting plants from herbivore attacks, they show biological activities (Ambrosio et al. 2004; Arakawa et al. 2008; Carvalho et al. 2011; Marquina et al. 2001; Nicolete et al. 2009; Porto et al. 2009). In order to improve the knowledge of belowground organs of Aldama species and to understand how these structures facilitate the survival of these plants, we selected four Aldama species occurring in Campos and Cerrado areas. This study aimed to evaluate, to our

knowledge, for the first time, their belowground system morphoanatomy and to describe the chemical profile of their essential oils.

Material and methods Plant species Belowground organs of Aldama anchusifolia (DC) E.E. Schill. & Panero (ESA135106; ESA135107; ESA135108), A. megapotamica (Malme) Magenta & Pirani (ESA135103; ESA135104; ESA135105), A. nudibasilaris (S.F. Blake) E.E. Schill. & Panero (ESA118188; ESA135101; ESA135102), and A. pilosa (Baker) E.E. Schill. & Panero (ESA135110; ESA135111; ESA135112) were collected from three different populations in 2013 during their flowering season. The species were identified by a Brazilian specialist, and the herbarium material was registered and incorporated into the collections of the Luiz de Queiroz College of Agriculture, University of Sao Paulo (ESA herbarium).

Anatomical analysis Samples of bud-bearing organs and roots of different diameters, as well as lateral roots (tuberized and non-tuberized portions), were analyzed. All the samples were fixed in FAA 50 (formalin, acetic acid, and 50% ethyl alcohol in a ratio of 1:1:18; Johansen 1940) or Karnovsky solution (Karnovsky, 1965), placed in a vacuum pump to remove the air from the tissue, dehydrated in a graded ethanol series, and stored in 70% ethanol. Some samples were dehydrated in a graded ethanol series and embedded in plastic resin (Leica Historesin®), as described by Paiva et al. (2011). The blocks were sectioned (6 µm thick) using a Leica RM 2045 rotary microtome, the slides were stained with 0.05% toluidine blue O in citrate– phosphate buffer, pH 4.5 (Sakai, 1973), and mounted in Entellan® synthetic resin (Merck, Darmstadt, Germany). Thicker sections (20–60 µm) from fixed samples were also prepared using Leica SN 2000 R sliding microtome. They were clarified in a commercial solution of 20% sodium hypochlorite (v/v) diluted to 2.5% (w/w), and then rinsed in distilled water, stained with safranin and Astra blue (Bukatsch 1972), and mounted in 50% glycerin. Some histochemical analyses were performed on sections obtained from fixed material and some on those embedded in Historesin. The following reagents and dyes were used: NADI reagent for the identification of essential and resin oils (David and Carde 1964); Sudan IV for lipophilic substances (Jensen 1962); Sudan black B for total lipids (Pearse 1968); PAS reaction for total polysaccharides (MacManus, 1948); zinc-chloride iodide for the detection of starch grains (Strasburger 1913); phloroglucinol in acid medium for the detection of lignin;

ferric chloride for phenolic compounds (Johansen 1940); and ruthenium red for pectic substances and mucilage (Gregory and Baas 1989). Digital photomicrographs were obtained using a Leica DM LB microscope and Leica DFC 310Fx camera, and LAS 4.0 software (Leica) was used for image analysis.

Essential oil extraction and analysis Two extractions were performed for each species, one for the bud-bearing organs and one for the roots, by using fresh matter that was fractionated to optimize the process. The essential oils were obtained by hydrodistillation for 3 h in a Clevenger-type apparatus. After cooling, the aqueous phase was collected, and the essential oil was obtained by washing the set-up with dichloromethane (50 mL). Each essential oil was dried over anhydrous sodium sulfate, weighed on an analytical balance to determine the yield, and stored at -5°C in a sealed amber glass flask. The essential oil constituents were determined by gas chromatography coupled to mass spectrometry (GC-MS) by using HP 5890 chromatograph Series II (Palo Alto, CA, USA) equipped with a Hewlett-Packard 5971 mass selective detector and an HP-5 capillary column (25 m × 0.20 mm × 0.33 µm). The GC-MS was performed with split/splitless injection by setting the injector at 220°C, the detector at 280°C, and the column at 60°C, with increments of 3°C·min-1 up to the final temperature of 240°C. The constituents were also determined by flame ionization detector (FID/DIC/ULTRA FAST) coupled to the Thermo Scientific TRACE GC Ultra gas chromatograph with an AS 3000 autosampler, split/splitless injection, HP-5 capillary column (30 m × 0.25 mm × 0.25 μm), temperatures equal to the aforementioned ones, and final temperature of 250°C. Helium was used as the carrier gas at 1 mL·min-1. The samples were dissolved in ethyl acetate at the concentration of 20 mg/mL. The constituents of the essential oils were identified by comparing their mass spectra with the NIST 11 Mass Spectral Library (Fabrication Varian Inc.), by co-injection of hydrocarbon patterns to calculate the Arithmetic index, as well as the data described by Adams (2007).

Results Morphoanatomy The belowground organs of Aldama analyzed comprised a thickened and bud-bearing stem structure that was located at the upper soil layers; it was responsible for producing all the adventitious tuberous and non-tuberous roots (Fig. 1A-E). The bud-bearing organs in A.

megapotamica and A. nudibasilaris were highly lignified and corresponded to an amplified portion of the stem that was located in the upper soil layers (Fig. 1A, C–D). They originated from axillary buds (Fig. 1F–G), developed tuberized roots (Fig. 1C–1D), and were characterized by a self-grafted structure (Fig. 1H–I). The adventitious roots of A. megapotamica exhibited some tuberized portions with a globular shape (Fig. 1C), whereas A. nudibasilaris exhibited uniform thickening along the tuberous roots (Fig. 1D). The bud-bearing organ showed similar morphology for A. anchusifolia and A. pilosa; however, unlike in other species, they develop an underground stem that grows horizontally and produces fusiform tuberous roots and new sprouts (Fig. 1B, E). The horizontal growth of these belowground stems was more pronounced in A. pilosa since they had longer and thicker structures and produced more new shoot sprouts than A. anchusifolia. The anatomical structure of the A. pilosa bud-bearing stem organ was similar to that of growth rings (Fig. 1H, 2A), which were defined by cells with thicker walls and by less and narrower vessel elements. A. megapotamica had the smallest belowground stems, which varied from 1 cm to 5 cm in size. The bud bank in this species showed the highest number of buds (75 buds) that were positioned throughout the structure unlike in the other species. The size of belowground organs in A. nudibasilaris, A. anchusifolia, and A. pilosa reached up to 15 cm, and the number of buds varied considerably among the species. The average number of buds was 25 for A. anchusifolia and A. nudibasilaris and 38 for A. pilosa. In these three species, the buds were located near the aerial stem base in the upper portion of the structure (Fig. 1F). All the buds observed in these organs were axillary and were characterized by the formation of a trace in the vascular tissue (Fig. 2A–B). The stem structure of the bud-bearing organs was confirmed by the centrifugal development of the primary xylem (Fig. 2A, inset) for the four species. The protective tissue, which gradually replaced the epidermis that was still present in some samples (Fig. 2C), was formed by suberized thick-walled cells, which had originated by periclinal divisions of the subepidermal cell layer with suberized thick walls (Fig. 2C–D). The cortical region comprised fibers, secretory spaces, and endodermal cells with Casparian strips (Fig. 2 D, inset) in all four species analyzed. In the vascular cylinder, secretory spaces were observed in the secondary phloem interspersed with vascular elements, fibers, and parenchyma cells (Fig. 2E); the lumen width varied among the species. Aldama nudibasilaris had more phloematic fibers than those in the other species (Fig. 2D), and secondary xylem was well developed and lignified in the four

species. Vessel elements were larger and greater in number in A. anchusifolia, A. nudibasilaris, and A. pilosa than in A. megapotamica (Fig. 2D–E). In A. megapotamica and A. pilosa, the cells of the secondary xylem located subjacent to the cambium were not completely differentiated and showed a dense cytoplasm content unlike the other xylem cells (Fig. 2E). The primary xylem was visible at the periphery of the medulla, in which secretory spaces were observed in all the species, and sclereids developed only in A. megapotamica (Fig. 2F). Inulin sphero-crystals were observed inside the cortical cells, vascular parenchyma, as well as in the medullary cells and inside the lumen of secretory structures (Fig. 2G–H). The root system consisted of tuberized and non-tuberized roots and their laterals, as mentioned before (Fig. 1B–E). In an incipient secondary structure, the epidermis was uniseriate, followed by exodermis, which was characterized by a layer of cells containing phenolic substances and suberin thickening (Fig. 3A). Fungi association was observed in the cortical parenchyma of some samples (Fig. 3A). The secretory ducts of lipophilic substances had an epithelium consisting of four cells: two cells from the cortical parenchyma and two endodermal cells (Fig. 3B). Pericycle was uniseriate, and the vascular system consisted of few protoxylem poles and fibers in the central region in the lateral roots (Fig. 3A) whereas, in adventitious roots, the number of protoxylem poles was higher and medulla was poorly developed (Fig. 3B). The non-tuberized roots, with established secondary structure, presented periclinal divisions in the parenchyma subjacent to the exodermis, which is eliminated with the epidermis. Anticlinal divisions occurred in the cortical parenchyma (Fig. 3C). In the secondary structure of tuberized roots, the epidermis and exodermis were replaced by several layers of suberized cell, resulting in the periclinal division of exodermis underlayer (Fig. 3D). Lenticels were identified in A. anchusifolia, A. nudibasilaris, and A. pilosa (Fig. 3D, inset), and new secretory ducts were formed in the inner cortex only in the last two species (Fig. 3E). In the larger roots of A. anchusifolia, A. nudibasilaris, and A. pilosa, secretory ducts, which originally consisted of four cells, had their epithelium and lumen extended (Fig. 3E). The pericycle increased the number of cell layers in all species and could reach up to five layers only in A. nudibasilaris and A. pilosa (Fig. 3G). The cambium was arranged in a continuous ring, which produced only radial parenchyma in some sectors and all elements of the axial and radial systems in other sectors (Fig. 3F–G). In A. megapotamica, the regions of radial parenchyma were wider than those in the other species (Fig. 3F–I). This cambium activity caused the displacement of tracheal elements to the inner portions of the structure owing to the formation of xylem parenchyma arranged in radial rows

(Fig. 3I). Secretory ducts were found in the secondary phloem of all species (Fig. 3G), but it was more frequent in A. pilosa and less frequent in A. nudibasilaris. Sclereids were present on the opposite sides of phloem and occurred only in A. anchusifolia and A. nudibasilaris. The medulla was characterized by an intense cell proliferation (Fig. 3H–I) and developed secretory cavities in three species except A. anchusifolia. The inulin crystals were also detected (Fig. 3J) inside cortical cells, in the parenchyma of secondary vascular tissues, inside vessel elements, in the medullary cells, and around and inside secretory ducts.

Essential oils The yield of essential oil (EO) from belowground organs was higher in the roots than in the belowground stem, except in Aldama megapotamica, in which yield was similar in both the organs (Table 1). The belowground stem of A. pilosa had the highest mean yield (0.63 ± 0.31% w/w), which was about seven times higher than that in A. megapotamica (0.09 ± 0.07% w/w) and A. nudibasilaris (0.08 ± 0.02% w/w). Considering the root essential oil yield, A. anchusifolia showed the highest mean, followed by A. pilosa and A. nudibasilaris (Table 1). In the species analyzed in this study, the composition of the essential oils revealed 22 monoterpenes, 42 identified sesquiterpenes, and 12 known diterpenes (Supplementary Table 1). Seventy-one constituents were identified in the essential oils from belowground stems and 68 in root EOs, 65 of which were common to both organs (Supplementary Table 1). Only one monoterpene (Limonene) occurred simultaneously on both the organs in the four species analyzed. The essential oil of A. anchusifolia and A. pilosa had monoterpene predominance, whereas A. megapotamica had the majority of sesquiterpenes, and A. nudibasilaris showed diterpenes as the major compounds. The major constituents were identified for each species, considering the ones with higher relative percentages (Table 2). Although α-Pinene was the major compound in A. anchusifolia and A. pilosa, the diterpenes Kaurene and Pimaral exhibited higher relative percentage in A. nudibasilaris and A. megapotamica, respectively (Table 2). The sesquiterpenes (epi-α)-Cadinol and Ciperene also were present in large amounts in A. megapotamica and A. anchusifolia EOs (Table 2). When a constituent was identified in the essential oils from at least two populations, in both or only one organ, it was considered as unique for that species (Table 2). A. anchusifolia showed three unique constituents (Allo-Aromadendrene, Abieta-8(14), 13(15)-diene, and

Metil-neoabietate); A. megapotamica presented nine (-Terpineol, Verbenona, Gurjunene, <4,8β-Epoxi>-Cariofilene, Guaiadiene, -Chamigrene, Aristolochene, β-Selinene, and 7-epi- -Selinene); A. nudibasilaris presented two unique constituents (Linalool and Eudesm-7(11)- en-4-ol); and A. pilosa also showed two unique constituents (Longiciclene and Filocladanol; Table 2).

Discussion The belowground systems of Aldama species evaluated in this study showed distinct morphological and chemical aspects related to the presence of subterranean stem extensions, carbohydrate storage, and essential oil profile, which might contribute to the better understanding of their survival strategies. According to Lindmann and Ferri (1974), the size of the belowground systems of plants from Campos tends to be smaller than the organs of species that grow in Cerrado. Our observations showed an opposite trend: A. anchusifolia and A. pilosa, which are among the tallest species of the genus (Magenta 2006), had larger belowground organs than those observed in Aldama from Cerrado areas (Bombo et al. 2014; Oliveira 2011; Silva et al. 2014). The size of belowground organs might be influenced by genetic features, soil constitution (Ramos et al. 1982), and water content (Franco and Inforzato 1946; Maina et al. 2002); further, Bizari et al. (2010) also reported that root density is directly related to plant height. Morphoanatomical studies have shown that Aldama species tend to develop xylopodium (Oliveira et al. 2013; Bombo et al. 2014; Silva et al. 2014), which is an extremely lignified and perennial structure, formed by the tuberization of hypocotyl or the root-stem transition region and the proximal portion of the main root (Rizzini 1965). Xylopodium is also characterized by a self-grafting process (Paviani 1987) by the predominance of woody tissues and the absence of storage tissues in addition to the vascular parenchyma (Appezzato- da-Glória, 2015). Although the four species develop a structure similar to xylopodium, it had some different aspects in these plants. The storage of carbohydrates (inulin-type fructan) and the formation of stem extensions were did not correspond to the general description of xylopodium; because of these traits, we classified them as belowground stems. Inulin-type fructans have been considered to be a plant strategy of species that withstand environmental disturbances such as prolonged drought, fire, and cold events (Figueiredo-Ribeiro 1993; Itaya et al. 2007; Melo-de-Pinna 2000; Portes and Carvalho 2006). This carbohydrate is widely reported in several Asteraceae from Cerrado (Dias-Tagliacozzo et

al. 1999; Figueiredo-Ribeiro et al. 1986; Tertuliano and Figueiredo-Ribeiro 1993); further, in addition to being a source of energy and carbon reserve, it also enables cells to rapidly change their osmotic potential and use this carbohydrate when environmental conditions become favorable (Valluru & Van den Ende 2008; Vilhalva et al. 2011). In Asteraceae species, inulin crystals were distributed in the storage parenchyma as well as in the cells of the vascular cylinder of tuberous roots, rhizophores, and xylopodium (Asega 2003; Hayashi and Appezzato-da-Glória 2005; Oliveira et al. 2013; Silva et al. 2014; 2015; Vieira and Figueiredo-Ribeiro 1993; Vilhalva et al. 2011). For the Aldama species analyzed in this study, we also identified inulin crystals inside secretory ducts, a feature that was already observed for other species of this genus (Bombo et al. 2014; Oliveira et al. 2013). The presence of growth rings in the stem extensions of Aldama pilosa was newly reported for the genus. False annual rings have already been identified in the xylopodium of Cerrado plants such as Mandevilla species (Appezzato-da-Glória and Estelita 2000) and Brasilia sickii G. M. Barroso (Paviani 1977). The formation of such increment zones in aerial branches might be influenced by favorable and unfavorable climate growth conditions of precipitation (Marcati et al. 2006; Worbes 1995) and temperature (Jump et al. 2006). For Aldama pilosa, which grows in Southern Brazil - a region characterized by hot summer, cold winters, and no dry season (Nimer 1990) - the development of such growth rings might be potentially influenced by these conditions. In the belowground organs of Aldama analyzed in this study, the internal secretory spaces were similar to those described previously for the genus (Bombo et al. 2014; Oliveira et al. 2013; Silva et al. 2014). Such structures produced EOs that contribute to the protection of belowground organs against microorganisms and herbivores (Rasmann et al. 2005; Unsicker 2009; Wink 1988). Further, we found that the EOs evaluated had higher yield values in the belowground organs of the species selected than in their aerial organs (unpublished data). The same trend was also reported for other species of the genus (Bombo et al. 2012, 2014; Oliveira 2011; Silva 2013). Variation of EO yield has been associated with environmental factors (Andrade 2000), which might change the plant metabolic pathway, and thus the biosynthesis of chemical compounds (Morais 2009), as well as the presence and frequency of secretory structures (Fahn 1979). Indeed, the number and size of secreting spaces in the belowground organs of A. anchusifolia, A. megapotamica, A. pilosa, and A. nudibasilaris were higher than those in their leaves and stems (unpublished data).

Some constituents detected in this study have biological activities that have already been reported for EOs in other plants. In Monticalia andicola (Asteraceae), the antibacterial action has been attributed to α-Pinene and Ciperene (Baldovido et al. 2009); in two species of Piper and in Viguiera dentata (Cav.) Sprenguel (Asteraceae), antifungal activity has been reported for α-Pinene (Canales et al. 2008; Constantin et al. 2001; Leite et al. 2007). Furthermore, besides their phytochemical importance, some chemical constituents were unique from each species, which allows them to be used as a tool to assist taxonomic studies in Aldama, as has been reported for this genus (Ambrosio et al. 2004; Bombo et al. 2012; Carvalho et al. 2011; Da Costa et al. 1996, 2001; Spring et al. 2001, 2003).

Conclusions

Based on morphoanatomical data, we classified the belowground system of Aldama anchusifolia, A. megapotamica, A. nudibasilaris, and A. pilosa as a subterranean stem bearing adventitious tuberous roots. The presence of buds in these organs, as well as the storage of fructans in all the belowground structures and the production of essential oils, is a trait that allows these species to survive and to resprout after unfavorable periods. Furthermore, the chemical composition of EOs revealed some constituents that have biological activities and others with a potential to be considered as chemical markers, which might assist in the identification of these species.

Acknowledgments This work was supported by the National Council for Scientific and Technological Development - CNPq (Proc. No. 303715/2014-6) and by the São Paulo Research Foundation - FAPESP (Thematic project number 2010/51454-3; Grant numbers 2012/02476-0 and 2012/01586-6). We would also like to thank Professor Mara Angelina Galvão Magenta for species identification.

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Figure 1. Belowground bud-bearing organs of Aldama anchusifolia (F), A. megapotamica (A, C), A. nudibasilaris (D, I), A. pilosa (B, F, G, H). A. Overview of the species. Bud-bearing organ at the upper soil layer (arrow, inset). B. Overview of the horizontal stem extension with new shoot sprouts (arrows) and tuberous roots (arrowhead, inset). C-D. Bud-bearing organs with tuberous roots (arrowheads). E. Belowground stem (arrow) with aerial branches and roots (arrowheads). F. Bud-bank at the swollen base of the aerial stem branch (arrows). G. Cross-section of the belowground organ showing an axillary bud connected to the center of the structure (dotted arrow). H. Stem branch with growth rings (arrows). I. Two self-grafted branches. Note the junction area between the two branches (arrows). Scale bars = 2.5 mm (G, H, I), 5 mm ( F), 2.5 cm (A, inset), 3 cm (B inset), 5 cm (C, D, E), 10 cm (A, B).

Figure 2. Cross-sections of belowground bud-bearing organs of Aldama anchusifolia (C, G, H), A. megapotamica (B, E), A. nudibasilaris (D), A. pilosa (A, F). A. Stem structure with endarch protoxylem (circled in inset) and growth rings (arrows). B. Detail of the axillary bud and part of its lacuna (arrow). C. Detail of the covering tissue with epidermis remains (arrow) and periclinal divisions of subepidermal cells (arrowhead). D-E. Secondary phloem (Sph) and xylem (Sxy). Detail of the secretory space (*, D inset) and endoderm with Casparian strips (arrow, D inset) Note the lower number and size of the vessel elements near the cambium (E), the secondary phloem with fibers (arrowhead) and secretory spaces (arrows), and xylem cells with denser cytoplasm (*, E). F. Medulla with sclereids (*). G-H. Inulin crystals observed under polarized light in the secondary phloem (G), inside secretory ducts (G, arrows), and in the medulla cells (H). Scale bars = 25 µm (A inset, D inset), 50 µm (C), 200 µm (B, D, E, F, G, H), 2,5 mm (A).

Figure 3. Cross-sections of the roots of Aldama anchusifolia (J), A. megapotamica (A, C, H, I), A. nudibasilaris (B, E, F), A. pilosa (D, G). A-C. Non-tuberous roots. A-B. Primary structure with epidermis (Ep), exodermis (Ex), cortical parenchyma (Cp), secretory ducts (arrows in B) with endoderm origin (En), pericycle (Pr). Note the presence of fungal hyphae (arrow in A). C. Secondary structure showing the beginning of periclinal divisions near the exodermis (arrow) and cambium establishment (arrowheads). Detail of the Casparian strips (inset). D-J. Tuberous roots. D. General view. The covering tissue may form lenticels (inset) and exodermis underlayer show periclinal division (arrowheads). The cambium may form wide xylem rays (*) E. Detail of the cortical region. Cortical cells (Cp) undergo anticlinal divisions. F. Root with maximum tuberization. G. The pericycle (Pr) with more than one layer. Cambium (*) originates only vascular parenchyma in some sectors of the cylinder. There are secretory ducts in the secondary phloem (Sph; arrows). H. Overview of the tuberized root. Note the size of the medulla (Me). I. Cambium (arrowheads) produces many parenchyma cells that change the primary xylem position (arrows). Detail of the medullar secretory cavity (inset). J. Inulin crystals (arrows) observed under polarized light. Scale bars = 25 µm (B), 50 µm (A, C, C inset, I inset), 100 µm (D, E, J) 200 µm (D inset, G), 500 µm (F, H, I).

Table 1. Fresh matter (W; g) and essential oil yield (Y; % w/w) from belowground organs of different populations of Aldama anchusifolia (AA), A. megapotamica (AM), A. nudibasilaris (AN) and A. pilosa (AP). Mean±standart deviation.

Species Organ Parameter Population Mean 1 2 3 Underground M 295.29 356.77 353.88 335.31 ± 34.69 AA stem Yeo 0.34 0.31 0.35 0.33 ± 0.02 Root M 549.61 122.48 308.11 220.78 ± 143.12 Yeo 0.19 0.35 0.49 0.34 ± 0.15 AM Underground M 102.78 84.67 70.54 86.00 ± 16.16

stem Yeo 0.01 0.16 0.11 0.09 ± 0.07 Root M 8.29 2.93 3.44 4.88 ± 2.95 Yeo 0.01 0.11 0.09 0.07 ± 0.05 AN Underground M 377.92 72.97 337.69 262.86 ± 165.67 stem Yeo 0.08 0.11 0.07 0.08 ± 0.02 Root M 294.29 138.42 66.49 166.40 ± 116.44 Yeo 0.24 0.30 0.08 0.20 ± 0.11 Underground M 124.11 126.25 139.44 358.50 ± 244.38 AP stem Yeo 0.40 0.51 1.00 0.63 ± 0.31 Root M 117.02 131.55 153.83 38.91 ± 37.35 Yeo 0.19 0.30 0.23 0.24 ± 0.05

Table 2. Chemical profile and relative percentage area (%) of the essential oils extracted from belowground organs of Aldama anchusifolia, A. megapotamica, A. nudibasilaris and A. pilosa. P = population, AIlit= arithmetic index from literature, AIcalc= Calculated arithmetic index.

IAC Aldama anchusifolia Aldama megapotamica Aldama nudibasilaris Aldama pilosa IALit al Composto Underground stem Root Underground stem Root Underground stem Root Underground stem Root

P P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 Monoterpenes α-Pineno 932 934 41.67 39.18 36.2 34.77 42.34 58.1 ------0.53 4.29 5.29 27.31 4.41 15.51 59.42 57.81 58.66 3.30 52.44 50.91 Linalool 991 995 ------2.28 10.97 3.64 ------δ Careno 1008 1011 3.46 1.20 1.20 5.42 3.39 8.76 - 3.73 - - - - 2.50 1.10 5.91 29.74 0.81 9.44 7.04 12.47 20.15 - 10.93 12.66 Limoneno 1024 1028 1.04 0.75 0.75 1.44 1.08 0.77 0.38 1.93 - 0.17 - - - 1.10 - 0.90 0.47 - 0.87 2.43 1.13 - 2.62 0.71 α-Terpineol 1186 1189 ------1.2 2.20 ------Verbenona 1204 1208 ------0.47 - 1.22 ------Timol,metil eter nc 1234 ------1.61 - - 2.12 - 0.70 ------Sesquiterpenes Longicicleno 1371 1369 ------0.31 0.54 - 0.36 0.21 Cipereno 1398 1398 - 0.26 - 1.10 1.10 0.47 14.4 18.45 25.33 9.98 5.29 ------α-Gurjuneno 1409 1405 ------0.70 - 0.93 0.61 ------Guaiadieno 1452 1449 ------0.70 - 0.93 0.34 ------Allo-Aromadendreno 1458 1459 0.75 0.75 0.83 0.70 0.69 0.70 ------Chamigreno 1476 1473 ------0.94 1.59 3.81 1.59 ------β Aristolocheno 1487 1483 ------0.41 - 1.15 ------β-Selineno 1489 1485 ------3.61 3.46 5.47 4.10 ------7-epi-α-selineno 1520 1516 ------0.89 - 2.11 0.44 ------<4,8β-Epoxi> Cariofileno 1423 1418 ------1.94 - 1.12 0.28 ------Humuleno epoxido II 1608 1608 ------0.5 - - 0.22 ------(epi α)-Cadinol 1638 1641 16.21 17.36 15.9 13.7 14.68 8.34 0.72 ------0.90 0.64 - 0.57 0.31 Eudesm-7(11)-en-4-ol, acetato 1839 1831 ------7.93 2.87 - 2.70 7.30 9.89 ------Diterpenes Kaureno 2042 2034 ------3.20 - 0.59 - - 52.02 70.87 42.85 3.50 37.11 29.93 ------Abieta-8(14),13(15)- dieno 2153 2142 - - - 3.27 5.06 ------Filocladanol 2209 2200 ------6.95 2.58 1.79 12.64 3.81 4.85 Metil neoabietato 2443 nc 2.50 3.53 - 3.17 3.30 ------Pimaral nc nc 2.08 0.82 - 2.78 - - 2.97 38.72 15.56 15.64 - - 0.47 - 2.91 0.98 1.12 1.28 ------

Supplementary Table 1. Chemical profile and relative percentage area (%) of the essential oils extracted from xylopodium and roots of Aldama anchusifolia, A. megapotamica, A.

nudibasilaris and A. pilosa. P = population, AIlit= arithmetic index from literature, AIcalc= Calculated arithmetic index. Constituent Aldama anchusifolia Aldama megapotamica Aldama nudibasilaris Aldama pilosa

AI lit AI calc Underground stem Root Underground stem Root Underground stem Root Underground stem Root P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3 Monoterpenes 56.10 45.54 42.12 55.79 53.39 73.10 6.71 14.54 4.98 0.76 2.11 - 9.03 17.46 17.67 69.05 6.57 30.60 74.84 87.69 88.28 12.87 83.07 69.15 α-Tujene 924 926 0.28 0.30 0.29 0.23 0.36 0.52 ------0.61 - - 0.47 0.65 0.91 - 0.55 0.37 α-Pinene 932 934 41.67 39.18 36.2 34.77 42.34 58.1 ------0.53 4.29 5.29 27.31 4.41 15.51 59.42 57.81 58.66 3.30 52.44 50.91 Canfene 946 948 2.32 0.91 0.44 3.21 2.00 0.91 ------0.89 1.76 0.38 - 3.86 0.31 Sabinene 969 972 0.69 0.57 0.74 0.90 0.73 1.12 ------1.00 0.95 1.04 - 1.10 0.82 β-Pinene 974 977 3.95 1.26 0.89 6.11 1.53 1.59 - 1.93 ------0.60 - 0.51 3.58 8.63 2.23 - 9.01 1.34 β-Mircene 988 990 0.63 - - 0.74 0.17 ------0.63 - - 0.79 0.71 0.94 - 0.77 0.65 Linalool 991 995 ------2.28 10.97 3.64 ------α-Felandrene 1002 1005 - - - 0.67 - 0.37 ------2.47 - 1.59 0.34 0.52 0.80 - 0.40 0.50 δ-Carene 1008 1011 3.46 1.2 1.2 5.42 3.39 8.76 - 3.73 - - - - 2.50 1.10 5.91 29.74 0.81 9.44 7.04 12.47 20.15 - 10.93 12.66 r-Cimene 1020 1021 ------0.69 - - 1.26 - 0.93 - 0.72 0.88 - 0.63 0.34 ρ-Cimene n.c. 1024 ------0.77 2.15 ------Limonene 1024 1028 1.04 0.75 0.75 1.44 1.08 0.77 0.38 1.93 - 0.17 - - - 1.10 - 0.90 0.47 - 0.87 2.43 1.13 - 2.62 0.71 β-Ocimene 1032 1035 0.46 0.39 0.20 1.59 0.72 0.68 0.40 2.33 1.46 0.15 2.11 ------α-Canfolenal 1122 1125 0.55 0.21 0.50 0.19 ------0.52 - 0.30 0.28 1.11 - - Sabinol 1137 1143 1.05 0.23 0.91 0.52 - 0.28 0.63 - 2.30 0.44 ------0.42 0.71 0.44 0.42 0.41 5.77 - 0.27 Mentha-1,5-dien-8-ol 1166 1166 ------1.84 ------0.71 0.46 ------1-Terpinen-4-ol 1174 1176 - 0.54 - - 1.07 - 0.92 - - - - - 0.60 ------0.32 0.47 1.27 0.76 0.27 m-Cimen-8-ol 1176 1180 ------0.23 2.42 - - - - 0.82 - 2.06 0.55 - 0.69 ------α-Terpineol 1186 1189 ------1.20 2.20 ------Mirtenol 1194 1195 ------0.64 ------1.42 - - Verbenona 1204 1208 ------0.47 - 1.22 ------Timol,metil eter n.c. 1234 ------1.61 - - 2.12 - 0.70 ------Sesquiterpenes 20.62 26.70 24.96 20.24 23.57 17.05 52.97 26.65 45.54 34.75 8.66 - 16.36 5.74 5.25 7.53 17.84 20.96 2.60 2.65 5.27 0.97 2.59 2.71 α-Longipinene 1350 1349 ------0.50 - 2.00 1.18 - - 0.83 1.07 1.87 - 1.29 0.74 Longiciclene 1371 1369 ------0.31 0.54 - 0.36 0.21 α-Copaene 1374 1375 ------0.98 - - 0.97 ------Modef-2-ene 1382 1379 ------0.98 - - 0.66 ------α-Isocomene 1387 1386 ------1.51 ------0.59 - - 0.42 Sativene 1390 1392 ------1.66 ------Ciperene 1398 1398 - 0.26 - 1.10 1.10 0.47 14.40 18.45 25.33 9.98 5.29 ------α-Gurjunene 1409 1405 ------0.70 - 0.93 0.61 ------n.i. n.c. 1435 ------1.02 - 1.56 0.62 ------Guaiadiene 1452 1449 ------0.70 - 0.93 0.34 ------α-Humulene 1452 1453 ------0.95 ------Allo-Aromadendrene 1458 1459 0.75 0.75 0.83 0.70 0.69 0.70 ------88

4-14,5diene,cis-Muurola 1465 1468 ------1.03 ------Chamigrene 1476 1473 ------0.94 1.59 3.81 1.59 ------β Germacrene D 1484 1480 ------1.48 - - - 3.37 - - - - 0.95 - 1.18 ------Aristolochene 1487 1483 ------0.41 - 1.15 ------β-Selinene 1489 1485 ------3.61 3.46 5.47 4.10 ------Amorfene 1495 1493 ------1.11 ------δ Valencene 1496 1491 ------0.99 ------Biciclogermacrene 1500 1495 - - 0.61 - 0.28 2.11 1.24 ------1.23 - 0.48 - - 0.31 -Bisabolene 1505 1507 - - - - 0.34 - 1.42 0.66 - - - - 1.05 - 2.06 ------β δ-Amorfene 1511 1514 2.56 2.79 3.19 2.52 2.53 2.63 - - - 0.52 ------7-epi-α-Selinene 1520 1516 ------0.89 - 2.11 0.44 ------δ-Cadinene 1522 1523 0.62 0.79 0.68 0.82 0.86 0.69 5.15 3.15 - 2.65 ------Zonarene 1528 1534 ------1.67 ------n.i. n.c. 1537 ------1.27 - - 0.38 ------Selina-3,7(11)-diene 1545 1541 ------1.01 - - 0.59 ------Germacrene B 1559 1556 ------1.38 - - 0.28 ------<4,8β-Epoxi> Cariofilene 1423 1418 ------1.94 - 1.12 0.28 ------ Nerolidol 1561 1561 ------0.67 - - 0.58 ------n.i. n.c. 1572 ------0.40 - - 0.90 ------Espatulenol 1577 1576 0.48 2.27 2.66 0.36 1.87 1.59 2.83 - 2.43 0.56 ------1.18 - 0.54 0.37 1.15 0.97 0.37 0.72 Oxido Cariofilene 1582 1582 ------1.81 - - 0.94 ------

Viridiflorol 1592 1590 - 1.13 - - 0.42 ------Carotol 1594 1587 ------0.58 - - 0.66 ------Guaiol 1600 1602 ------4.16 - - 0.48 ------Humuleno epoxido II 1608 1608 ------0.50 - - 0.22 ------n.i. n.c. 1612 ------0.54 - 0.66 1.07 ------1-epi-Cubenol 1627 1627 ------0.64 ------(epi α)-Cadinol 1638 1641 16.21 17.36 15.90 13.70 14.68 8.34 0.72 ------0.90 0.64 - 0.57 0.31 α-Cadinol 1644 1653 - - 1.09 1.04 - 0.52 0.50 ------Muurolol 1644 1645 ------1.08 - - 0.44 ------β-Eudesmol 1649 1648 ------0.36 - - 0.66 ------Selin-11-en-4-α-ol 1658 1653 - 1.35 - - 1.14 ------7-epi-α-Eudesmol n.c. 1656 ------0.84 0.59 ------Mustacona 1676 1679 ------2.2 ------Eudesm-7(11)-en-4-ol, acetate 1839 1831 ------7.93 2.87 - 2.70 7.30 9.89 ------Diterpenes 4.58 9.40 5.59 9.22 13.38 3.03 4.91 41.92 15.56 46.62 - - 44.56 68.00 45.76 1.78 30.93 21.32 7.46 2.58 1.79 12.64 4.12 6.30 n.i n.c. 1922 0.48 0.55 - 0.50 0.49 ------Pimaradiene 1948 1940 - - 2.65 - - 2.04 ------0.51 - - - 0.31 0.45 n.i. n.c. 1944 ------7.16 1.72 3.20 0.55 4.66 1.77 ------

n.i. n.c. 1953 ------1.78 - 2.01 - 0.97 ------n.i. n.c. 1982 ------0.40 - - - 0.71 1.30 - - - 3.54 0.51 0.28 Kaurene 2042 2034 ------3.20 - 0.59 - - 52.02 70.87 42.85 3.50 37.11 29.93 ------Manool 2056 2057 - 1.91 - - 1.34 ------1.01 Manool <13-epi> 2059 2067 - 3.14 - - 3.68 ------Abietadiene 2085 2090 - - 2.94 - - 0.99 ------n.i. n.c. 2134 ------15.39 1.68 6.10 - 1.01 ------n.i. n.c. 2140 2.39 4.46 0.60 ------3.15 0.95 1.17 Abieta-8(14), 13(15)-diene 2153 2142 - - - 3.27 5.06 ------n.i. n.c. 2154 - - 12.45 - - 5.78 ------1.50 ------n.i. n.c. 2196 - - 1.87 ------Filocladanol 2209 2200 ------6.95 2.58 1.79 12.64 3.81 4.85 n.i. n.c. n.c. 3.74 1.84 - 4.59 2.05 ------2.19 3.08 2.43 ------n.i. n.c. n.c. 2.01 2.01 - 2.24 1.80 ------1.02 3.71 6.68 1.33 4.23 3.00 3.04 1.49 1.09 18.06 1.72 4.21 n.i. n.c. n.c. 2.94 2.70 0.41 3.4 2.42 ------1.82 - 11.12 2.80 8.01 5.62 4.45 2.20 1.64 44.44 4.58 6.00 n.i. n.c. n.c. 2.30 1.37 - 2.70 1.22 ------3.69 1.93 1.25 - - 4.81 n.i. n.c. n.c. ------9.67 ------Metil neoabietato 2443 n.c. 2.50 3.53 - 3.17 3.30 ------n.i. n.c. n.c. - - 5.37 ------1.95 0.95 0.37 - 1.72 2.23 3.35 n.i. n.c. n.c. ------2.97 1.09 0.67 - - - Pimaral n.c. n.c. 2.08 0.82 - 2.78 - - 2.97 38.72 15.56 15.64 - - 0.47 - 2.91 0.98 1.12 1.28 ------n.i. n.c. n.c. ------0.47 1.68 2.2 - 0.96 ------n.i. n.c. n.c. - 0.65 1.36 ------n.i. n.c. n..c ------3.39 3.00 ------n.i. n.c. n.c. ------0.23 2.01 n.i. n.c. n.c. ------0.66 16.09 6.55 ------Ent-8 (14)15-pimaradien-3 β- n.c. n.c. ------6.77 ------ol Diterpenic acid n.c. n.c. ------1.94 - - 23.62 ------n.i. n.c. n.c. ------2.61 - -

Total identified (%) 81.3 81.64 72.67 85.25 90.34 93.18 64.59 83.11 66.08 82.13 10.77 - 69.95 91.20 68.68 78.36 55.34 72.86 84.90 92.92 95.34 26.48 89.77 78.15

______Capítulo 4

Underground organs of Brazilian Asteraceae: testing CLO-PLA database traits

“Órgãos subterrâneos de Asteraceae do Brasil: teste do bando de dados CLO-PLA”

Filartiga AL1, 2, Klimešová J3, Appezzato-da-Glória B1,2

(manuscript accepted by Folia Geobotanica)

1 Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brasil. 2 Departamento de Ciências Biológicas, Escola Superior de Agricultura ‘Luiz de Queiroz’, Piracicaba, SP, Brasil. 3 Instituto de Botânica, Acadeia de Ciências da República Tcheca, Třeboň, República Tcheca.

Abstract: The standardization of plant traits and databasing are not available for all regions and all plant traits. Some ecosystems as tropical grasslands are being underrepresented in such databases due to the difficulty to the assessment of bud bank and evaluation of clonal growth. The two aims of this works are: (i) to test if Brazilian morphological traits of belowground organs can be translated into categories used in CLO-PLA database and (ii) to test the applicability of clonal and bud bank traits standardized in CLO-PLA database for Brazilian Aldama. A total of 165 species, including herbs, subshrubs and shrubs of 37 genera from different Brazilian ecosystems were evaluated. The translation of traditional Brazilian morphological categories into CLO-PLA traits was not fully successful and resulted in lower number of categories and loss of information about plant morphology. Furthermore, we succeed only partially when assessing clonal and bud bank traits for Aldama encountering difficulties when dealing with traits based on seasonal development. Nevertheless, the results are promising for future comparative studies across ecosystems and biomes for which common trait standardization is necessary. However, it is clear that further research is needed on functional morphology of clonal and bud-bank traits in tropical regions.

Keywords Aldama, Brazilian grasslands, bud bank, Campos, Cerrado

Resumo: A padronização dos caracteres das plantas e banco de dados não estão disponíveis para todas as regiões e todas as características vegetais. Alguns ecossistemas como os campos tropicais estão sendo sub-representados em tais bancos de dados devido à dificuldade de acesso ao banco de gemas e avaliação do crescimento clonal. Os dois objetivos deste trabalho foram: (i) testar se as características morfológicas dos órgãos subterrâneos das plantas brasileiras podem ser traduzidas em categorias usadas no banco de dados CLO-PLA, e (ii) testar nas Aldama brasileiras a aplicabilidade das características padronizadas no CLO-PLA para plantas clonais e para o banco de dados. Foi avaliado um total de 165 espécies, incluindo ervas, subarbustos e arbustos de 37 gêneros, provenientes de diferentes ecossistemas brasileiros. A tradução de categorias morfológicas tradicionalmente brasileiras em parâmetros do CLO-PLA não foi totalmente bem sucedida, e resultou em um menor número de categorias e na perda de informações sobre a morfologia vegetal. Além disso, conseguimos avaliar apenas parcialmente os caracteres clonais e do banco de gemas de Aldama, e encontramos dificuldades para lidar com características baseadas no desenvolvimento sazonal. No entanto, os resultados são promissores para futuros estudos comparativos nos ecossistemas e biomas, para isso a normalização das características é necessária. No entanto, está clara a necessidade de maiores investigações sobre a morfologia funcional de características clonais e do banco de gemas nas regiões tropicais.

Pavavras chave Aldama, banco de gemas, campos brasileiros, Cerrado

Introduction Functional ecology has broaden its focus geographically in the last decade thanks to standardized approaches to plant traits measurements (Cornelissen et al. 2003; Violle et al. 2007; Pérez-Harguindeguy et al. 2013) and databasing of collected data (Kleyer et al. 2008; Kattge et al. 2011; Hudson et al. 2014). These activities are not, however, equally successful for all ecosystems and all plant traits. Some ecosystems, for example, tropical grasslands, are being underrepresented in such databases as their knowledge is still rudimentary (Kattge et al. 2011). Moreover, some traits are difficult to standardize and thus we have rather local data for them. An example of such difficult to standardize traits is morphology of belowground plant organs, assessment of bud bank and evaluation of clonal growth (Klimešová and de Bello 2009). As a consequence, we have the only standardized description on clonal and bud bank traits for Central European flora (see CLO-PLA database, Klimešová and Klimeš 2006). This situation persists despite the knowledge of clonal plants regarding the diversity of clonal organs and a role of the bud banks in plant regeneration and population maintenance increased over the past 20 years. The new information was gained for different environments such as temperate prairies (e.g. Hendrickson and Briske 1997; Rogers and Hartnett 2001; Benson et al. 2004; Dalgleish and Hartnett 2006; Carter et al. 2012; Vanderweide 2013), tropical savannas (e.g. Midgley 1996; Luoga et al. 2004; Neke et al. 2006; Maurin et al. 2014; Hoffmann 1999; Damascos et al. 2005; Hayashi and Appezzato-da-Glória 2007; Overbeck and Pfadenhauer 2007; Appezzato-da-Glória et al. 2008; Fidelis et al. 2014), and Mediterranean region (Pausas et al. 2004; Paula et al. 2009; Paula and Pausas 2011). Nevertheless, the authors differ in methods of bud bank assessment and description of clonal traits and categorization of bud-bearing clonal organs. Depending on local tradition some morphological categories such as rhizome and stolon can have different meanings with different authors (see overview in Klimešová and Klimeš 2008) and studies from different ecosystems or done by different authors are not readily comparable. Prerequisite for understanding a role of bud-bearing organs in resprouting after disturbance and in vegetative spreading over large geographical gradients is their unified classification. For example, analysis of bud-bearing clonal organs of Ladakh (an arid mountainous region in Northwest Himalaya, India; Klimešová et al. 2011) and of North Billjefjorden (a high polar region in central Spitsbergen, Svalbard; Klimešová et al. 2012) are two examples of a successful analysis based on methods established for central-European

species in CLO-PLA database (Klimešová and Klimeš 2008; Klimešová and de Bello 2009). Methods of evaluation of clonal and bud bank traits from the CLO-PLA database, therefore, seems to have a potential to be used over large geographical region or biomes and may become a tool for world-wide comparisons and applications. In order to understand plant species on Brazilian savanna and grasslands, aim of our study is to test the applicability of methods from CLO-PLA database for Asteraceae that grows in these environments. These ecosystems are subjected to accelerated degradation and fragmentation (Klink and Machado 2005; Almeida et al. 2005; Overbeck et al. 2005, 2006; Fidelis 2008; Appezzato-da-Glória et al. 2008) and understanding their functional diversity and strategies can contribute to their protection. Here we have two aims (i) to test whether we can translate morphological categories described according to Brazilian tradition into categories used in CLO-PLA database and (ii) to test the applicability of clonal and bud bank traits standardized in CLO-PLA database for herbaceous species from Brazilian tropical grasslands. In order to achieve our goals, we excerpted available literature data on the morphology of belowground organs for herbs and subshrubs from the Asteraceae family characterized by high species diversity in subtropical and tropical regions in Brazil. We know that species from the family have specialized belowground organs (Appezzato-da-Glória 2015 and references therein) and they are therefore suitable for seeking parallels in CLO-PLA classification of bud-bearing clonal organs. For testing a suitability of CLO-PLA standards for assessment of other clonal and bud bank traits we used representatives of genus Aldama La Llave (Asteraceae). The genus is composed of 112 species, of which 35 are perennial herbs and subshrubs (Magenta et al. 2010; Magenta and Pirani 2014) that are able to resprout after fire and occur in fire-prone ecosystems (Cerrado and Campos) of South America (Ratter et al. 1997; Silva et al 2014).

Material and Methods Asteraceae species – clonal growth organ Data concerning belowground organs of Asteraceae species from different Brazilian ecosystems were obtained from published papers, books, dissertations, doctoral thesis and lists of endangered species. Search yielded 136 species including herbs, subshrubs and shrubs of 36 genera (Table 1) from Campos (subtropical grasslands), Cerrado (tropical savanna), Caatinga, Campo rupestre (rocky field), Campo de altitude (high altitude grassland), Restinga, Atlantic forest and Pantanal. The classification of belowground organs traditionally

used in Brazil (Appezzato-da-Glória 2015) was compared with the classification according to CLO-PLA database according to Klimešová and Klimeš (2008) (Table 2).

Aldama genus – clonal and bud bank traits Field data were obtained during expeditions since 2010 to Brazilian areas of Cerrado (tropical savanna) and Campos (subtropical grasslands). Aerial and subterranean organs of 20 species of Aldama during flowering stages were collected (Table 3). A total of 147 individuals were excavated, cleaned, fixed in FAA 50 (formaldehyde, acetic acid and ethanol 1:1:18 in 50% ethanol) and preserved in 70% ethanol. For these plants we recorded information on many traits including the persistence of tap root, the reproduction type, the presence of storage organs, the number and seasonality of bud bank, the type and role of clonal growth organ (CGO), the number of offspring shoots per parent shoot per year, the lateral spread, the branching type, the root position along the CGO, and the relation between plant reproduction and clonal events. The plant classification was made according to Klimešová and Klimeš (2008) and to Klimeš and Klimešová (2005) (Table 4). The number of buds was determined using a stereo microscope and digital photographs were obtained using a digital camera, Nikon, Coolpix P520. The vouchers of these species are stored in ESA herbarium (Luiz de Queiroz College of Agriculture) for future comparison and analyses. Moreover, we also included in this analysis some data of belowground organ of another nine species of Aldama which were previously described by Brazilian Aldama specialist (Magenta 2006; Tables 1, 3).

Results

Asteraceae species – clonal growth organ Among the 136 Asteraceae described in published works we distinguished six categories of the belowground organ: xylopodium, rhizome, rizophore, gemmiferous roots, stem tuber and non-thickened roots (Table 1). About 73% of the species had xylopodium, followed by 12% with rhizome, 7% with non-thickened root and 6% of plants developing rhizophore. Only 2 species have gemmiferous roots and just one a stem tuber. According to CLO-PLA standards these organs were grouped into four categories (Table 1): xylopodium were classified as hypogeogenous rhizome when originated from stem/hypocotyl or as perennial main root when originated from primary root, rhizome was considered epigeogenous rhizome, rhizophore and stem tuber were both defined as hypogeogenous

rhizome, bud-bearing organs described as gemmiferous roots (roots with adventitious buds) were maintained with the same classification and the non-thickened main root were considered as perennial main root. In most species (89) the classification of the type of belowground organ was not possible to be performed accurately. In this case, the organs were classified as perennial main root, hypogeogenous rhizome or hypocotyle tuber because the consulted literature contained only information about morphology and no anatomical data, of which is essential to differentiate both organs. Hypogeogenous rhizome and epigeogenous rhizome were both identified in 16 plants, followed by perennial main root (13) and root with adventitious buds (2) (Table 1).

Aldama genus – clonal and bud bank traits The detailed clonal and bud bank trait assessment of Aldama species was possible for 13 CLO-PLA traits (Table 5). All Aldama are sympodial and have a lignified belowground organ, classified here as necessary hypogeogenous rhizome which does not maintain the tap root but has tuberous adventitious roots along its whole length. In these species, the reproduction period occurs after the development of the hypogeogenous stem, of which generally show a lateral spread lower than 0.01 cm per year (Fig. 1), except for Aldama anchusifolia and Aldama pilosa which spread laterally from 0.01 cm to 0.25 cm per year (Fig. 2). The subterranean bud bank is perennial and concentrated at 0 cm to -10 cm from the soil layer. Bud numbers vary from 1 to over 10 depending on the species. Other trait values (connection persistence, the number of offspring shoots per parent shoot and offspring size) assessed on adult specimens in the field are listed in Table 5) on the other hand, we were not able to assess traits like shoot cyclicity. Shoot cyclicity denotes to shoot lifespan from bud sprouting to fruiting and death in number of seasons (cycles). In temperate plants the number of seasons is possible to be assessed according to morphological marks in the shoot. In tropical grasslands from which Aldama species come and where there is seasonality thanks to dry periods we were not able to distinguish cyclicity solely using morphological characters (see comments in Table 4).

Discussion We were able to translate traditionally used Brazilian morphological categories for description of belowground bud-bearing organs into categories used in CLO-PLA database. The translation resulted in lower number of categories implying a loss of information about plant morphology. We also succeed when assessing clonal and bud bank traits for selected

genus with the exception of traits based on seasonal development like shoot cyclicity. Those results are promising for future comparative studies across ecosystems and biomes for which common trait standardization is necessary. Nevertheless, it is also clear that regional differences should be taken into account and new morphological categories or plant traits identified and potentially also implemented into comparative scheme.

Translation of morphological categories of belowground organs Belowground organs of Brazilian species can be classified into categories which are found not only in Central Europe (Klimešová and Klimeš, 2008) but also in arid mountains in India (Klimešová et al. 2011) and in the high polar region in Svalbard (Klimešová et al. 2012). This fact clearly enables clonal growth organs morphologies across geographical scales and ecosystems. For example, from our study follows that while 37% of 316 species belonging to Asteraceae recorded on CLO-PLA database developed epigeogenous rhizome, the majority of Brazilian species (65%) has not had an exact belowground classification due to lack of anatomical data, the organs were considered perennial main root or hypogeogenous rhizome. Second most represented clonal growth organ in CLO-PLA database is hypogeogenous rhizome (21%) and in Brazilian flora it is epigeogenous rhizome and hypogeogenous rhizome with 12% each. Rare in both floras are roots with adventitious buds (1% in Brazilian species and 3% in temperate plants), perennial main root (10 and 12%, respectively) and horizontal aboveground stem (0 and 3%). This comparison is highly preliminary as we assessed morphology only a small fraction of Asteraceae occurring in Brazil. The analyzed 165 species (37 genera) that composes the Brazilian group represent less than 10% of the total number of species occurring in Brazil (Souza and Lorenzi 2008). Despite successful translation of morphological categories, we feel that there are morphological (and therefore also probably functional) differences in paralleled organs. The main difference is a high proportion of plants with secondary thickening and prominent storage in belowground organs of Brazilian tropical ecosystems especially those subjected to fire (Overbeck and Pfadenhauer 2007). In CLO-PLA classification the occurrence of secondary thickening is not considered and is certainly less often present due to more humid climate. In CLO-PLA classification is major stress put on placement of the organ in relation to soil surface (above-ground versus belowground), in which organ a storage carbohydrates are located (leaves, stem or roots), which organ provide connection between mother and daughter shoot (stem of root) and what is an origin of buds from which daughter shoot sprout

(axillary or adventitious). On the other hand, in Brazilian tradition anatomical features or early ontogeny of the organ are very important while consequence for plant architecture is less explored. As the main aim of morphological descriptions in Brazil is to describe all peculiarities of examined species, the stress in classification is put on them. In the CLO-PLA classification, on the other hand, there was a goal to generalize over details in order to allow comparative approach. Therefore, CLO-PLA classification is unable to reflect all details to characterize the organs of tropical flora (Troll 1956; Aubréville 1963; Rizzini 1965). For example xylopodium has hypocotyl-root origin, is extremely lignified and dry, characterized by self-grafting and predominance of woody tissues (Lindman 1906; Appezzato-da-Glória 2015) while non-thickened main root has no profound storage or secondary thickening, both those categories would fall into one CLO-PLA category in the case xylopodium is formed by root tissue but their ecological role is rather different when considering probability to survive fire in tropical grassland (Simon and Pennington 2012; Fidelis et al. 2014). In the case xylopodium is made by hypocotyl tissue, it should be categorized as hypocotyl tuber according to CLO-PLA standards. From gross morphology of xylopodium, however, it is not clear what organ participate on its body the most and whether older parts decay enabling the potential for clonal growth. This question will need further study focused on observation of organ development in ontogeny and its function in plant architecture and survival. Rhizomes according to CLO-PLA are simply stem belowground organs with adventitious roots further classified according to their formation in relation to the soil surface (Table 2). In turn, belowground stems according to Brazilian tradition are split to different categories according to traits which probably are not paralleled in temperate flora (see Table 2). Although Brazilian rhizophore resemble hypogeogenous rhizome from CLO-PLA database standards it is not clear whether belowground stems produce aerial stem or not. Similarly, we cannot be sure whether secondary thickening woody rhizomes ever lost their oldest parts and consequently show prerequisite for clonal growth as is usually in rhizomes from temperate flora. Although morphological and anatomical studies in Brazil have long tradition and there are numerous literature data available about the morphology of belowground plant organs, their role in resprouting of plant shoots after injury or seasonal drought and in clonal growth are not sufficiently known.

Assessment of clonal and bud bank traits

When assessing clonal and bud bank traits for Aldama species we entered problems in those defined for years/seasons like lateral spread per season, production of offspring shoot per mother shoot per year and shoot lifespan. Although it is clear that woody belowground organs of Aldama species are perennial for many years, no data of annual increments in underground structures has been published yet. Indeed, the presence of growth rings has been verified in branches of different Cerrado woody species (Marcati et al. 2006 and references therein) but it is still necessary to determine if the growth rings are annual. It is, therefore, clear that for future usage of clonal and bud bank traits we need deepen our knowledge on their ecology in tropical ecosystems.

Conclusions The initiative to test the methodology of CLO-PLA database by using Brazilian species was considered an important step to improve the tropical flora evaluation. It is clear that the assessment of the diversity and trends of the belowground organs of Asteraceae species should be further represented by increasing the number of plants and also by monitoring specimens. Moreover, it is essential to complement CLO-PLA standards by adding some traits to represent tropical species appropriately. Necessary for this step, however, is to understand more properly a role of organs like rhizophore in the architecture of plant and understanding effect of seasonal dryness and fires on the development of bud- bearing organs. Establishment of categories of bud-bearing organs and standardization of clonal and bud bank traits will enable comparative studies with other ecosystems and enable understanding of plant strategies under recurrent disturbance. Despite contributing to the solution of those general ecological questions, such research will be useful for prediction of regeneration after disturbance, population dynamics, risk assessment in endangered species and conservation managements of threatening ecosystems.

Acknowledgments We thank The National Council for Scientific and Technological Development (CNPq) for grant (Proc. nº 303715/2014-6) and the São Paulo Research Foundation (FAPESP) (Thematic Project Proc. nº 2010/51454-3 and Proc. nº 2014/09401-0) for providing financial support and grants for the first author.

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Table 1. Brazilian Asteraceae species evaluated. Original Classification according Specie classification CLO-PLA traits Reference perennial main root or Aldama anchusifolia xylopodium hypogeogenous rhizome Magenta, 2006 Aldama arenaria xylopodium hypogeogenous rhizome Oliveira et al. 2013 perennial main root or Aldama aspilioides xylopodium hypogeogenous rhizome Magenta, 2006 perennial main root or Aldama bakeriana xylopodium hypogeogenous rhizome Magenta, 2006 perennial main root or Aldama bracteata xylopodium hypogeogenous rhizome Magenta, 2006 perennial main root or Aldama corumbensis xylopodium hypogeogenous rhizome Field observation perennial main root or Aldama discolor xylopodium hypogeogenous rhizome Magenta, 2006 Aldama filifolia xylopodium hypogeogenous rhizome Bombo et al. 2014 perennial main root or Aldama gardneri xylopodium hypogeogenous rhizome Magenta, 2006 perennial main root or Aldama goyazii xylopodium hypogeogenous rhizome Magenta, 2006 perennial main root or Aldama grandiflora xylopodium hypogeogenous rhizome Magenta, 2006 Aldama kunthiana xylopodium hypogeogenous rhizome Silva et al 2014 Aldama linearifolia xylopodium hypogeogenous rhizome Bombo et al. 2014 perennial main root or Aldama macrorhiza xylopodium hypogeogenous rhizome Magenta, 2006 perennial main root or Aldama megapotamica xylopodium hypogeogenous rhizome Magenta, 2006 perennial main root or Aldama nudibasilaris xylopodium hypogeogenous rhizome Magenta, 2006 perennial main root or Aldama nudicaulis xylopodium hypogeogenous rhizome Magenta, 2006 perennial main root or Aldama oblongifolia xylopodium hypogeogenous rhizome Magenta, 2006 perennial main root or Aldama paranensis xylopodium hypogeogenous rhizome Magenta, 2006 perennial main root or Aldama pilosa xylopodium hypogeogenous rhizome Magenta, 2006 Aldama robusta xylopodium hypogeogenous rhizome Oliveira et al. 2013 perennial main root or Aldama rubra xylopodium hypogeogenous rhizome Magenta, 2006 perennial main root or Aldama santacatarinensis xylopodium hypogeogenous rhizome Magenta, 2006 perennial main root or Aldama squalida xylopodium hypogeogenous rhizome Magenta, 2006 Aldama tenuifolia xylopodium hypogeogenous rhizome Silva et al., 2014 Aldama trichophylla xylopodium hypogeogenous rhizome Bombo et al., 2014 perennial main root or Aldama tuberosa xylopodium hypogeogenous rhizome Magenta, 2006 perennial main root or Aldama veredensis xylopodium hypogeogenous rhizome Magenta, 2006 perennial main root or Aldama vernonioides xylopodium hypogeogenous rhizome Magenta, 2006 perennial main root or Angelphytum grisebachii xylopodium hypogeogenous rhizome Mondin, 2007 perennial main root or Aspilia foliácea xylopodium hypogeogenous rhizome Bringel Jr., 2007 perennial main root or Aspilia reflexa xylopodium hypogeogenous rhizome Rachid, 1947 perennial main root or Baccharis cognata xylopodium hypogeogenous rhizome Fidelis et al., 2009

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perennial main root or Baccharis crispa xylopodium hypogeogenous rhizome Monteiro Jr., 2013 perennial main root or Baccharis curitibensis xylopodium hypogeogenous rhizome Monteiro Jr., 2013 Baccharis dracunculifolia non-thickened root perennial main root Tertuliano and Figueiredo-Ribeiro, 1993 perennial main root or Baccharis pentaptera xylopodium hypogeogenous rhizome Schneider, 2009 perennial main root or Baccharis pentodonta xylopodium hypogeogenous rhizome Fernandes, 2009 perennial main root or Baccharis pseudotenuifolia xylopodium hypogeogenous rhizome Beretta et al., 2008 perennial main root or Baccharis rufescens xylopodium hypogeogenous rhizome Rachid, 1947; Beretta et al., 2008 perennial main root or Baccharis sessiliflora xylopodium hypogeogenous rhizome Tertuliano and Figueiredo-Ribeiro, 1993 perennial main root or Baccharis subdentada xylopodium hypogeogenous rhizome Vilhalva, 2004 perennial main root or Baccharis tridentada xylopodium hypogeogenous rhizome Beretta et al., 2008 Bidens gardneri non-thickened root perennial main root Tertuliano and Figueiredo-Ribeiro, 1993 Bidens speciosa non-thickened root perennial main root Tertuliano and Figueiredo-Ribeiro, 1993 perennial main root or Calea abbreviata xylopodium hypogeogenous rhizome Bringel Jr., 2007 perennial main root or Calea acaulis xylopodium hypogeogenous rhizome Red list/CNCFLORA perennial main root or Calea clausseniana xylopodium hypogeogenous rhizome Almeida, 2008 perennial main root or Borges - personal com - Red Calea cuneifólia xylopodium hypogeogenous rhizome list/CNCFLORA perennial main root or Rachid, 1947; Tertuliano and Figueiredo- Calea platylepsis xylopodium hypogeogenous rhizome Ribeiro, 1993 perennial main root or Calea uniflora xylopodium hypogeogenous rhizome Fernandes, 2009 Calea verticillata xylopodium hypogeogenous rhizome Vilhalva and Appezzato-da-Glória, 2006b Chresta sphaerocephala gemmiferous roots roots with adiventitious buds Appezzato-da-Glória et al., 2008 perennial main root or Borges - personal com - Red Chromolaena costatipes xylopodium hypogeogenous rhizome list/CNCFLORA Chromolaena squalida xylopodium perennial main root Appezzato-da-Glória, 2008 perennial main root or Borges - personal com - Red Chrysolaena nicolackii xylopodium hypogeogenous rhizome list/CNCFLORA perennial main root or Dimerostemma bichopii xylopodium hypogeogenous rhizome Moraes and Semir, 2009 perennial main root or Dimerostemma brasilianum xylopodium hypogeogenous rhizome Moraes and Semir, 2009 perennial main root or Dimerostemma episcopale xylopodium hypogeogenous rhizome Moraes and Semir, 2009 perennial main root or Dimerostemma grazielae xylopodium hypogeogenous rhizome Moraes and Semir, 2009 Dimerostemma perennial main root or humboldtianum xylopodium hypogeogenous rhizome Moraes and Semir, 2009 perennial main root or Tertuliano and Figueiredo-Ribeiro, 1993; Dimerostemma lippioides xylopodium hypogeogenous rhizome Moraes and Semir, 2009 perennial main root or Dimerostemma myrtifolium xylopodium hypogeogenous rhizome Moraes and Semir, 2009 perennial main root or Dimerostemma oblongum xylopodium hypogeogenous rhizome Moraes and Semir, 2009 perennial main root or Dimerostemma paneroi xylopodium hypogeogenous rhizome Moraes and Semir, 2009 Dimerostemma perennial main root or pseudosilphioides xylopodium hypogeogenous rhizome Moraes and Semir, 2009 perennial main root or Dimerostemma reitzii xylopodium hypogeogenous rhizome Moraes and Semir, 2009

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perennial main root or Dimerostemma retifolium xylopodium hypogeogenous rhizome Moraes and Semir, 2009 perennial main root or Moraes and Semir, 2009; Bringel Jr., Dimerostemma vestitum xylopodium hypogeogenous rhizome 2007 perennial main root or Dimerostemma virgosum xylopodium hypogeogenous rhizome Moraes and Semir, 2009 non-thickened Elephantopus mollis roots perennial main root Tertuliano and Figueiredo-Ribeiro, 1993 perennial main root or Elephantopus racemosus xylopodium hypogeogenous rhizome Tertuliano and Figueiredo-Ribeiro, 1993 Eupathorium horminioides non-thickened root perennial main root Tertuliano and Figueiredo-Ribeiro, 1993 perennial main root or Eupatorium ascendens xylopodium hypogeogenous rhizome Beretta et al., 2008 perennial main root or Eupatorium chlorolepsis xylopodium hypogeogenous rhizome Tertuliano and Figueiredo-Ribeiro, 1993 perennial main root or Eupatorium commersonii xylopodium hypogeogenous rhizome Beretta et al., 2008 perennial main root or Eupatorium congestum xylopodium hypogeogenous rhizome Beretta et al., 2009; Almeida, 2008 perennial main root or Eupatorium laevigatum xylopodium hypogeogenous rhizome Tertuliano and Figueiredo-Ribeiro, 1993 perennial main root or Eupatorium lanigerum xylopodium hypogeogenous rhizome Beretta et al 2009 ; Fernandes, 2009 perennial main root or Eupatorium ligulaefolium xylopodium hypogeogenous rhizome Fidelis, 2008 perennial main root or Eupatorium maximilianii xylopodium hypogeogenous rhizome Vilhalva, 2004 Eupatorium molissimum non-thickened root perennial main root Tertuliano and Figueiredo-Ribeiro, 1993 perennial main root or Eupatorium oblongifolium xylopodium hypogeogenous rhizome Beretta et al., 2008 perennial main root or Eupatorium squalidum xylopodium hypogeogenous rhizome Tertuliano and Figueiredo-Ribeiro, 1993 Eupatorium tanacetifolium rizophore hypogeogenous rhizome Fidelis et al., 2009 perennial main root or Eupatorium xylorhizum xylopodium hypogeogenous rhizome Almeida, 2008 perennial main root or Gochnatia barrosii xylopodium hypogeogenous rhizome Tertuliano and Figueiredo-Ribeiro, 1993 perennial main root or Gochnatia pulchra xylopodium hypogeogenous rhizome Tertuliano and Figueiredo-Ribeiro, 1993 Graphistylis argyrotricha rhizome epigeogenous rhizome Teles, 2008 Graphistylis dichroa rhizome epigeogenous rhizome Teles, 2008 Graphistylis itatiaiae rhizome epigeogenous rhizome Teles, 2008 Graphistylis organensis rhizome epigeogenous rhizome Teles, 2008 Graphistylis riopretensis rhizome epigeogenous rhizome Teles, 2008 Gyptis lanigera gemmiferous roots roots with adiventitious buds Appezzato-da-Glória, 2008 Azevêdo-Gonçalves and Matzenbacher Hypochaeris catharinensis rhizome epigeogenous rhizome 2007; Reck et al., 2011 Isostigma megapotamicum xylopodium hypogeogenous rhizome Vilhalva and Appezzato-da-Glória, 2006b perennial main root or Rachid, 1947; Tertuliano and Figueiredo- Isostigma peucedanifolium xylopodium hypogeogenous rhizome Ribeiro, 1993 perennial main root or Borges - personal com - Red Lepidaploa almasesis xylopodium hypogeogenous rhizome list/CNCFLORA Lessingianthus bardanoides xylopodium perennial main root Appezzato-da-Glória, 2008 perennial main root or Borges - personal com - Red Lessingianthus exiguus xylopodium hypogeogenous rhizome list/CNCFLORA perennial main root or Borges - personal com - Red Lessingianthus reitzianus xylopodium hypogeogenous rhizome list/CNCFLORA perennial main root or Lucilia lycopodioides xylopodium hypogeogenous rhizome Vilhalva, 2004 Mikania anethifolia xylopodium perennial main root or Ritter and Miotto, 2005

108

hypogeogenous rhizome

Mikania cordifolia xylopodium hypogeogenous rhizome Appezzato-da-Glória and Cury, 2011 perennial main root or Mikania oblongifolia xylopodium hypogeogenous rhizome Tertuliano and Figueiredo-Ribeiro, 1993 perennial main root or Mikania parodii xylopodium hypogeogenous rhizome Beretta et al., 2008 perennial main root or Ritter and Miotto, 2005; Beretta et al., Mikania pinnatiloba xylopodium hypogeogenous rhizome 2008 Mikania sessilifolia xylopodium hypogeogenous rhizome Appezzato-da-Glória and Cury, 2011 perennial main root or Minasia ramosa xylopodium hypogeogenous rhizome Loeuille et al., 2011 Noticastrum calvatum rhizome hypogeogenous rhizome Beretta et al., 2008 Orthopappus angustifolius rizophore hypogeogenous rhizome Appezzato-da-Glória et al., 2008 Panphalea cardaminifolia rhizome epigeogenous rhizome Cabrera and Klein, 1989 perennial main root or Panphalea commersonii xylopodium hypogeogenous rhizome Fernandes, 2009 Panphalea smithii rhizome epigeogenous rhizome Cabrera and Klein, 1973 Perezia catharinensis rhizome epigeogenous rhizome Katinas, 2012 Perezia eryngioides rhizome epigeogenous rhizome Katinas, 2012 Perezia multiflora rhizome epigeogenous rhizome Katinas, 2012 Perezia squarrosa subs cubataensis rhizome epigeogenous rhizome Katinas, 2012 Perezia squarrosa subs squarrosa rhizome epigeogenous rhizome Katinas, 2012 perennial main root or Porophyllum bahiense xylopodium hypogeogenous rhizome Hind, 2002 Pterocaulon alopecuroides xylopodium perennial main root Appezzato-da-Glória and Cury, 2011 Pterocaulon angustifolium xylopodium perennial main root Appezzato-da-Glória et al., 2008 perennial main root or Pterocaulon interruptum xylopodium hypogeogenous rhizome Rizzini and Heringer, 1961 perennial main root or Pterocaulon lorentzii xylopodium hypogeogenous rhizome Lima, 2008 perennial main root or Pterocaulon polypterum xylopodium hypogeogenous rhizome Lima, 2008 perennial main root or Pterocaulon rugosum xylopodium hypogeogenous rhizome Lima, 2008 perennial main root or Pterocaulon virgatum xylopodium hypogeogenous rhizome Lima, 2008 perennial main root or Pteurocaulon cordobense xylopodium hypogeogenous rhizome Lima, 2008 perennial main root or Richterago riparia xylopodium hypogeogenous rhizome Roque, 2001 Riencourtia oblongifolia non-thickened root perennial main root Tertuliano and Figueiredo-Ribeiro, 1993 Senecio ceratophylloides rhizome epigeogenous rhizome Beretta et al., 2008 Senecio oleosus rhizome epigeogenous rhizome Teles, 2008 Solidago chilensis rhizome epigeogenous rhizome Mercandeli et al., 2012 perennial main root or Trichocline catharinensis xylopodium hypogeogenous rhizome Pasini and Ritter, 2012 perennial main root or Trichocline cisplatina xylopodium hypogeogenous rhizome Pasini and Ritter, 2012 perennial main root or Trichocline humilis xylopodium hypogeogenous rhizome Pasini and Ritter, 2012 perennial main root or Trichocline incana xylopodium hypogeogenous rhizome Pasini and Ritter, 2012 perennial main root or Trichocline macrocephala xylopodium hypogeogenous rhizome Pasini and Ritter, 2012 perennial main root or Trichocline maxima xylopodium hypogeogenous rhizome Pasini and Ritter, 2012

109

Trixis nobilis stem tuber hypogeogenous rhizome Appezzato-da-Glória and Cury, 2011 non-thickened Vernonia apiculata roots perennial main root Tertuliano and Figueiredo-Ribeiro, 1993 perennial main root or Rachid, 1947; Tertuliano and Figueiredo- Vernonia bardanoides xylopodium hypogeogenous rhizome Ribeiro, 1993 Rachid, 1947; Tertuliano and Figueiredo- Vernonia brevifolia rhizophore hypogeogenous rhizome Ribeiro, 1993 perennial main root or Vernonia brevipetiolata xylopodium hypogeogenous rhizome Tertuliano and Figueiredo-Ribeiro, 1993 Vernonia elegans xylopodium hypogeogenous rhizome Appezzato-da-Glória and Cury, 2011 perennial main root or Vernonia ferruginea xylopodium hypogeogenous rhizome Tertuliano and Figueiredo-Ribeiro, 1993 Vernonia flexuosa rhizophore hypogeogenous rhizome Fidelis, 2008 perennial main root or Borges - personal com - Red Vernonia glabrata xylopodium hypogeogenous rhizome list/CNCFLORA perennial main root or Vernonia grandiflora xylopodium hypogeogenous rhizome Rachid, 1947 Hayashi, 2003; Hayashi and Appezzato- Vernonia herbacea rhizophore hypogeogenous rhizome da-Glória, 2005 perennial main root or Vernonia intermedia xylopodium hypogeogenous rhizome Beretta et al., 2008 perennial main root or Vernonia lepidifera xylopodium hypogeogenous rhizome Beretta et al., 2008 Vernonia linearifolia rhizophore hypogeogenous rhizome Menezes et al., 1979 Vernonia megapotamica xylopodium hypogeogenous rhizome Appezzato and Cury, 2011 perennial main root or Vernonia nudiflora xylopodium hypogeogenous rhizome Beretta et al., 2008 perennial main root or Vernonia oxydonta xylopodium hypogeogenous rhizome Beretta et al., 2008 Vernonia oxylepis xylopodium perennial main root Tertuliao and Figueiredo-Ribeiro, 1993 Hayashi, 2003; Hayashi and Appezzato- Vernonia platensis rhizophore hypogeogenous rhizome da-Glória, 2005 perennial main root or Vernonia squarrosa xylopodium hypogeogenous rhizome Beretta et al., 2008 perennial main root or Vernonia warmingiana xylopodium hypogeogenous rhizome Monteiro Jr., 2013 perennial main root or Vernonia zuccariniana xylopodium hypogeogenous rhizome Tertuliano and Figueiredo-Ribeiro, 1993 Vernonia psolophylla rhizophore hypogeogenous rhizome Menezes et al. 1979 perennial main root or Wedelia pallida xylopodium hypogeogenous rhizome Bringel Jr., 2007 perennial main root or Wedelia regis xylopodium hypogeogenous rhizome Bringel Jr., 2007 Wulffia stenoglossa non-thickened root perennial main root Tertuliano and Figueiredo-Ribeiro, 1993

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Table 2. Translation of Brazilian categories of belowground bud-bearing plant organs (Appezzato-da-Gloria 2015) into CLO-PLA categories of clonal growth organs (Klimešová and Klimeš 2008).

Brazilian categories Description CLO-PLA categories Description Gemmiferous root Root forming adventitious Root with adventitious Root forming adventitious buds buds buds Lignotuber Perennial secondarily Perennial hypocotyle tuber Tuberously thickenned thickened cotyledonary hypocotyle prerennating region of a stem and for the whole plant lifespan hypocotyle with adventitious buds, usually in shrubs and trees Non-thickened main root Perennial main root Perennial main root Main root growing from without prominent storage root pole of embryo and perennating for the whole plant lifespan, storage placed usually in root Rhizome Growing from plumule, Epigeogenous rhizome Belowground stem formed monopolar system of stems at the soil surface with (growing belowground), adventitious roots and bearing adventitious roots, decaying from oldest parts visible nodes Rhizophore Growing from Probably hypogeogenous Belowground stem formed cotyledonary buds, etc., rhizome belowground with bipolar growth (plant has adventitious roots and two types of shoots, one decaying from oldest parts growing belowground and having storage function and one forming aboveground shoots), bearing adventitious roots, visible nodes Sobole Secondary thickening Probably both, See above belowground stems bearing epigeogenous and adventitious roots, usually hypogeogenous rhizomes in shrubs and trees Stem tuber Tuberous cauline structure Probably hypogeogenous See above rhizome Xylopodium Perennial secondarily Perennial main root when See above system. May be either root originated from primary (primary root origin) or root and hypogeogenous stem structures (hypocotyl rhizome when originated origin) or both when from hypocotyl originate from hypocotyl and primary root

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Table 3. List of Aldama species analyzed.

Plant Analyzed Expedition Specie Origin habit plants year

Aldama anchusifolia Subshrub 14 2012 Rio Grande do Sul Aldama arenaria Herb-subshrub 7 2010 São Paulo Aldama aspilioides Herb-shrub 6 2015 Paraná Aldama bakeriana Herb-subshrub 10 2012 Minas Gerais Herb-shrub 8 2015 Aldama bracteata Goiás Aldama corumbensis Herb-shrub 3 2015 Mato Grosso do Sul Aldama discolor Herb-subshrub 9 2012 São Paulo; Minas Gerais Aldama filifolia Herb 3 2010 Goiás

Aldama gardneri Herb-shrub 7 2015 Goiás Aldama grandiflora Herb-shrub 4 2012 Goiás Aldama goyazii* Subshrub-shrub - - Minas Gerais Aldama kunthiana Herb 5 2012 Goiás Aldama linearifolia Shrub 6 2010 Mato Grosso do Sul

Aldama macrorhiza* Subshrub-shrub - - São Paulo Aldama megapotamica Shrub 17 2012 Minas Gerais Aldama nudibasilaris Shrub 9 2013 Minas Gerais Aldama nudicaulis* Herb-subshrub - - Rio Grande do Sul Herb-subshrub - - Aldama oblongifolia* Goiás Aldama paranensis* Herb-subshrub - - Paraná Aldama pilosa Subshrub-shrub 6 2013 Rio Grande do Sul Aldama robusta Herb-subshrub 4 2010 São Paulo

Aldama rubra Herb-subshrub 9 2014 São Paulo Aldama santacatarinensis* Subhrub - - Santa Catarina Aldama squalida Herb-subshrub 7 2012 Mato Grosso do Sul Aldama tenuifolia Herb-subshrub 8 2012 Minas Gerais Aldama trichophylla Herb 5 2010 Paraná

Aldama tuberosa* Herb-subshrub - - Rio Grande do Sul Aldama veredensis* Herb-subshrub - - Minas Gerais Aldama vernonioides* Shrub - - Mato Grosso

* Specie evaluated by data from literature (Magenta 2006)

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Table 4. CLO-PLA traits (Klimeš and Klimešová 2005; Klimešová and Klimeš 2008) used to characterize Aldama species. CGO = clonal growth organ.

CLO-PLA trait Description Remarks Persistence of tap root Presence of main root Sometimes difficult to identify only by field observations being necessary to analyze anatomically Reproduction type Reproduction may be vegetative, Data from field observations and from generative or both literature Presence of storage organ Structure developed to storage Identification by field observations, compounds, of which participate anatomical analyses and literature data indirectly in the regrowth Bud bank numbers All belowground buds per shoot Identification using stereomicroscope Bud bank seasonality May be perennial if persists two or Identify by annual field observations more years, seasonal if persists less than two years and potential if it has the ability to form buds on leaves and roots. Type of CGO Delimited by morphology, origin Difficult to classify based on CLO- and placement PLA types Role of CGO May be necessary if all the adult Easy to identify by field observations plant possess it, additive if it is absent in some plants and regenerative if it develops after an injury Persistence of connection Persistence of connection between Identification only by annual field parent and offspring shoots in year observations or potentially by herbochronological methods Number of offspring per Number of offspring shoots per Identification only by annual field parent parent shoot per year observations or potentially by herbochronological methods Lateral spread Increment in horizontal direction Difficult do identify since increments per year have no evidence of annual growth activity or potentially by herbochronological methods Branching type May be monopodial or sympodial Easy to identify by field observations and data from literature Root position along the Development on oldest part, on Easy to identify by field observations CGO youngest part or along the organ and CGO illustration from literature Start of CGO formation CGO may be formed before, after Identify by field observations and data versus reproduction or at the time of flowering from literature

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Table 5. Evaluation of Aldama species analyzed based on CLO-PLA parameters (for definitions of traits see Table 1). CGO=clonal growth organ; HR=hypogeogenous rhizome.

Connection Number of Lateral Specie Tap root Reproduction Storage Bud bank Bud bank Type of CGO persistence offspring spread Branching Roots Clonal vs persistence type organs number§ seasonality§ CGO role (years)# per parent# (cm/year)# type position reproduction Aldama anchusifolia - generative root 1-10 perennial HR necessary >2 1-5 >0.01 sympodial along CGO before Aldama arenaria no generative root >10 perennial HR necessary >2 1-4 <0.01 sympodial along CGO before Aldama aspilioides - generative root 1-10 perennial HR necessary >2 1-5 >0.01 sympodial along CGO before Aldama backeriana - generative root 1-10 perennial HR necessary >2 1-2 <0.01 sympodial along CGO before Aldama bracteata - generative root >10 perennial HR necessary >2 1 <0.01 sympodial along CGO before Aldama corumbensis - generative root >10 perennial HR necessary >2 1 <0.01 sympodial along CGO before Aldama discolor - generative root 1-10 perennial HR necessary >2 1-2 <0.01 sympodial along CGO before Aldama filifolia no generative root >10 perennial HR necessary >2 1-3 <0.01 sympodial along CGO before Aldama gardneri - generative root 1-10 perennial HR necessary >2 1 <0.01 sympodial along CGO before Aldama goyazii* - generative root - perennial HR necessary >2 - <0.01 sympodial - before Aldama grandiflora - generative root >10 perennial HR necessary >2 1 <0.01 sympodial along CGO before Aldama kunthiana no generative root 1-10 perennial HR necessary >2 1-2 <0.01 sympodial along CGO before Aldama linearifolia no generative root 1-10 perennial HR necessary >2 1-2 <0.01 sympodial along CGO before Aldama macrorhiza* - generative root - perennial HR necessary >2 - <0.01 sympodial - before Aldama megapotamica - generative root >10 perennial HR necessary >2 1-3 <0.01 sympodial along CGO before Aldama nudibasilaris - generative root 1-10 perennial HR necessary >2 1-2 >0.01 sympodial along CGO before Aldama nudicaulis* - generative root - perennial HR necessary >2 - <0.01 sympodial - before Aldama oblongifolia* - generative root - perennial HR necessary >2 - <0.01 sympodial - before Aldama paranensis* - generative root - perennial HR necessary >2 - <0.01 sympodial - before Aldama pilosa - generative root >10 perennial HR necessary >2 1-5 >0.01 sympodial along CGO before Aldama robusta no generative root 1-10 perennial HR necessary >2 1-2 <0.01 sympodial along CGO before Aldama rubra - generative root >10 perennial HR necessary >2 1-4 <0.01 sympodial along CGO before Aldama santacatarinensis* - generative root - perennial HR necessary >2 - <0.01 sympodial - before Aldama squalida - generative root 1-10 perennial HR necessary >2 1-2 <0.01 sympodial along CGO before Aldama tenuifolia no generative root >10 perennial HR necessary >2 1-2 <0.01 sympodial along CGO before Aldama trichophylla no generative root >10 perennial HR necessary >2 1-2 <0.01 sympodial along CGO before Aldama tuberosa* - generative root - perennial HR necessary >2 - <0.01 sympodial - before Aldama veredensis* - generative root - perennial HR necessary >2 - <0.01 sympodial - before Aldama vernonioides* - generative root - perennial HR necessary >2 - <0.01 sympodial - before

* Species evaluated by pictures and published description (Magenta 2006) § Trait related only to belowground bud bank # Educated guess according to long perenniality and low lateral spread of organs in horizontal direction which was visible from gross morphology

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Figure 1. Hypogeogenous rhizomes (HR) (according to CLO-PLA database classification) of Aldama grandiflora (a-d) and A. nudibasilaris (e-f). (a) HR with thick roots with storage tuberous areas. (b-d) Detail of the buds. e. HR with thin roots with uniform storage tuberous areas. (f) Detail of the bud bank. Arrows = buds. Scale bars = 0.25 cm (B, D), 0.32 cm (B), 0.5 cm (F), 1.5 cm (A), 3.0 cm (E).

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Figure 2. Hypogeogenous rhizome (according to CLO-PLA database) of A.pilosa. (a) Overview of the organ with the ends showing aerial stems and leaves. (b) Detail of organ with thin and tuberous roots (arrowheads) and new sprouts (arrows). (c) Detail of the bud bank (arrows). Scale bar = 1.0 cm (C), 2.0 cm (B), 5.0 cm (A).

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CONSIDERAÇÕES FINAIS

Neste estudo foram levantados dados relacionados à morfoanatomia, histoquímica e composição dos óleos essenciais de folhas, caules e órgãos subterrâneos de espécies de Aldama, com a intenção de identificar novas características com valor diagnóstico e caracterizar as particularidades e a capacidade de rebrotamento dos órgãos subterrâneos deste grupo de plantas. As análises dos órgãos vegetativos aéreos de Aldama anchusifolia, A. megapotamica, A. nudibasilaris e A. pilosa (Capítulo 1) demonstraram que certas características anatômicas das folhas e dos caules podem ser úteis para a distinção dessas espécies, uma vez que estas características se mantiveram constantes entre os indivíduos analisados. Neste capítulo também foram registradas algumas novidades para o gênero, como a presença de frutanos do tipo inulina no caule aéreo de Aldama pilosa e a descrição anatômica do pecíolo de três das quatro espécies. Além disso, a análise dos óleos essenciais de folhas e caules revelou o valor quimiotaxonômico de determinados terpenos. A avaliação do número e do posicionamento de canais secretores na nervura central indicou que na maior parte das espécies tais parâmetros são altamente variáveis ao longo de uma mesma folha, entre folhas de um mesmo indivíduo e em folhas de indivíduos diferentes, indicando que estas características devem ser utilizadas com cautela na distinção de espécies e/ou em conjunto com outros fatores (Capítulo 2). Por outro lado, o padrão de venação foi constante entre os indivíduos analisados, e assim mostrou ser uma ferramenta útil para contribuir com a distinção das espécies. Nos capítulos 3 e 4 os órgãos subterrâneos de espécies de Aldama foram analisados detalhadamente. O estudo morfoanatômico revelou adaptações macro e microscópicas que auxiliam a sobrevivência destas plantas após distúrbios, assim como o desenvolvimento de um banco de gemas subterrâneo, a presença de frutanos do tipo inulina e a produção de óleos essenciais que contribuem para a proteção dessas estruturas contra microrganismos e herbívoros. Além disso, o desenvolvimento de extensões horizontais do caule subterrâneo em Aldama anchusifolia e A. pilosa foi o primeiro registro feito para o gênero. Por fim, no Capítulo 4 a aplicabilidade do banco de dados CLO-PLA (clonal plants) foi testada para um grupo de plantas brasileiras, o qual não só incluiu espécies de Aldama, mas também de outros gêneros de Asteraceae. Esta iniciativa foi o primeiro

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passo para a construção de um banco de dados focado nas características dos órgãos subterrâneos das plantas brasileiras, o qual está em elaboração e será uma ferramenta útil para levantar e responder questões mais abrangentes relacionadas às estratégias adaptativas dessas plantas. Os dados gerados nesse estudo representam uma significativa contribuição para o conhecimento sobre as espécies brasileiras de Aldama, uma vez que fornecem informações com potencial para auxiliar a distinção das espécies e novidades para o grupo, as quais serão úteis para futuras pesquisas em diversas áreas da botânica.

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ANEXO 1

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ANEXO 2