Fernanda Maria Cordeiro De Oliveira Contribuições Aos Estudos Anatômicos De Bromeliaceae

Fernanda Maria Cordeiro de Oliveira

Contribuições aos estudos anatômicos de Bromeliaceae (Poales) sob uma perspectiva filogenética Contribution to the anatomical studies of Bromeliaceae (Poales) under a phylogenetic perspective

São Paulo 2017

Fernanda Maria Cordeiro de Oliveira

Contribuições aos estudos anatômicos de Bromeliaceae (Poales) sob uma perspectiva filogenética Contribution to the anatomical studies of Bromeliaceae (Poales) under a phylogenetic perspective

Tese apresentada ao Instituto de Biociências da Universidade de São Paulo, para a obtenção de Título de Doutora em Ciências Biológicas, na Área de Botânica. Orientadora: Profa. Dra. Gladys Flavia de Albuquerque Melo de Pinna Co-Orientadora: Profa. Dra. Maria das Graças Wanderley.

São Paulo 2017

Oliveira, Fernanda Maria Cordeiro Contribuições aos estudos anatômicos de Bromeliaceae (Poales) sob uma perspectiva filogenética 161 páginas

Tese (Doutorado) - Instituto de Biociências da Universidade de São Paulo. Departamento de Botânica.

1. Reconstrução de caracteres ancestrais; 2. Evolução; 3. Complexo Nidularióide; 4. Anatomia floral; 5. Tricomas glandulares I Universidade de São Paulo. Instituto de Biociências. Departamento de Botânica.

Comissão Julgadora

______Prof(a). Dr(a). Prof(a). Dr(a).

______Profa. Dra. Gladys Flavia de Albuquerque Melo de Pinna (Orientadora)

Àquela que me amou desde o primeiro olhar.

“A humanidade é parte de um vasto universo em evolução. A Terra, nosso lar, está viva com uma comunidade de vida única. As forças da natureza fazem da existência uma aventura exigente e incerta, mas a Terra providenciou as condições essenciais para a evolução da vida. (...) O meio ambiente global com seus recursos finitos é uma preocupação comum de todas as pessoas. A proteção da vitalidade, diversidade e beleza da Terra é um dever sagrado. ”

Carta da Terra (1992)

Agradecimentos

Quatro anos podem passar muito devagar quando não se gosta do que faz. Mas quando você gosta, passam rápido... Para mim, no final das contas, quatro anos passaram voando e deixam ótimas lembranças. E, se passou rápido, foi devido a todo o apoio que recebi, desde a minha chegada em São Paulo até agora.

Agradeço primeiramente ao Instituto de Biociências da Universidade de São Paulo

(IB-USP), instituição que me recebeu e forneceu toda a infraestrutura para a realização deste trabalho e também a todos os professores e funcionários.

Ao CNPq, pela bolsa concedida, que possibilitou que este trabalho fosse realizado.

Agradeço com muito carinho, minha querida orientadora, Professora Dra. Gladys

Flavia de Albuquerque Melo-de-Pinna. Agradeço por todos os momentos de orientação, pela confiança desde o primeiro dia, quando este projeto ainda era um sonho. Pelos ensinamentos- de botânica e de vida. Agradeço também por todos os momentos deamizade, todas as conversas e conselhos!

À professora Dra. Maria das Graças Lapa Wanderley, que tão prontamente aceitou me coorientar. Obrigada pelas discussões sobre morfologia e pela colaboração com o doutorado.

Ao professor Dr. Rafael Batista Louzada, que tão bem me acolheu em seu laboratório em Recife. Obrigada pela ajuda com o mapeamento dos caracteres, pelos almoços com muitas discussões sobre Bromeliaceae e pela colaboração neste trabalho! Não posso deixar de agradecer aqui, também, à Dra. Maria Claudia Medeiros pela ajuda com o R e por todos os bons momentos em Recife. Agradeço aos membros que fizeram parte da minha banca de qualificação: Profa. Dra.

Nanuza Luiza de Menezes, Profa. Dra. Vera Lúcia Scatena e Prof. Dr. Benoît Loeille.

Obrigada por todos os conselhos e recomendações referentes ao projeto desta tese.

Aos professores do Laboratório de Anatomia Vegetal da USP: Profa. Dra. Nanuza

Luiza de Menezes, Prof. Dr. Diego Demarco, Prof. Dr. Gregório Ceccantini, Profa. Dra.

Gladys Flavia de Albuquerque Melo-de-Pinna, Profa. Dra. Verônica Angyalossy e Dra.

Berta Lange de Morretes. Obrigada por todos os ensinamentos botânicos, pelas disciplinas ministradas, pelas referências, artigos, livros. Agradeço também por todas as vezes em que tive dúvidas e vocês estavam sempre prontos a saná-las.

Às técnicas do Laboratório de Anatomia Vegetal, Gilese Costa e Tássia dos Santos, por toda a ajuda que recebi desde o meu primeiro dia na USP. Obrigada pelas dicas sobre as técnicas, pelos conselhos, pela ajuda com a parte prática. Obrigada por todas as vezes em que se dispuseram a me ajudar e que me incentivaram quando as coisas não davam certo. Por todos os “tenta outra vez Fê, vai dar certo!”. E, claro, por todos os momentos na copinha para o café...

Aos meus queridos colegas do Laboratório de Anatomia Vegetal. Seria impossível nomear todos, mas vocês fazem parte desta história.

Ao técnico Irwandro Pires pelas imagens em microscopia eletrônica de varredura.

Aos professores do Laboratório de Morfo-Taxonomia Vegetal da UFPE, Prof. Dr.

Rafael Batista Louzada, Prof. Dr. Benoît Loeille e Prof. Dr. Marccus Alves. Obrigada por me permitirem fazer parte deste laboratório, por me apresentarem a Caatinga!

Aos meus queridos irmãos de orientação, à equipe Melo-de-Pinna. Obrigada Aline

Ogura, Bruno Edson, Carlos Valério, José Hernandes, Juliana Brasileiro, Leyde Nunes, Rafael Cruz, Renata Lemos, Ricardo Vita pela colaboração no laboratório, pelas sugestões e por todo o carinho.

Aos colegas do Laboratório de Morfo-Taxonomia Vegetal: Beta Ferralc, Beth

Córdula, Camila Alcantara, Danielly Lucena, Débora Cavalcanti, Edlley Pessoa, Marcio

Lucas, Marily Jhullis, Naédja Kaliéri, Regina Carvalho e Thales Coutinho.

Ao Des. Elton Leme, que abriu as portas do seu refúgio dos Gravatás em Teresópolis, permitindo que eu coletasse o material necessário para o desenvolvimento do primeiro capítulo desta tese.

Agradeço em especial ao Rafael Cruz, por toda a paciência em me ensinar a usar o mesquite! Por me ajudar com os alinhamentos e com o “terrível” Mr. Bayes. Também por todos os momentos agradáveis e de descontração.

À Aline Ogura, por todos os cafézinhos, onde a gente colocava a conversaem dia.

Obrigada por todo o carinho!

À Renata Lemos –Renatiiiiinhaaaa! Obrigada por estar “juntinho” todos os momentos! Obrigada pela paciência, por todas as jantinhas maravilhosas, por me escutar e me aconselhar. Obrigada pela amizade, sempre!

Aos amigos de SP, nossa galerinha reunida de todos os meses! Obrigada Bruno Sales,

Gustavo Burin, José Hernandes, Tássia Santos e Renata Lemos por fazerem de São Paulo uma cidade tão acolhedora! Obrigada por todas as gargalhadas até o ar me faltar, pelas discussões de política, pelos filmes, pelas pipocas, pelas gordices! Obrigada por estarem sempre pertinho! Às minhas queridas Viseiras: Carolina Bastos, Luíza Teixeira, Mariana Victório,

Yasmin Hirao e Thaíla Vieira. Obrigada por todos os momentos de risos, todos os encontros e obrigada pela motivação sempre!

Aos queridos Karla Figueiredo e Mário Lima que me receberam em sua casa no período em que estive em Recife. Obrigada por me fazerem me apaixonar pelo Nordeste e por todo o carinho que me deram.

À minha família, que me apoiou incondicionalmente para que esse sonho se tornasse realidade. Minha Mãe Olga Oliveira, Irmão Darley Oliveira e minha pequena Gabriela

Oliveira! Obrigada por todas as ligações, por todas as vezes que me buscaram na rodoviária, todos os momentos de carinho!

E ao Weslley Dalcol, que me apoiou desde o início. Obrigada por compreender os momentos de ausência!

Muito Obrigada!

Resumo Bromeliaceae ocupa posição basal na Ordem Poales e é considerada monofilética, tendo sinapomorfias morfológicas e moleculares. Atualmente é subdividida em oito subfamílias, sendo Bromelioideae a subfamília com maiores problemas na delimitação genérica. Nesse contexto encontra-se o Complexo Nidularióide, formado pelos gêneros Nidularium Lem., Wittrockia Lindm., Neoregelia L.B.Sm., Canistropsis (Mez) Leme e Edmundoa Leme. A dificuldade na delimitação destes gêneros se dá pelo uso de um grande número de caracteres não exclusivos, que mostram o íntimo relacionamento entre estes gêneros. Embora estes gêneros não sejam monofiléticos, como indicam as filogenias recentes, o Complexo Nidularióide como um todo sempre emerge em um clado, indicando que formam uma unidade taxonômica. Neste contexto, analisamos caracteres morfológicos e anatômicos a fim de estabelecer novas sinapomorfias para o Complexo. Nossos resultados indicam que os principais caracteres morfológicos utilizados na delimitação dos gêneros do complexo Nidularióide são homoplásticos. A maior parte dos caracteres anatômicos também representam homoplasias. No entanto a anatomia foliar mostrou-se útil, provendo novas sinapomorfias para o grupo, tais como a presença de células da ala alongadas nos tricomas, e presença de células epidérmicas adaxiais com paredes levemente espessadas na lâmina foliar. No segundo capítulo, analisamos a anatomia floral de 16 espécies de Bromeliaceae, pertencente a três das oito subfamílias, a fim de estabelecer caracteres anatômicos florais úteis na sistemática do grupo, bem como discuti- los sob uma perspectiva ecológica e filogenética. Uma nova sinapomorfia é proposta para Pitcairnoideae a partir de dados sobre a vascularização do gineceu. No terceiro capítulo, analisamos o desenvolvimento foliar de três espécies de Tillandsia L. (Tillandsoideae). Neste estudo, foi possível registrar a presença de coléteres nas bainhas foliares próximas aos ápices caulinares de T. tricholepis (L.) L. Estas estruturas são responsáveis pela proteção do meristema apical caulinar (MAC) por meio de seu exsudado. Este estudo é o primeiro registro destas estruturas em Bromeliaceae.

Abstract Bromeliaceae ocupies a basal position in Poales Order and is considered monophyletic by morphological and molecular sinapomorphies. The family is currently subdivided into eight subfamilies. Bromelioideae has major problems in generic delimitation. In this context is included Nidularioid Complex, formed by the genera Nidularium Lem., Wittrockia Lindm., Neoregelia L.B.Sm., Canistropsis (Mez) Leme e Edmundoa Leme. The difficulty to generic delimitation in these genera occurs by the presence of a great number of non-exclusive character, which demonstrates their intimate relashioship. Although recent phylogenies indicate these genera are not monophyletic, Nidularioid Complex always emerges as a clade, which suggests they are a true taxonomic entity. In this context, we analyzed morphological and anatomical characters to establish new sinapomorphies for the Complex. Our results indicates that the main morphological characters used in genera delimitation on Nidularioid Complex are homoplastic. The major part of anatomical characters are also homoplastic. However, leaf anatomy shows to be useful, providing new synapomorphies for this group, such as the presence of trichomes with elongated wing’s cells and the presence of adaxial epidermal cells with lightly thickened walls on leaf blade. On the second chapter, we analyzed floral anatomy of 16 species of Bromeliaceae, belonging to three out of eight subfamilies, to establish anatomical floral characters useful in the group sistematics, as well as discuss it under an ecological and phylogenetic perspective. A new synapomorphy for Pitcairnoideae is established, by data of gynoicea vascularization. On the third chapter, we analyzed leaf development of three species of Tillandisia L. (Tillandsoideae). In this study, it was possible to describe the presence of colleters in leaf sheath, next to shoot apices of T. tricholepis (L.) L. These structures are responsible by shoot apical meristem’s (SAM) protection by their secretion. This is the first record of colleters in Bromeliaceae.

Índice

Introdução geral...... 1

1.1 Caracterização morfológica de Bromeliaceae...... 3

1.2 Aspectos anatômicos em Bromeliaceae...... 4

1.3 Importância econômica e ecológica de Bromeliaceae...... 8

1.4 Estrutura Geral da Tese...... 10

Referências...... 12

Capítulo I: “Morphoanatomical characters in the Nidularioid Complex (Bromeliaceae:

Bromelioidae) on a phylogenetic perspective”……………………………….………………17

Abstract………………………………………………………..…………………….19

Resumo…………………………………………………………………….………..20

1. Introduction………………………………………………………..…………….21

2. Materials and methods………………………………………..…………………23

2.1 Taxon sampling………………………………………………..…………..23

2.2 Sequence alignment and phylogenetic analyses……………...……………23

2.3 Morphological and anatomical data………...... ………………………24

2.3 Ancestral state reconstruction………………………………………….….24

3. Results………………………………...………………….………………………25

4. Discussion………………………...…………………………..…………………..26

4.1 Morphological characters……………………..…………………………...26

4.2 Anatomical characters…………………………..…………………………28

5. Conclusions………………………………………………….…………………...35

6. Acknowledgements………………………………………..………..……………36

Appendix I...... 55

Appendix II: Character delimited...... 57 Appendix III: Characters and their state of character………………...…………63

References……………………………………..…………………………………....70

Capítulo II: “Floral anatomy of Bromeliaceae with special reference on androecium and gynoecium ………….....…...... 77

Abstract...... 79

Resumo...... 80

1. Introduction...... 81

2. Materials and methods...... 82

3. Results...... 83

3.1 Androecium...... 83

3.2 Gynoecium...... 84

4. Discussion...... 86

4.1 General characteristics ...... 86

4.2 Vascularization...... 89

4.3 Secretory structures...... 91

5. Conclusions...... 95

6. Acknowledgements...... 95

References...... 123

Capítulo III: “New record of colleters in Monocots: Bromeliaceae (Poales)”……………....129

Abstract...... 131

Resumo...... 132

1. Introduction...... 133

2. Materials and methods...... 134

2.1 Plant material...... 134 2.2 Light microscopy...... 134

2.3 Scanning Electron Microscopy (SEM)...... 135

3. Results...... 135

4. Discussion...... 137

5. Conclusion...... 139

6. Acknowledgements...... 139

7. References...... 153

Conclusões gerais...... 157

Introdução Geral

Introdução Geral F.M.C. Oliveira

Introdução Geral Bromeliaceae possui cerca de 3.350 espécies, distribuídas em 58 gêneros (Luther, 2012), sendo a sua distribuição predominantemente neotropical (Smith & Downs, 1974). A exceção é

Pitcairnia feliciana (A. Chev.) Harms & Mildbr. que ocorre na costa oeste da África, resultado de uma dispersão a longa distância (Jacques-Felix, 2000; Givnish et al., 2004). A família possui dois grandes centros de diversidade: o Escudo das Guianas e a Costa Leste do Brasil (Givnish et al. 2007). Para o Brasil, são citados 40 gêneros ca. de 1300 espécies, sendo que ca. de 1100 espécies são consideradas endêmicas (BFG, 2015) e alguns táxons são encontrados exclusivamente na Mata Atlântica (Martinelli et al., 2008).

Durante muito tempo, graças a características singulares e de fácil reconhecimento,

Bromeliaceae foi considerada a única família pertencente à ordem Bromeliales (Cronquist,

1981; Dahlgren et al. 1985; Gilmartin & Brown, 1987). Atualmente incluída na ordem Poales, a família tem como grupo-irmão Typhaceae, ocupando a posição basal da ordem (APG IV,

2016). Seu monofiletismo é sustentando tanto por caracteres moleculares - sequência de ndhF

(Givnish et al. 2007; Givnish et al., 2011), morfológicos - presença de tricomas peltados absorventes (Benzing, 2000), presença de estigmas tipo espiral conduplicado (Brown &

Gilmartin, 1989a) e citogenéticos - número cromossômico básico x=25 (Brown & Gilmartin,

1989b).

É tradicionalmente dividida em três subfamílias: Bromelioideae, Tillandsoideae e

Pitcairnoideae s.l., cujas circunscrições levam em conta principalmente a morfologia foliar, posição do ovário, tipo de fruto e de semente (Smith & Downs, 1974, 1977, 1979). No entanto, por meio de estudos moleculares, verificou-se que Pitcairnoideae não representava um grupo monofilético. Assim, a família foi então reorganizada em oito subfamílias, a saber:

Bromelioideae, Tillandsoideae, Pitcairnoideae s.s., Navioideae, Puyoideae, Brocchinioideae,

Hechtioideae e Lindmanioideae (Fig. 1) (Givnish et al. 2007; Givnish et al. 2011).

Introdução Geral F.M.C. Oliveira

Figura 1: Árvore de máxima parcimônia de consenso stricto, baseada na variação nas sequências plastidiais ndhF, com as relações propostas entre as subfamílias de Bromeliaceae. Grupos externos de sete famílias de Poales não são mostrados. Números acima dos ramos são os valores de bootstrap; números em parênteses após os nomes das subfamílias indicam o número de táxons utilizados nas análises. Retirado de Givnish et al. (2011).

Apesar dos avanços obtidos com o advento da sistemática molecular, muito ainda há para ser feito em relação à sistemática de Bromeliaceae. Segundo Escobedo-Sarti et al. (2013), dos 58 gêneros descritos em Bromeliaceae, apenas 14 são considerados monofiléticos, a saber:

Brocchinia Schult. f., Alcantarea Harms, Catopsis Griseb., Glomeropitcairnia Mez, Guzmania

Ruiz & Pav., Werauhia J. R. Grant, Hechtia Klotzsch, Steyerbromelia L. B. Sm., Fosterella L.

B. Sm., Puya Molina, Ananas Mill., Araeococcus Brongn., Bromelia L., e Acanthostachys Link,

Klotzsch & Otto. Estes gêneros têm em comum poucas espécies e fácil reconhecimento morfológico.

Introdução Geral F.M.C. Oliveira

Segundo Wanderley & Moreira (2000) a questão conceitual genérica desta família como um todo tem se mostrado muito complexa. Assim, as autoras recomendam o uso de estudos mais aprofundados nestes grupos, a fim de não tornar a taxonomia da família ainda mais artificial e complexa. As autoras ressaltam ainda a necessidade de uma revisão genérica, utilizando-se de caracteres morfológicos e estudos de populações em seu ambiente natural.

Dentre as subfamílias supracitadas, Bromelioideae é detentora da maior diversidade morfológica refletindo na inclusão de mais da metade dos gêneros de Bromeliaceae (Benzing,

2000). É caracterizada por possuir a margem foliar espinescente, ovário ínfero e fruto tipo baga

(Smith & Downs, 1979). Nesta subfamília é notória a precariedade de delimitação de vários gêneros, sendo que, muitas vezes, apesar de se ter a descrição completa de uma espécie, é difícil decidir a qual gênero pertence (Smith & Downs, 1979).

Algumas características chaves, tais como a presença de tricomas peltados absorventes ao longo da folha, metabolismo Ácido das Crassuláceas (CAM), e de um tanque formado pela sobreposição das bainhas foliares que acumula água, permitiram a família ocupar variados habitats, ocorrendo desde o nível do mar até altas altitudes (ca. de 4000 metros), em ambientes mésicos e xéricos (Smith & Down, 1974; Benzing, 1976; Benzing, 2000; Silvestro et al., 2014).

1.1 Caracterização morfológica de Bromeliaceae

Bromeliaceae é formada por plantas herbáceas perenes, terrestres, epífitas ou rupícolas, com caule curto ou longo, rizomatoso ou estolonífero com raízes reduzidas absortivas nas plantas terrestres, fixadoras nas epífitas e rupícolas ou ausentes. As folhas são simples, alternas, polísticas à dísticas, recobertas por tricomas peltados absorventes. Por vezes o imbricamento da bainha foliar forma uma cisterna que acumula água, minerais e detritos orgânicos. Bainha foliar geralmente aberta, pouco ou muito distinta da lâmina foliar. Lâmina foliar pode apresentar margem lisa ou espinescente. As inflorescências são terminais indeterminadas, simples ou compostas. Flores solitárias são raras, como em Tillandsia usneoides L. As flores

Introdução Geral F.M.C. Oliveira são bissexuadas com perianto diferenciado em cálice e corola, dispostas na axila de brácteas, frequentemente coloridas. As sépalas podem ser livres ou conadas. As pétalas, livres ou conadas, muitas vezes apresentam um par de apêndices em sua base e algumas vezes calosidades longitudinais. O androceu é formado por duas séries de três estames cada, filetes livres ou conados, as vezes adnatos às pétalas. O ovário pode ser súpero, ínfero ou semi-ínfero, tricarpelar e trilocular, com placentação axial, apresentando nectários septais; o estigma é trífido, podendo ser classificado em simples-ereto, espiral-conduplicado ou laminar-convuluto.

O fruto pode ser do tipo cápsula septícida ou baga; sementes sem apêndices, aladas ou plumosas

(Smith & Downs, 1974; Cronquist,1981; Wanderley & Martins, 2007).

1.2 Aspectos anatômicos em Bromeliaceae

Sem dúvida alguma os tricomas peltados absorventes, frequentemente chamados de escamas, são as estruturas anatômicas mais estudadas na família (Benzing, 2000). Diversos pesquisadores buscaram compreender sua organização anatômica e funcionamento (Mez, 1896;

Benzing, 1976; Tomlinson, 1969). Estes tricomas, na ausência de raízes (como em algumas espécies de Tillansia spp.), ou quando estas estão presentes mas têm função de fixação no substrato (como em espécies saxícolas ou epífitas), são responsáveis pela absorção de água de nutrientes. Atualmente, sabe-se que a presença de tricomas peltados em Bromeliaceae é uma das características que permitiram a adaptação radiativa da família (Benzing, 2000).

Estes tricomas são compostos, geralmente, de uma célula basal, um pedicelo (que pode ter um número variado de células), célula domo, disco e ala. As células do disco, também chamadas de centrais, conectam a ala ao pedicelo (Krauss, 1949; Tomlinsom, 1969) (Fig. 2 A-

B, 3A).

Sendo a maior parte da família epífita ou saxícola, as Bromeliaceae sofrem estresse hídrico, aproveitando-se da umidade do ar ou de água acumulada em suas cisternas, também

Introdução Geral F.M.C. Oliveira chamadas de fitotelma (Benzing, 2000). A rápida absorção de água dá-se por meio de um mecanismo complexo, com envolvimento ativo das escamas presentes nas folhas e foi descrito por Benzing (1976). Além da absorção de água por meio dos tricomas, e da presença de uma cisterna que formada pelo imbricamento de suas folhas, as Bromeliaceae, em geral, contam com um reservatório interno de água proporcionado pela suculência de suas folhas. Tal suculência

é dada pela presença de um tecido armazenador de água, denominado hipoderme aquífera

(Tomlinson, 1969).

Outra característica da família é a presença de células epidérmicas, nas folhas, com paredes anticlinais sinuosas em vista frontal e contendo um cristal esférico de sílica

(Krauss,1949; Tomlinsom, 1969) (Fig. 2D, Fig. 3B). Muito já foi discutido sobre a função de tais cristais, e acredita-se que estejam envolvidos na reflexão da luz (Prychid et al. 2004).

As folhas de Bromeliaceae possuem, ainda, canais de aeração longitudinais preenchidos por células do parênquima braciforme, intercalados com os feixes vasculares Tais canais têm comunicação direta com as câmaras subestomáticas. (Krauss, 1949; Tomlinson, 1969; Benzing,

2000) (Fig. 2C, Fig. 3C-D).

Introdução Geral F.M.C. Oliveira

Figura 2: Características anatômicas foliares de Bromeliaceae. A: Tricoma peltado absorvente em visão frontal. B: Tricoma peltado em secção transversal. C: Secção transversal da lâmina foliar de Ananas comusus L. D: Epiderme adaxial em vista frontal de A. comusus. A-B: Modificado de Benzing (2000), redesenhado de Tomlinson (1969). C-D: Modificado de Krauss (1949).

Introdução Geral F.M.C. Oliveira

Figura 3: Características anatômicas das folhas de Bromeliaceae. A: Tricoma peltado de Vriesea platynema Gaudich. em vista frontal B: Epiderme adaxial da bainha foliar de Neoregelia compacta (Mez) L.B.Sm. em vista frontal C: Secção transversal da lâmina foliar de Wittrockia superba Lindm. D: Detalhe da secção transversal da lâmina foliar de W. superba, evidenciando um canal de aeração. Al= Células da ala do tricoma; Ca= Canais de aeração; D= células do disco do tricoma; Setas indicam os cristais de sílica.

Características anatômicas relacionadas às suas flores também tornam a família um interessante alvo de estudo. A presença de nectários septais, tributo pouco comum na ordem

Poales, é descrita para Bromeliaceae, sendo possíveis duas conformações: infraloculares, quando os ovários são súperos ou semi-ínferos e os nectários ocorrem abaixo dos lóculos; e septais interloculares, quando os ovários são ínferos e os nectários ocorrem nos septos das folhas carpelares, entre os lóculos do ovário (Sajo et al.2004a; Linder & Rudall, 2005) (Fig.

Introdução Geral F.M.C. Oliveira

4A-B). Tais estruturas já foram estudadas em relação à composição química de seus produtos

(Bernardello et al. 1991; Stahl et al., 2012), sua ultraestrutura e anatomia (Fiordi & Palandri,

1982; Kulkarni & Pai, 1982; Sajo et al. 2004a) e também sobre a sua posição na flor relacionando-a com a evolução da epiginia no grupo (Sajo et al. 2004a).

Outro aspecto amplamente estudado é a estrutura anatômica do óvulo em Bromeliaceae.

Estudos descrevem que os óvulos podem apresentar estruturas características denominadas de apêndices calazais e micropilares (Sajo et al. 2004b; Fagundes & Mariath, 2014; Nogueira et al. 2015; Kuhn et al. 2016;) (Fig.4C-D). Os apêndices calazais podem ter origem dérmica ou subdérmica e podem ser reconhecidos como um crescimento pronunciado na região da calaza

(Fig. 4C-D). Já os apêndices micropilares podem ser reconhecidos pelo crescimento dos tegumentos (interno e externo), formando uma conspícula protrusão (Sajo et al. 2004b).

1.3 Importância econômica e ecológica de Bromeliaceae

Bromeliaceae é popularmente conhecida como a família do abacaxi. Mas sua relevância econômica vai muito além da comercialização dos saborosos frutos de Ananas comosus (L.)

Merr. Devido à beleza de suas folhas e ao colorido de duas inflorescências, espécies de

Bromeliaceae são utilizadas como plantas ornamentais (Reitz, 1983). A grande procura pelas

Bromeliaceae de valor ornamental intensificou o extrativismo destas plantas de seu ambiente natural nos últimos anos, colocando alguns táxons em maior grau de ameaça.

Introdução Geral F.M.C. Oliveira

Figura 4: A-B Secção transversal do ovário evidenciando o nectário septal (Ns). A: Aechmea distichantha. Lem. B: Canistropsis billbergioides. (Schult & Schult. f.) Leme C: Microscopia Eletrônica de Varredura do óvulo de Billbergia nutans H. Wendl. Ex. Regel evidenciado o apêndice calazal (Ca). D: Secção longitudinal do óvulo de B. nutans. ca= apêndice calazal, fg= megagametófito; oi= tegumento externo; ii= tegumento interno; Hi= hipoderme; Ns= Nectário septal; Pl= placenta. A e B: Retirado de Oliveira et al. (2016). C e D: Retirado de Fagundes & Mariath (2014).

Introdução Geral F.M.C. Oliveira

Devido a água acumulada armazenada em seu tanque central (fitotelma), as

Bromeliaceae têm importância ecológica fundamental, sendo um elemento importante para a ampliação da diversidade. (Oliveira, 2004). Numerosas espécies de animais utilizam essas plantas para a sua reprodução (como é o caso dos anfíbios), forrageamento e refúgio contra seus predadores (Rocha et al., 1997). Algumas espécies de plantas germinam e se desenvolvem no fitotema, possuindo um papel importante no estabelecimento de outras espécies vegetais, como por exemplo em ambientes de restinga. (Fialho & Furtado, 1993; Zaluar & Scarano, 2000).

Todavia, a importância ecológica não se restringe à água acumulada em suas rosetas.

As flores das Bromeliaceae, por meio do néctar produzido, atraem uma gama de polinizadores que nelas obtêm recursos alimentares. Assim, durante o período de floração, essas plantas constituem fonte importantíssima de alimento para diversos animais, como abelhas, mariposas, beija-flores, vespas e borboletas. Suas flores também são reduto para diversas espécies de

ácaros (Rocha et al., 1997).

1.4 Estrutura geral da tese

No primeiro capítulo, o trabalho aborda o estudo da anatomia em espécies pertencentes ao Complexo Nidularióide com enfoque filogenético. Este grupo é formado pelos gêneros

Nidularium Lem., Neoregelia L.B.Sm., Edmundoa Leme e Wittrockia Lindm. (Leme, 1998). O interesse surgiu ao perceber que os gêneros pertencentes a este Complexo possuem difícil delimitação e, provavelmente, não são monofiléticos. No entanto, o Complexo como um todo emerge sempre como um grupo monofilético nas filogenias recentes de Bromelioideae. Para tanto, foram selecionadas espécies pertencentes ao Complexo Nidularióide abordadas por

Silvestro et al. (2014), buscando estabelecer sinapomorfias anatômicas para o grupo. Neste sentido, este capítulo aborda aspectos morfoanatômicos foliares sob uma perspectiva filogenética.

Introdução Geral F.M.C. Oliveira

No segundo capítulo, selecionamos 16 espécies, pertencentes as subfamílias

Bromelioideae, Pitcairnoideae e Tillandsoideae, para o estudo comparativo do androceu e gineceu. Nosso objetivo, neste trabalho, foi descrever caracteres que possam ser utilizados na sistemática do grupo, assim como discutí-los sob uma perspectiva ecológica e filogenética.

Assim, descrevemos caracteres referentes a anatomia geral do androceu e ginegeu, caracteres referentes a vascularização destes verticilos e também referentes às estruturas secretoras.

Adicionalmente, propusemos uma nova sinapomorfia anatômica, referente à vascularização do gineceu, para Pitcairnoideae.

No terceiro capítulo, ao observar o desenvolvimento foliar em espécies pertencentes ao gênero Tillandsia L. (Tillandsoideae), notamos a presença de tricomas glandulares próximos ao meristema apical caulinar. Assim, neste capítulo, nosso objetivo foi descrever estes tricomas glandulares e realizar testes histoquímicos pertinentes para a classificação dos principais compostos presentes no exudado destas estruturas.

Introdução Geral F.M.C. Oliveira

Referências

APG IV. 2016. An update of the Angiosperm phylogeny group classification for the orders and families of flowering plants: APG IV. Botanical Journal of Linnean Society, 181: 1-20.

Benzing, D.H. 1976. Bromeliad trichomes: structure, function and ecological significance. Selbyana. 1:330-348.

Benzing, D.H. 2000. Bromeliaceae: profile of an adaptative radiation. New York: Cambridge University Press.

Bernardello, L.M.; Galetto, L.; Juliani, H.R. 1991. Floral nectar, nectary structure and pollinators in some Argentinean Bromeliaceae. Annals of Botany 67 (5): 401-411.

BFG. 2015. Growing knowledge: an overview of Seed Plant diversity in Brazil. Rodriguésia, v.66, n.4, p.1085-1113. (DOI: 10.1590/2175-7860201566411)

Brown, G. K.; Gilmartin, A. J. 1989a. Stigma types in Bromeliaceae- a systematic survey. Systematic Botany. 14: 110-132.

Brown, G.K.; Gilmartin, A.J. 1989b. Cromosssomes number in Bromeliaceae. American Journal of Botany 76 (5):657-665.

Cronquist, A. 1981. An Integrated System of Classification of Flowering Plants. New York: Columbia University Press.

Dahlgren, R.M.T.; Clifford, H.T.; Yeo, P.F. 1985. The families of the monocotyledons: structure, evolution and taxonomy. Berlin: Springer-Verlag.

Escobedo-Sarti, J.; Ramírez, I.; Leopardi, C.; Carnevali, G.; Magallón, S.; Duno, R.; Mongradón, D. 2013. A phylogeny of Bromeliaceae (Poales, Monocotyledonaee) derived from an evaluation of nine methods. Journal of Systematics and Evolution. 51 (6): 743-757.

Fagundes, N.F.; Mariath, J.E.A. 2014. Ovule ontogeny in Billbergia nutans in the evolutionary contex of Bromeliaceae (Poales). Plant. Systematics and Evolution 300:1323-1336.

Fialho, R.F.; Furtado, A.L.S. 1993. Germination of Erythroxylum ovalifolium (Erythroxylaceae) seeds within the terrestrial bromeliad Neoregelia cruenta. Briotropica. 25:359-362.

Introdução Geral F.M.C. Oliveira

Fiordi, A.C.; Palandri, M.R. 1982. Anatomic and ultrastructural study of the septal nectary in some Tillandsia (Bromeliaceae) species. Caryologia 35(4): 477-489.

Gilmartin, A.J.; Brown, G.K. 1987. Bromeliales, Related monocots, and resolution of relationships among Bromeliaceae Subfamilies.Systematic Botany 12 (4): 493-500.

Givnish, T.J.; Millam, K.C.; Evans, T.M.; Hall, J.C.; Pires, J.C.; Berry, P.E.; Sytsma, K.J. 2004. Anciant vicariance or recent long distance dispersal? Inferences about phylogeny and south american-african disjunctions in Rapateaceae and Bromeliaceae based on ndhF sequence data. International Plant Science. 4 (Supplem.), S35-S54.

Givnish, T.J.; Millan,K.C.; Berry, P.E.; Systma, K.J. 2007. Phylogeny, adaptive radiation, and historical biogeography of Bromeliaceae inferred from ndhF sequence data. Aliso, n. 23, p. 3- 26.

Givnish, T.J.; Barfuss, M.H.J.; Van Ee, B.; Riina, R.; Schulte, K.; Horres, R.; Gonsiska, P.A.; Jabaily, R.S.; Crayn, D.M.; Smith, A.C.; Winter, K.; Brown, G.K.; Evans, T.M.; Holst, B.K.; Luther, H.; Till, W.; Zizka, G.; Berry, P.E.; Systma, K.J. 2011. Phylogeny, adaptive radiation, and historical biogeography in Bromeliaceae: insights from an eight-locus plastid phylogeny. American Journal of Botany, n.98, v.5, p 1-24.

Jacques-Felix, H. 2000. The discovery of a bromeliad in Africa: Pitcairnia feliciana. Selbyana 21 (1,2): 118-124.

Krauss, B.H. 1949. Anatomy of the vegetative organs of the pineapple Ananas comosus (L.) Merr.: II The leaf. Botanical Gazette. 110 (3): 333-404.

Kuhn, S.A.; Nogueira, F.M.; Fagundes, N.F.; Mariath, J.E. 2016. Morphoanatomy of the ovary and ovule in Bromeliaceae subfamily Tillandsoideae and its systematic relevance. Botanical Journal of Linnean Society 181:343-361.

Kulkarni, R.A.; Pai, R.M. 1982. The floral anatomy of Puya spathacea Mez. (Bromeliaceae) with special reference to nectaries. Proceedings of the Indian Academy of Science 91 (6): 473- 478.

Leme, E.M.C. 1998. Canistropsis - Bromélias na Mata Atlântica. Rio de Janeiro: Salamandra

Linder, H.P.; Rudall, P.J. 2005. Evolutionary history of Poales. Annual Review of Ecology, Evolution and Systematics 36: 107-124.

Introdução Geral F.M.C. Oliveira

Luther, H. 2012. An alphabetical list of bromeliad binomials. 12th ed. The Bromeliad Society International. The Marie Selby Botanical Gardens, Sarasota & Bromeliad Society International, 44 p.

Martinelli, G., Vieira, C.M., Gonzalez, M., Leitman, P., Piratininga, A., Costa, A.F.; Forzza, R.C. 2008. Bromeliaceae da Mata Atlântica brasileira: lista de espécies, distribuição e conservação. Rodriguésia, 59 (1): 209-258.

Mez, C. 1896. Bromeliaceae. In: Candolle, A. L.P.P. de et Candolle A.C.P.de (Eds.). Monographiae Phanerogamarum… Paris: G. Masson.

Nogueira, F.M.; Fagundes, N.F.; Kuhn, S.A.; Fregonezi, J.N.; Mariath, J.E.A. 2015. Ovary and ovule anatomy in the nidularioid complex and its taxonomic utility (Bromeliaceae: Bromelioideae). Botanical Journal of Linnean Society 177: 66-77.

Oliveira, R.R. 2004. Importância das bromélias epífitas na ciclagem de nutrientes na Floresta Atlântica. Acta Botânica Brasílica. 18(4): 793-799.

Oliveira, F.M.C.; Souza, A. M.; Corrêa, B. B. R.; Maeda, T. M.; Melo-de-Pinna, G. F. A. 2016. Anatomia floral de Aechmea distichantha Lem. e Canistropsis billbergioides (Schult. & Schult. f.) Leme (Bromeliaceae). Hoehnea 43 (2), 183-193.

Prychid, C.J.; Rudall, P.; Gregory, M. 2004. Systematics and biology of silica bodies in Monocotyledons. The Botanical Review, 69: 377-440.

Reitz, R. 1983. Bromeliáceas e a malária – bromélia endêmica. Flora ilustrada Catarinense.

Rocha, C.F.D.; Cogliatti-Carvalho, L.; Almeida, D.R.; Freitas, A.F.N. 1997. Bromélias: ampliadoras da diversidade. Bromélia. 4: 7-10.

Sajo, M.G; Rudall, P.J.; Prychid, C.J. 2004. Floral anatomy of Bromeliaceae, with particular reference to the evolution of epigyny and septal nectaries in comelinid monocots. Plant Systematics and Evolution 247: 215-231.

Sajo, M.G.; Prychid, C.J.; Rudall, P.J. 2004b. Structure and development of the ovule in Bromeliaceae. Kew Bulletin. 59: 261-267.

Silvestro, D.; Zizka, G.; Schulte, K. 2014. Desentangling the effects of key innovations on thediversification of Bromelioideae (Bromeliaceae). Evolution 68 (1): 163-175.

Introdução Geral F.M.C. Oliveira

Smith L.B. & Downs R.J. 1974. Pitcairnioideae (Bromeliaceae). Flora Neotropica Monograph 14: 1-662.

Smith L.B. & Downs R.J. 1977. Tillandsioideae (Bromeliaceae). Flora Neotropica Monograph 14: 663-1492.

Smith L.B. & Downs R.J. 1979. Bromelioideae (Bromeliaceae). Flora Neotropica Monograph 14: 1493-2142.

Stahl, J.M.; Nepi, M.; Galetto, L.; Guimarães, E.; Machado, S.R. 2012. Functional aspects of floral néctar secretion os Ananas ananassoides, an ornithophilous bromeliad from the Brazilian savanna. Annals of Botany 109 (7): 1243-1252.

Tomlinson, P. 1969. Commelinales- Zingiberales. In: Metcalf, CR. (ed.) Anatomy of the Monocotyledons: III. Claredon Press, Oxford. Pp: 193-294.

Zaluar, H.L.T.; Scarano, F.R. 2000. Facilitação em restingas e moitas: um século de buscas por espécies focais. In: Esteves, F.A. & Lacerda, L.D. (Eds.). Ecologia de restingas e lagoas costeiras do Brasil. Rio de Janeiro: NUPEM, UFRJ.

Wanderley, M.G.L.; Moreira, B.A. 2000. Notas taxonômicas sobre Nidularium Lem. e Wittrockia Lindm. (Bromelioideae, Bromeliaceae). Acta Botanica Brasilica, 14 (1): .1-9.

Wanderley, M.G.L.; Martins, S.E. 2007. Bromeliaceae. In: Wanderley, M.G.L.; Shepherd, G.J.; Melhem, T.S.; Giulietti, A. M. (Org.). Flora Fanerogâmica do Estado de São Paulo. São Paulo: Instituto de Botânica. 5: 1-476.

Capítulo I

Morphoanatomical characters in the Nidularioid Complex (Bromeliaceae: Bromelioidae) on a

phylogenetic perspective

Fernanda Maria Cordeiro de Oliveira1,2, Rafael Batista Louzada3, Maria das Graças Lapa

Wanderley4 e Gladys Flávia Melo-de-Pinna1

¹Universidade de São Paulo, Instituto de Biociências, Departamento de Botânica, Laboratório de

Anatomia Vegetal, Rua do Matão 321 Travessa 14, 05508-090 São Paulo, SP, Brasil.

2Corresponding author: [email protected]

3Universidade Federal de Pernambuco, Centro de Ciências Biológicas, Departamento de Botânica,

Laboratório de Morfo-Taxonomia Vegetal, Av. Moraes Rego s/n, Recife, PE, Brasil.

4 Instituto de Botânica, Av. Miguel Estéfano 3687, 04301-902, Água Funda, São Paulo, SP, Brasil.

Submetido ao periódico Flora

Capítulo I Oliveira, F.M.C.

Abstract

The Nidularioid complex, comprised by the genera Nidularium Lem., Wittrockia Lindm.,

Neoregelia L.B.Sm., Canistropsis (Mez) Leme and Edmundoa Leme, is one of the groups with the most difficult generic delimitation in Bromelioideae. This difficulty is attributed to the large number of non-exclusive characters that are used in the abovementioned genera, which demonstrates their intimate relationship. In current phylogenies, the genera belonging to the

Nidularioid complex always emerge as a unity, being referred to as 'Nidularioid Clade'. This study aims to reconstruct usual morphological characters in Bromelioideae, as well as anatomical characters from the leaf sheath and blade of species from the Nidularioid complex, in order to propose new synapomorphies for the group. To accomplish that we propose a phylogenetic hypothesis that used chloroplastidial atpB-rbcL, matK, trnL-trnF and nuclear

PhyC gene sequences obtained from the NCBI portal in a bayesian analysis that resulted in a consensus tree. We also used parsimony and bayesian methods to reconstruct previously delimited morpho-anatomical characters. Our results indicate that the morphological characters typically used in the group's taxonomy represent homoplasies. Some anatomical characters are also homoplastic, such as the number of layers in the abaxial mechanical hypoderm from the leaf sheath. However, leaf anatomy provided new synapomorphies for the group, such as the presence of trichomes with elongated cells from the wing, and the presence of adaxial epidermal cells with lightly thickened walls on the leaf blade. Thus, this paper presents new perspectives for future studies on the evolution of characters in the Nidularioid complex.

Key-words: Ancestral State Reconstruction, Bromelioideae, evolution, synapomorphies.

Capítulo I Oliveira, F.M.C.

Resumo

O Complexo Nidularióide, formado pelos gêneros Nidularium Lem. Wittrockia Lindm.,

Neoregelia L.B.Sm., Canistropsis (Mez) Leme e Edmundoa Leme, é um dos grupos de maior dificuldade de delimitação genérica em Bromelioideae. Atribui-se a isso a grande quantidade de caracteres não exclusivos utilizados na circunscrição dos gêneros citados, demostrando o seu

íntimo relacionamento. Nas filogenias atuais, os gêneros pertencentes ao Complexo

Nidularióide sempre emergem como uma unidade, sendo referido como ‘Clado Nidularióide’.

Este estudo tem como objetivo reconstruir os caracteres morfológicos tradicionais utilizados em Bromelioideae, bem como caracteres anatômicos da bainha e lâmina foliar de espécies do

Complexo Nidularióide, a fim de propor novas sinapomorfias para o grupo. Para tanto, foi proposta uma hipótese filogenética a partir de sequências gênicas cloroplastidiais de atpB-rbcL, matK, trnL-trnF e nuclear PhyC, adquiridas no portal NCBI, e posterior análise Bayesiana, resultando em uma árvore de consenso. A reconstrução dos caracteres morfo-anatômicos delimitados previamente foi realizada pelo método de parcimônia e Bayesiana. Nossos resultados indicam que os caracteres morfológicos tradicionais na taxonomia do grupo representam homoplasias. Alguns caracteres anatômicos mostraram-se homoplásticos, tais como o número de camadas da hipoderme mecânica abaxial na lâmina foliar, e o espessamento das paredes das células da hipoderme mecânica abaxial na bainha foliar. No entanto, a anatomia foliar proveu novas sinapomorfias para o grupo, como a presença de tricomas com as células da ala alongadas e presença de células epidérmicas adaxiais com paredes levemente espessadas na lâmina foliar. Assim, este estudo apresenta nova perspectiva para futuros trabalhos de evolução de caracteres no Complexo Nidularióide.

Palavras-chave: Reconstrução de Caracteres Ancestrais; Bromelioideae, evolução, sinapomorfias.

Capítulo I Oliveira, F.M.C.

1. Introduction

The family Bromeliaceae belongs to the order Poales (APG IV, 2016) and its monophyletism is supported by morphological and molecular characters (Brown and Gilmartin,

1989; Benzing, 2000; Givnish et al., 2007, Givnish et al., 2011). Traditionally Bromeliaceae is subdivided into three subfamilies, Pitcairnoideae, Tillandsoideae and Bromelioideae, the three being distinguished from the one another by the leaf margin, ovary position, fruit and seed types

(Smith and Downs, 1974, 1977, 1979). With the advances of molecular systematics

Pitcarnoideae was shown not to be a monophyletic group, and Bromeliaceae was reorganized into eight subfamilies: Bromelioideae and Tillandsoideae (that were maintained), Navioideae was recircumscripted and Pitcairnoideae was split into Pitcairnoideae s.s., Puyoideae,

Brocchinioideae, Hechtioideae and Lindmanioideae (Givnish et al., 2007; Givnish et al., 2011).

Among the aforementioned subfamilies Bromelioideae shows the greater morphological diversity representing more than half of the family's genera with its 33 genera and ca. 936 species (Smith and Downs, 1979; Benzing, 2000; Butcher and Gouda, cont. Updated). Species from this subfamily are characterized by having aculeate leaf margin, inferior ovary, fruits as berries, and seeds without appendices (Smith and Downs, 1979). Its monophyletism is supported both by morphological, cytogenetic and molecular characters (Givnish et al., 2007;

Givnish et al., 2011). However according to Smith and Downs (1974) genus delimitation is fragile due to great morphological diversity. The morphological traits commonly used to circumscribe the genera in this subfamily such as the presence of petal appendages, branched inflorescence, and presence of pedicles are homoplastic (Sousa et al., 2007; Schulte and Zizka,

2008; Aguirre-Santoro et al., 2016).

The Nidularioid complex represents one of Bromelioideae's groups with difficult delimitation (Leme, 1997). According to Leme (1997, 1998, 2000) the complex comprises the

Capítulo I Oliveira, F.M.C. genera Nidularium Lem., Wittrockia Lindm., Neoregelia L.B.Sm, Canistropsis (Mez) Leme and Edmundoa Leme, and is characterized by inflorescences with developed superior scape bracts and primary bracts that accumulate water and resemble a nest over the foliar rosette.

Nevertheless, the author highlights that the diagnostic characters for genera delimitation are too fragile and non-exclusive.

According to Wanderley et al. (2007) the challenges to the delimitation of genera within the Nidularioid complex reflects the intimate relationship between them. The authors also highlight that when analyzing different species described in Canistrum, Wittrockia and

Edmundoa it is possible to notice a continuum of the characters considered as diagnostic, not supporting a separation of these genera. Leme (1997) further highlights that, although

Canistrum is assigned to the Nidularioid complex due to its superior involucral peduncle bracts and primary bracts, this genus is morphologically more similar to Aechmea. Among these similarities, the presence of asymmetrical sepals with membranous lateral wing and the presence of a pungent apex in its sepals are worth mentioning. This positioning can be verified in phylogenies of the subfamily Bromelioideae where species of Canistrum often emerge along with species of Aechmea in clades closely related to the Nidularioid clade (Silvestro et al., 2014,

Heller et al., 2015).

Phylogenetic studies indicate that genera belonging to the Nidularioid complex are artificial (Silvestro et al., 2014; Evans et al., 2015). However these genera always emerge related to one another (with the exception of Canistrum) (Silvestro et al., 2014; Evans et al.,

2015; Heller et al., 2015), which suggests that they are a true taxonomic entity that is commonly referred to as “Nidularioid Clade” or “Nidularioid Complex”.

Morphological and anatomical studies have shown to be useful in the delimitation of taxonomic groups in Bromeliaceae (Hornung-Leoni and Sosa, 2008; Gomes-da-Silva et al.,

2012), such as the presence of spiraled petals that are twined after anthesis and persistent in the

Capítulo I Oliveira, F.M.C. apex of Puya fruits (Hornung-Leoni and Sosa, 2008); the presence of clorophilian parenchyma cells elongated anticlinally, and the presence of a single-layered mechanical hypoderm in the abaxial portion in the Vriesia corcovadensis group (Gomes-da-Silva et al., 2012), the presence of mechanical hypoderm with intense wall thickening, and the presence of aquiferous hypoderm in the abaxial and adaxial portions of the leaf in the clade enclosed by Encholirium + Dyckia

(Santos-Silva et al., 2013).

In this sense this study examines possible morphoanatomical synapomorphies of the

Nidularioid complex and its subclades, allowing new perspectives for studies in the phylogenetic relationships within this group.

2. Materials and methods

2.1 Taxon sampling -

We sampled fifteen species belonging to the genera that compose the Nidularioid complex sensu Leme (1997) for the phylogenetic and morphoanatomical analyses (table 1 and

2). Acanthostachys strobilacea (Schult. f.) Link, Klotzsch & Otto was included as external group.

2.2 Sequence alignment and phylogenetic analyses

The chloroplast gene sequences of atpB-rbcL (797 pb), matK (1747 pb), trnL-trnF (796 pb) and nuclear PhyC (1212 pb) were obtained in genBank's NCBI portal using the accession numbers provided by Silvestro et al. (2014) (Table 1), and were alligned using the software

Muscle (Edgar, 2004). The sequences were manually checked using Mesquite 3.01 (Maddison and Maddison, 2015).

We used the AIC (Akaike Information Criterion) (Akaike, 1973) to select the best evolutionary model for each sequence, being GTR +G+I the best applicable. For the bayesian

Capítulo I Oliveira, F.M.C. analysis we built a unique, concatenated matrix, and each partition was named. The best evolutionary model (GTR +G+I) was applied to each partition of the matrix. Bayesian analysis were run in Mr. Bayes 3.2.6 (Ronquist et al., 2012) on the Cipres Gateway online server

(http://www.phylo.org) (Miller et al., 2011). We run the MCMC (Markov Chain Monte Carlo) chain for 10,000,000 generations sampling every 1000th generation. The resulting phylogenies were summarized in a consensus tree after removing the first 500,000 generations as burnin

(5%) (Appendix I)

2.3 Morphological and anatomical data

We obtained the morphological data from samples fixed in ethanol, as well as from the analysis of exsiccatae (SP, SPF, HUPG, MBM). For the delimitation of anatomical characters, we used three individuals from each species from which we sampled three fully expanded leaves. The samples were fixed in FAA 50 (Johansen, 1940) and stored in ethanol 70º. We prepared anatomical slides using longitudinal and transversal sections of the medial third of the leaf sheath and blade, which were stained using Astra Blue and Fuchsin (Kraus and Arduin,

1997) and mounted in glicerinated agar (Kaiser, 1880 apud Kraus and Arduin, 1997).

We used Scanning Electron Microscopy (SEM) for the leaf surface analysis. The samples were dehidrated in a gradual ethilical series up to absolute ethanol, submitted to critical

CO2 point, and lastly submitted to vacuum gold metalization. After the metalization the samples were analysed in the Scanning Electron Microscope SSV-550 (Silveira, 1989).

2.4 Ancestral state reconstruction

We used two methods for ancestral state reconstruction: the first using parsimony in

Mesquite (Maddison and Maddison, 2015), and the second using bayesian analysis using RASP

(Yu et al., 2012). Both analyses were run using the consensus tree (the same topology proposed by Silvestro et al., 2014) and the characters were treated as unordered.

Capítulo I Oliveira, F.M.C.

The 10 MCMC chains for the bayesian analysis in RASP were run for 50000 generations, sampled every 100th generation, and the first 100 generations were discarded as burnin (Yu et al., 2012).

3. Results

The Bayesian tree obtained with Mr. Bayes are available in appendix I. In total 87 characters were described (Appendix II and III). From those 20 characters were selected for ancestral state reconstruction, being four of them regarding the morphology of reproductive structures commonly used for the delimitation of the Nidularioid complex characters, and 16 leaf anatomical characters, which are described below. All character states described in the leaf anatomy are presented in Figures 1 and 2.

Character 1: Inflorescence, type: (0) simple; (1) compound. Character 2: Flowers: (0) sessile; (1) pedicellate. Character 3: Petal appendages: (0) absent; (1) present. Character 4:

Petal, longitudinal callosities: (0) absent; (1) present. Character 5: Leaf sheath, abaxial surface, contour: (0) smooth to lightly wavy (Fig.1B); (1) furrowed (Fig. 1A). Character 6: Leaf sheath, cell wall thickening of abaxial mechanical hypodermal cells: (0) absent (Fig. 1C); (1) present

(Fig. 1D). Character 7: Leaf sheath, number of layers of adaxial mechanical hypodermal cells:

(0) one (Fig. 1E); (1) more than one (Fig.1F); Character 8: Leaf sheath, number of layers of abaxial mechanical hypodermal cells: (0) one (Fig. 1C); (1) more than one (Fig. 1D); Character

9: Leaf sheath, bigger vascular bundle, pericyclic fibers: (0) in caps (Fig 1G); (1) rounded all bundle (Fig. 1H). Character 10: Leaf sheath, cells of brachiform parenchyma: (0) with short arms (Fig. 1I); (1) with long arms (Fig. 1J). Character 11: Leaf sheath, brachiform parenchyma, raphides: (0) absent (Fig. 1I); (1) present (Fig. 1J). Character 12: Leaf sheath, water storage parenchyma: (0) absent (Fig. 1A); (1) present (Fig. 1B). Character 13: Leaf sheath, wing’s cells of trichome: (0) rounded (Fig. 1K); (1) elongated (Fig. 1L and M).

Capítulo I Oliveira, F.M.C.

Character 14: Leaf blade, adaxial epidermal cells, thickening in leaf cross section: (0) periclinal wall strongly thick (Fig. 2A); (1) periclinal wall slightly thick (Fig. 2B). Character

15: Leaf blade, subestomatic camara occlusion: (0) absent (Fig. 2D); (1) present (Fig 2C).

Character 16: Leaf blade, cell wall thickening of abaxial mechanical hypodermal cells: (0) absent (Fig. 2E); (1) present (Fig 2F). Charcter 17: Leaf blade, number of layers of abaxial mechanical hypodermal cells: (0) one (Fig. 2E); (1) more than one (Fig. 2F); Character 18:

Leaf blade, cells of brachiform parenchyma: (0) with short arms (Fig. 2G); (1) with long arms

(Fig. 2H). Character 19: Leaf blade, brachiform parenchyma, raphides: (0) absent (Fig. 2H);

(1) present (Fig 2G). Character 20: Leaf blade, water storage parenchyma: (0) absent (Fig. 2J);

(1) present (Fig. 2I).

Both methods for ancestral state reconstruction yielded similar results for the 20 studied characters. Most characters used have shown to be homoplastic. However, synapomorphies are discussed throughout this paper (figures 3-7).

4. Discussion

4.1 Morphological characters

The presence of simple inflorescence is the main character that differentiates Neoregelia from all other genera that compose the Nidularioid complex. The most recent common ancestor to all species in this complex probably had compound inflorescence, which is considered a synapomorphy for the group (99% probability, figure 3, character 1). However, compound inflorescences are also seen in Neoregelia subg. Hylaeaicum, a subgenus with amazonic distribution (Ramirez, 2000), as seen in Neo. eleuteropetala, constituting an autapomorphy for this taxon.

The presence of sessile or pedicelled flowers is a broadly used character for the delimitation of genera in Bromelioideae, with the most recent common ancestor to the

Capítulo I Oliveira, F.M.C.

Nidularioid complex clade having most likely sessile flowers (ca. 70% probability - figure 3 character 2). The genus Neoregelia is characterized by having pedicellated flowers (Smith and

Downs, 1979), although this is not a character exclusive of the clade to which they belong, but also shared with its sister clade formed by W. superba and N. procerum and by species of

Canistropsis. Despite the fact that the presence of subsessile flowers is a diagnostic character in Canistropsis (Leme, 1998), in this study this type of flower was considered pedicellated. For reconstruction only two states were considered for this character: pedicellated or sessile flowers. This means that we considered as pedicelled flowers with a short pedicel, like the flowers from Canistropsis.

The petal appendages are frequently used in genera delimitation in Bromeliaceae (Smith and Downs, 1979), and were considered as the most important taxonomic characters in the subfamilies Tillandsioideae and Bromelioideae for a long period (Brown and Terry, 1992). The same authors argue that this character should be reevaluated for the usage in generic circumscription, since it shows late development being the last structure to be formed in the flower. The same observations were made by Oliveira et al. (2016) when analyzing completely formed floral buds from Aechmea distichantha that did not yet have those structures in its petals.

Schult and Zizka (2008) reconstructed the ancestral state of presence/absence of petal appendages for the subfamily Bromelioideae, and concluded that this character is homopolastic, and should not be used in genera delimitation in Bromelioideae. The same pattern is observed when reconstructing this character for the Nidularioid clade, where the common ancestor probably had petals without appendices (57% probability) and where the presence of petal appendages have evolved at least three times (figure 3, character 3). It is also possible to notice that, despite Neoregelia being characterized by having petals without these structures, Neo. eleutheropetala (that belongs to the subgenus Hylaeaicum) does have appendages in its petals, corroborating the study by Ramirez (1994). This author describes the presence of appendages

Capítulo I Oliveira, F.M.C. in nine species of this subgenus, also questioning the use of this character for genus dellimitaion, especially in Neoregelia.

Leme (1997, 1998, 2000) used the presence of longitudinal callosities in the petals to differentiate the genera in the Nidularioid complex. According to the author, Wittrockia,

Canistropsis, and Edmundoa are characterized by having these callosities. This character was also shown to be homoplastic, with its absence emerging at least twice in the Nidularioid clade: once in the most recent common ancestor of the Neoregelia genus and another in W. cyathiformis (Figure 3, character 4).

4.2 Anatomical characters

The presence of peltate absorbant trichomes (scales) is a synapomorphy of

Bromeliaceae, being one of the traits that allowed the adaptive radiation of this family (Givnish et al. 2014; Males, 2016). Since the absorption of water and nutrients is performed by the peltate trichomes, the roots began to have a key role in the anchorage of epiphytic bromeliads (Benzing,

2000). In addition to water and nutrient absorption, the trichomes also lower evapotranspiration through its wings that cover stomata (Benzing, 1976; Benzing et al. 1976). The peltate trichome is composed by a peduncle that connects the trichome to the surrounding epidermal cells and to the mesophyll, and by a wing that is subdivided into peripheral wing and central cells of the disc (Strehl, 1983). The frontal view organization of cells is different among subfamilies, varying from simple in Pitcairnioideae to extremely complex as in Tillandsioideae (Tomlinson,

1969; Benzing, 1976). In the Pitcairnioideae there is no clear distinction between the wing and the central cells of the disc. In Tillandsioideae the trichomes are well organized and symmetrical in frontal view, allowing the four central cells of the disc, the eight pericentral cells and one 32-

64 celled wing to be clearly distinguished. In Bromelioideae the frontal view organization of trichomes is simple, consisting of four central cells of the disc and the wing. However, the distinction between these two regions can be inconspicuous (Tomlinson, 1969; Strehl, 1983).

Capítulo I Oliveira, F.M.C.

The wing is usually formed by dead cells (Benzing, 1976), yet with living cells being reported for Brocchinia reducta (Benzing et al., 1976) and for Vrisea platynema (Kowalski et al., 2016).

The peltate trichomes of species belonging to the Nidularioid clade are simple, although with some organization, which means that it is possible to distinguish the wing from the central cells. It is also possible to notice that in these species the wing cells are elongated, differing from the trichome wing cells present in species from the Nidularioid complex's sister group.

Thus this trait is likely a synapomorphy for the Nidularioid clade (ca. 99% probability, Figure

4, character 5). In the sister group the trichome wing cells are round and show no distinction between disc and wing cells.

In atmospheric species trichomes are present along the foliar surface, whereas in tank- forming species these trichomes tend to occur concentrated in the leaf sheath (Benzing, 1976;

Benzing et al., 1978). In juvenile, non-tank-forming phases of heteroblastic species, trichomes are distributed along the leaf. In the adult phase, when the tank is already formed, the trichomes are concentrated mainly in the leaf sheath (Adams and Martin, 1986). Since in tank-forming species water and nutrients accumulate in the leaf sheath, the high density of trichomes in this foliar region facilitate the absorption of water and nutrients (Benzing, 1970, 1976). This scenario of a leaf sheath fully covered by trichomes a leaf blade with fewer of these structures is observed in all analyzed species.

The presence of a “U-shaped” thickening in the anticlinal and periclinal internal walls of epidermal cells is one of the most striking traits of Bromeliaceae (Krauss, 1949; Tomlinson,

1969, Ayoama and Sajo, 2003; Proença and Sajo, 2007; Monteiro et al., 2011; Faria et al., 2012;

Santos-Silva et al., 2013; Saraiva et al., 2015). Along with the thickening of the cells of the mechanical hypoderm, this U-shaped thickening lowers water evaporation from the tissues, avoiding cell collapse by shrivelling (Scatena and Segecin, 2005). The most recent common ancestor of the Nidularioid clade likely had adaxial epidermal cells with lightly thickened walls

Capítulo I Oliveira, F.M.C. in the leaf blade, being considered an anatomical synapomorphy of the clade (83% probability).

The presence of cells with highly thickened walls represent a reversion for the clade formed by

W. superba and N. procerum, as well as for C. microps and Neo.binotii (Figure 4, character 6).

Stomata in all species are inserted at the same level as other epidermal sells, with their substomatal chamber occluded. Stomata with these occlusions are common in Bromeliaceae, being registered in the subfamily Bromelioideae in Aechmea (Ayoama and Sajo, 2003) and

Quesnelia (Almeida et al., 2009). This occlusion is due to the presence of hypodermal cells

(substomatal ring) that point towards the interior of the chamber. This allows stomata to be kept open even when the leaf is flacid, ensuring mechanical support (Krauss, 1949). According to

Benzing (2000) this stomata type is prevalent in the Bromelioideae subfamily, with this arrangement (hypodermal cells occluding the substomatal chamber) considered the most complex in the family. Among all species of the Nidularioid complex only C. microps, E. perplexa and Neo. binotii do not present substomatal chambers occluded by hypodermal cells, with this condition being homoplastic for these taxa (Figure 4, character 7). If we consider that taxa belonging to the Nidularioid complex are epiphytic species in humid environments, this trait can be attributed to the genetic variability rather than to an adaptation to the environment.

The contour of the leaf surface may be sulcated due to the insertion of the trichome pedicles and to the presence of stomata. This character has already been used for species distinction by Tomlinson (1969) and in the characterization of the genus Bromelia by Monteiro et al. (2011). The most recent common ancestor likely had leaf sheath with smooth abaxial surface, and the sulcated surface is homoplastic for C. billbergioides, Neo. eleutheropetala and

Neo. binotii. Thus the common ancestor of the Nidularioid clade has 99% probability of having the abaxial surface of the leaf sheath lightly sulcated (Figure 4, character 8).

Several authors described the presence of a differentiated hypoderm in Bromeliaceae

(Krauss, 1949; Tomlinsom, 1969; Ayoama and Sajo, 2003; Scatena and Segecin, 2005; Sousa

Capítulo I Oliveira, F.M.C. et al., 2005; Proença and Sajo, 2007; Monteiro et al., 2011). This hypoderm may or may not have cells with thickened and lignified cell walls.

Species belonging to the Nidularioid clade show, in its majority, abaxial mechanical hypodermal cells without thickening in the leaf sheath, with this condition likely being the condition of the most common recent ancestor of these taxa (ca. 72% probability). Within the

Nidularioid clade the presence of hypoderm with thickened cells can be considered a homoplasy for the clade formed by W. superba and N. procerum and for Neo. eleutheropetala and C. billbergioides (figure 5, character 9). In contrast, the prevalent character state for the leaf blade is the presence of cells of the abaxial mechanical hypoderm with wall thickening. This is probably also the condition of the most recent common ancestor to the Nidularioid clade (ca.

99% probability), with the absence of this thickening being considered a homoplasy for Neo. laevis and C. microps (figure 5, character 10).

Besides having cells with thickened walls, the mechanical hypoderm can be uni- or multisseriated (Tomlinson, 1969). The only species that shows more than one layer of cells in the adaxial face of the leaf sheath is Wittrockia superba, which is an autapomorphy in relation to the Nidularioid clade (figure 5 character 11). A multi-layered abaxial hypoderm of the leaf sheath is a homoplastic character for W. superba and Neo. eleutheropetala (figure 5 character

12). Regarding the leaf blade the common ancestor for the Nidularioid clade shows a unisseriated hypoderm on the abaxial face (ca. 97% probability), and the presence of more than one layer is a homoplastic character for the clade formed by W. cyathiformis and E. lindenii, and for E. perplexa and W. superba (figure 6 character 13).

In addition to providing mechanical support, the hypoderm can also function on the lowering of water evaporation through the leaf surface (Krauss, 1949; Tomlinson 1969).

According to Monteiro et al. (2011) the presence of the hypoderm is not necessarily related to

Capítulo I Oliveira, F.M.C. water availability, since species from humid environments can also show lignified mechanical hypoderm, as it was observed in the present study.

Another common character in Bromeliaceae is the occurrence of extensions of the vascular bundle sheath, which can fully circle the bundle or can form calottes (Krauss, 1949;

Tomlinson, 1969; Scatena and Segecin, 2005; Sousa et al., 2005; Proença and Sajo, 2007;

Versieux et al., 2010; Faria et al., 2012; Santos-Silva et al., 2013). According to our results, the most recent common ancestor of the Nidularioid complex probably had fibers surrounding completly the larger vascular bundles in the leaf sheath region (ca. 55% probability). However, the pericyclic fibers arranged in caps constitute a reversion for the clade formed by W. superba,

N. procerum, C. microps, C. billbergioides and Neo. laevis (figure 6 character 14).

In the studied species, the brachiform parenchyma belongs to a system of aeration channels that occurs longitudinally through all of the leaf, which in Bromeliaceae has the function of gas accumulation. This tissue also acts as gas exchange regulators helping the foliar ventilation, especially in the basal portion of the leaf (leaf sheath) where there is no gas exchange trhough the epidermis due to water accumulation in the foliar rosette. All studied species accumulate water in their tanks, and the presence of aeration channels is a key adaptation for the gas exchange to occur in the leaf sheath of these species, representing an adaptation for the mesic environment. Regarding cell shape, Santos-Silva et al. (2013) argue that species with Crassulacean Acid Metabolism (CAM) usually have intercellular spaces in the larger aeration channels. Species with CAM reduce evapotranspiration by opening their stomata only at night (Lutge et al., 1983; Crayan et al., 2004). Thus, the aeration channels may also assure that all other mesophyll cells exchange gases even when stomata are closed. This is corroborated by the studied species, since all of them have CAM (Silvestro et al., 2014) and larger cellular spaces in their aeration channels.

Capítulo I Oliveira, F.M.C.

The most recent common ancestor to the Nidularioid clade showed cells with long arms in the leaf sheath region (ca. 93% probability - figure 6 character 15). Within the Nidularioid clade only species from the Neoregelia genus possess brachiform cells with short arms, and this is a reversion for the clade. For the leaf blade region, ancestral state reconstruction shows that the common ancestor to the Nidularioid clade had brachiform parenchyma cells with short arms

(ca. 99% probability). Long-armed brachiform parenchyma cells can be considered homoplastic to W. cyathiformis, Neo. laevis and W. superba (figure 6 character 16).

Despite the presence of raphides being common in Bromeliaceae (Krauss, 1949;

Tomlinson, 1969; Prychid and Rudall, 1999; Sousa et al., 2005; Proença and Sajo, 2007;

Monteiro et al., 2011), this character also has taxonomic importance by distinguishing species belonging to the genera Aechmea and Bromelia (Monteiro et al., 2011; Ayoama and Sajo, 2003;

Sousa et al., 2005). Crystals of calcium oxalate can be related to calcium regulation, detoxification, protection against herbivory, and even light reflection (Franceschi and Horner,

1980; Nakata, 2003; Franceschi and Nakata, 2005; Nakata 2012; Doege and Korth, 2003).

The most recent common ancestor to the Nidularioid clade did not have idioblasts containing raphides in the brachiform parenchyma neither in the leaf sheath region (ca 97% probability) nor in the leaf blade region (ca. 99% probability). The presence if raphides in the brachiform parenchyma of the leaf sheath is a synapomorphic character in the clade formed by

W. cyathiformis and E. lindenii, and homoplastic for E. ambigua and W. superba (figure 7 character 17). Conversely, the presence of raphides in the brachiform parenchyma of the leaf blade can be considered a homoplasy for the taxa E. lindenii, C. microps and Neo. binotii (figure

7 character 18).

During eventual or seasonal dry periods, the tank-forming epiphytic Bromeliaceae species have two important water resources: the tank itself (phytotelm) and the aquiferous parenchyma (Freschi et al., 2010) that is responsible for the succulency of the leaves in this

Capítulo I Oliveira, F.M.C. family (Krauss, 1949; Tomlinson, 1969; Benzing, 2000; Givnish et al., 2004; Monteiro et al.,

2011; Ayoama and Sajo, 2003). The aquiferous parenchyma can be considered therefore a key innovation for the survival of individuals during times of drought (Freschi et al. 2010), since the water that accumulates in the tank is available only for less than a week in natural conditions.

According to Givnish et al. (2004), the combination of the presence of water-accumulating tissue and the CAM metabolism provides a way of reducing transpirations when in dry periods, although with an associated cost for the photosynthetic capacity.

Despite the presence of aquiferous tissue being a common character in Bromeliaceae, its terminology it is still controversial. Some authors name as aquiferous hypoderm the layer that underlies the mechanical hypoderm, which accumulates water and has anticlinally elongated, thin-walled cells that have corrugated anticlinal walls in transversal sections

(Tomlinson, 1969; Sousa et al., 2005; Scatena and Segecin, 2005; Proença and Sajo, 2007;

Versieux et al. 2010). Nevertheless, according to Krauss (1949) and Monteiro et al. (2011), this tissue is described only as aquiferous parenchyma without referring to its hypodermal origin, since ontogenetic studies to determine this origin for the aquiferous parenchyma are still to be done.

Fahn and Cutler (1992) define succulent plants as plants that are tolerant to drought, and that have the ability to store water in its tissues, which is easily mobilized and used to maintain the processes essential to life. Fahn (1974) affirms that in succulent plants such as the

Bromeliaceae the aquiferous parenchyma is responsible for the leaf succulence. This parenchyma is characterized by having large, thin-walled cells without chloroplasts. In the studied species we opted to use the term "aquiferous parenchyma" since we did not study the foliar ontogeny for these species. Therefore we only considered as aquiferous parenchyma the tissue whose cells had the following characteristics: lack of chloroplasts, thin cell walls, and anticlinal elongation in transversal or longitudinal sections. When comparing this character in

Capítulo I Oliveira, F.M.C. the leaf sheath and leaf blade, we observe that the occurrence of aquiferous parenchyma is more frequent in the leaf blade, and is absent in this region only in E. perplexa and E. ambigua. This constitutes a synapomorphy for this clade (figure 7 character 20). In the leaf sheath, the aquifererous parenchyma is present in W. superba, W. cyathiformis, C. billbergioides, and Q. arvensis, being considered a homoplastic character by parallelism (figure 7 character 19).

According to Monteiro et al. (2011) the cells from the water-storage tissue in species from the genus Bromelia have thin cellulosic walls, and its cellular content usually consists of mucilaginous substances. These mucilaginous substances keep water inside the cells, as well as having a role in avoiding its loss during dry periods. Furthermore those substances are responsible for rapidly absorbing water once it is available in the environment (Krauss, 1949).

Krauss (1949) still reinforces that this tissue can absorb heat, lowering the light incidence in the chlorophyllian tissue, which usually is located in the abaxial portion of the mesophyll.

Given that all studied species show tanks in their bases, and that the highest concentration of peltate absorbent trichomes is located in the same region, we can infer that the water that is absorbed in this region is likely accumulated in the aquiferous hypoderm of the leaf blade. Thus, this could be a possible explanation for the lack of this aquiferous hypoderm in the leaf sheath in the majority of studied species.

5. Conclusions

In this study we could observe through ancestral state reconstruction that the morphological characters typically used for genera delimitation in Bromelioideae, such as inflorescence type, presence of pedicellate flowers, presence of petal appendages, and presence of longitudinal callosities in the petal are homoplastic and should be avoided in the circumscription of the genera from the Nidularioid complex.

Capítulo I Oliveira, F.M.C.

The ancestral state reconstruction for the anatomical characters of the leaf sheath and leaf blade is useful, being the presence of peltate absorbent trichomes with elongated wing cells a new synapomorphy for the Nidularioid clade, as well as the presence of cells with lightly thickened walls in the adaxial epidermis in the leaf blade.

For the subclades of the Nidularioid complex, we suggest some anatomical synapomorphies such as the presence of raphides in the brachiform parenchyma from the leaf sheath for the clade W. cyathiformis and E. lindenii; brachiform parenchyma in the leaf sheath composed by short-branched cells in the clade Neoregelia.

In summary, studies on character evolution in Bromelioideae are still scarce, especially the ones that analyze foliar anatomical characters. In that sense the present study proposes leaf anatomical synapomorphies for the Nidularioid complex, which can help future studies on the evolution of this group, as well as for the subfamily Bromelioideae.

6. Acknowledgements

The authors thank Elton M. C. Leme for providing the plant material for this research. The authors also acknowledge the National Counsel of Technological and Scientific Development

(CNPq - process number 140115/2013-7 and 308070/2012-7) for the funding.

Capítulo I Oliveira, F.M.C.

Table 1: List of numbers accessed on GenBank (NCBI portal) to download the sequences used on this research.

atpB-rbcL partial trnL / trnL-trnF Species author matK gene phyC gene spacer spacer Acanthostachys pitcairnioides (Mez) Rauh & Barthlott JX649328 JX649495 / JX649285 JX649231 JX649382 Aechmea distichantha Lemaire EU219714 DQ084579 AY950041 JX649388 Aechmea turbinocalyx Mez JX649340 JX649505 / JX649294 JX649240 JX649407 Canistropsis billbergioides (Schultes f.) Leme JX649349 JX649512 / JX649300 JX649248 JX649424 Canistropsis microps (E. Morren ex Mez) Leme JX649350 JX649513 / JX649301 JX649230 JX649425 (Wanderley & Leme) Edmundoa ambigua Leme JX649359 JX649521 / JX649309 JX649257 JX649439 Edmundoa lindenii (Regel) Leme EU219685 DQ084631 AY950012 JX649440 Edmundoa perplexa (L.B. Smith) Leme JX649360 JX649522 / JX649310 JX649258 JX649441 Neoregelia binotii (Antoine) L.B. Smith EU219682 DQ084613 AY950009 JX649459 Neoregelia laevis (Mez) L.B. Smith EU219681 DQ084612 AY950008 JX649460 Nidularium procerum Lindman EU219686 DQ084628 AY950013 JX649461 Neoregelia eleutheropetala. (Ule) L.B.Sm EU219710 DQ084592 AY950037 JX649403 Quesnelia arvensis (Vellozo) Mez EU780886 EU780874 EU780850 - Quesnelia quesneliana (Brongniart) L.B. Smith JX649377 JX649537 / JX649325 JX649274 JX649482 Wittrockia cyathiformis (Vellozo) Leme JX649381 JX649494 / JX649284 JX649279 JX649428 Wittrockia superba Lindman EU219698 DQ084611 AY950025 JX649489

Table 2: List of vouchers for taxa used for anatomical analyses.

Specie Number of collector or herbarium number Aechmea disticantha Lem. Leme 8.146 (HB), S.N.A.Miyamoto s/n (HUPG) Aechmea turbinocalyx Mez Leme 7.053 (HB) Quesnelia arvensis (Vell.) Mez Leme s/n (HB), F.M.C. Oliveira 39 (HUPG) Quesnelia quesneliana (Brong.) L. B. Sm. Leme s/n (HB), F.M.C. Oliveira 38 (HUPG) Nidularium procerum Lindm. Leme 990 (HB) Wittrockia superba Lindm. Leme 4.751(HB) Neoregelia laevis (Mez) L.B.Sm. Leme 4.752 (HB) Neoregelia binotii (Mez) L.B.Sm. Leme 3.482 (HB) Neoregelia eleutheropetala (Ule) L.B.Sm. Leme 1.976 (HB) Edmundoa ambígua (Wand &Leme) Leme Leme 756 (HB) Edmundoa perplexa (L.B.Sm) Leme Leme 2.956 (HB) Canistropsis billbergioides (Schult. f.) Leme Leme 4.918 (HB) Canistropsis microps (E. Morren ex Mez) Leme Leme 759 (HB) Edmundoa lindenii (Regel) Leme Leme 1.307 (HB) Wittrockia cyathiformis (Vell.) Mez Leme 6.411 (HB)

Capítulo I Oliveira, F.M.C.

Figure 1: A-J: Cross section of leaf sheath. A: Neoregelia binotii (Mez) L.B.Sm.; B: Wittrockia superba Lindm.; C: Detail of abaxial epidermis in Edumundoa lindenii (Regel) Leme; D: Detail of abaxial epidermis in W. superba; E: Detail of adaxial epidermis in Nidularium procerum Lindm; F: Detail of adaxial epidermis in W. superba; G: Detail of vascular bundle in E. lindenii; H: Detail of vascular bundle in W. superba; I: Detail of armed parenchyma in Neoregelia binotii; J:Detail of armed parenchyma in W. superba. K-M: Scanning electron microscopy of leaf sheath. K: Detail of peltate trichome of Quesnelia arvensis (Vell.) Mez; L: Detail of peltate trichome in Canistropsis billbergioides (Shult. f.) Leme; M: Detail of peltate trichome in Wittrockia cythiformis (Vell.) Leme.

Capítulo I Oliveira, F.M.C.

Figure 2: A-K: Cross section of leaf blade. A: Detail of adaxial epidermis in Wittrockia superba Lindm.;B: Detail of adaxial epidermis in Edmundoa lindenii (Regel) Leme; C: Detail of abaxial epidermis in showing a stomata with occluded substomatic camara in W. superba; D: Detail of abaxial epidermis showing a stomata without occlusion in substomatic camara in Canistropsis microps (E. Morren ex Mez) Leme; E: Detail of abaxial hypodermis in Neoregelia laevis (Mez)L.B.Sm.; F: Detail of abaxial hypodermis in W. superba. G: Detail of brachiform parenchyma in E. lindenii; H: Detail of brachiform parenchyma in W. superba; I: Detail of brachiform parenchyma in Neoregelia binotii; J: Detail of Edmundoa perplexa (L.B.Sm.) Leme. K: Detail of W. superba.

Capítulo I Oliveira, F.M.C.

Figure 3: Morphological ancestral character reconstruction. Lines represent the reconstruction by parcimony method. Graphics represent the reconstruction by baeysian method. Graphics also show the probability of ancestral reconstruction. Character 1: Inflorescense; Character 2: Flowers; Character 3: Petal appendages; Character 4: Petal longitudinal callosities.

Capítulo I Oliveira, F.M.C.

Figure 4: Anatomical ancestral character reconstruction. Lines represent the reconstruction by parcimony method. Graphics represent the reconstruction by baeysian method. Graphics also show the probability of ancestral reconstruction. Character 5: Trichome’s wing cells; Character 6: Leaf blade, periclinal thickening of abaxial epidermal cells; Character 7: Leaf blade, occlusion of subestomatal chamber; Character 8: Leaf sheath, abaxial surface contour.

Capítulo I Oliveira, F.M.C.

Figure 5: Anatomical ancestral character reconstruction. Lines represent the reconstruction by parcimony method. Graphics represent the reconstruction by baeysian method. Graphics also show the probability of ancestral reconstruction. Character 9: Leaf sheat, abaxial hypodermis thickening; Character 10: Leaf blade, thickening of abaxial mecanical hypodermis cell walls; Character 11: Leaf sheat, number of layers of adaxial mecanical hypodermis cells; Character 12: Leaf sheat, number of layers of abaxial mecanical hypodermis cells.

Capítulo I Oliveira, F.M.C.

Figure 6: Anatomical ancestral character reconstruction. Lines represent the reconstruction by parcimony method. Graphics represent the reconstruction by baeysian method. Graphics also show the probability of ancestral reconstruction. Character 13: Leaf blade, number of layers of abaxial mecanical hypodermis cells; Character 14: Leaf sheath, larger vascular bundle, fibers; Character 15: Leaf sheath, brachiform parenchyma, cell shape; Character 16: Leaf blade, brachiform parenchyma, cell shape.

Capítulo I Oliveira, F.M.C.

Figure 7: Anatomical ancestral character reconstruction. Lines represent the reconstruction by parcimony method. Graphics represent the reconstruction by baeysian method. Graphics also show the probability of ancestral reconstruction. Character 17: Leaf sheath, brachiform parenchyma, raphids; Character 18: Leaf blade, brachiform parenchyma, raphids; Character 19: Leaf sheath, water storage tissue; Character 20: Leaf blade, water storage tissue.

Capítulo I Oliveira, F.M.C.

Appendix I: Tree 1: Consensus tree of Bayesian analysis of combined nuclear and plastid data (atpB-rbcL, matK, trnL-trnF and PhyC). Numbers on the branches indicate bootstrap percentage.

Capítulo I Oliveira, F.M.C.

Appendix II: Character delimited

Leaf morphology

1. Leaf, apex, pungent projection: (0) absent, (1) presente;

2. Leaf, spines, orientation: (0) one orientation, antrose; (1) one orientation, retrose; (2)

two orientations, retrose and antrose at same leaf.

3. Leaf, spines, color: (0) brown; (1) green.

4. Leaf, transition between leaf sheath and leaf blade: (0) abrupt; (1) tenuous.

5. Leaf, foliar texture: (0) thin to moderate thin; (1) sub-rigid to leathery.

6. Leaf, concolor leaf at anthesis: (0) absent; (1) present.

7. Leaf, sheath with purple or brown coloration: (0) absent; (1) present;

8. Leaf, apex: (0) acuminate; (1) acute; (2) rounded.

9. Leaf, length: of the spines: (0) 0,5-1mm; (1)1,1-5mm; (2) bigger than 5mm.

Peduncle morphology

10. Peduncle, length: (0) exceeding the leaf sheath lenght; (1) smaller than leaf sheath

length;

11. Peduncle, indumenta: (0) absent; (1) present;

Morphology of peduncle bracts

12. Peduncle bracts, morphology: (0) equal; (1) dimorphic;

13. Peduncle bracts, shape: (0) oval; (1) oblong; (2) elliptic; (3) lanceolate.

14. Peduncle bracts, apex: (0) attenuate; (1) acute; (2) apiculate.

15. Peduncle bracts, apex projection: (0) absent; (1) present.

16. Peduncle bracts, margin: (0) entire; (1) spinose.

17. Peduncle bracts, disposition: (0) lax; (1) imbricate;

18. Peduncle bracts, indumenta: (0) absent; (1) present.

57 Capítulo I Oliveira, F.M.C.

Inflorescence

19. Inflorescence, type: (0) simple; (1) compound.

20. Inflorescence, indumenta: (0) absent; (1) present.

21. Inflorescence, length proportion comparing with the leaf sheath: (0) smaller than leaf

sheath; (1) bigger than leaf sheath.

22. Inflorescence, primary bracts: (0) absent; (1) present.

23. Inflorescence, primary bracts, shape: (0) oval; (1) oblong; (2) elliptic; (3) lanceolate.

24. Inflorescence, primary bracts, apex: (0) atemuate; (1) acute; (2) obtuse.

25. Inflorescence, primary bracts, apex projection: (0) absent; (1) present.

26. Inflorescence, primary bracts, indumenta: (0) absent; (1) present.

27. Inflorescence, primary bracts, margin: (0) entire, (1) spinose.

Morphology of floral bracts

28. Floral bracts, morphology: (0) carinate; (1) ecarinate.

29. Floral bracts, apex: (0) acute, (1) acuminate; (2) mucronate; (3) obtuse.

30. Floral bracts, hyaline sheath: (0) absent; (1) present.

31. Floral bracts, shape: (0) oval; (1) triangular; (2) oblong; (3) lanceolate.

32. Floral bracts, margin: (0) entire; (1) spinose;

33. Floral bract, apex projection: (0) absent; (1) present.

34. Floral bract, indumenta: (0) absent; (1) present;.

Flowers

35. Flowers: (0) sessile; (1) pedicellate.

Sepal morphology

36. Sepal, symmetry: (0) asymmetric; (1) symmetric.

58 Capítulo I Oliveira, F.M.C.

37. Sepal, concrescence: (0) absent; (1) present.

38. Sepal, morphology: (0) carinate; (1) ecarinate;

39. Sepal, apex morphology: (0) acute to sub-acute; (1) acuminate; (2) rounded; (3) obtuse

tp sub-obtuse.

40. Sepal, apex: (0) apiculate; (1) attenuate.

41. Sepals, indumenta: (0) absent; (1) present.

42. Sepal, shape: (0) oval; (1) oboval; (2) elliptic.

Petal morphology

43. Petal, concrescence: (0) absent (1) present.

44. Petal, apex: (0) acute to rounded; (1) acuminate; (2) sub-acute; (3) retuse.

45. Petal during the anthesis: (0) erect; (1) sub-erect;

46. Petal appendages: (0) absent; (1) present.

47. Petal, longitudinal callosities: (0) absent; (1) present.

48. Petal, shape: (0) linear; (1) spatulated; (2) lanceolate; (3) oblong; (4) oboval.

49. Petal, concrescence: (0) until 1/3 of its length; (1) more than1/3 of its length.

Filament morphology

50. Filament: (0) free; (1) one whorl adnate to petal;

Ovary morphology

51. Ovary, shape: (0) oblong; (1) elliptic; (2) trigonal; (3) oval; (4) clavate.

Leaf anatomy- the sheath

52. Leaf sheath, adaxial surface, contour: (0) smooth to lightly wavy; (1) furrowed.

53. Leaf sheath, abaxial surface, contour: (0) smooth to lightly wavy; (1) furrowed.

54. Leaf sheath, shape of adaxial epidermal cells: (0) square; (1) elongated.

59 Capítulo I Oliveira, F.M.C.

55. Leaf sheath, shape of abaxial epidermal cells: (0) square; (1) elongated.

56. Leaf sheath, adaxial epidermal cells, thickening in leaf cross section: (0) periclinal wall

strongly thick; (1) periclinal wall slightly thick.

57. Leaf sheath, abaxial epidermal cells, thickening in leaf cross section: (0) periclinal wall

strongly thick; (1) periclinal wall slightly thick.

58. Leaf sheath, cell wall thickening of adaxial mechanical hypodermal cells: (0) absent;

(1) present.

59. Leaf sheath, cell wall thickening of abaxial mechanical hypodermal cells: (0) absent;

(1) present.

60. Leaf sheath, cell wall thickening of adaxial mechanical hypodermal cells: (0) strongly

thick; (1) slightly thick.

61. Leaf sheath, cell wall thickening of abaxial mechanical hypodermal cells: (0) strongly

thick; (1) slightly thick.

62. Leaf sheath, number of layers of adaxial mechanical hypodermal cells: (0) one; (1) more

than one;

63. Leaf sheath, number of layers of abaxial mechanical hypodermal cells: (0) one; (1) more

than one;

64. Leaf sheath, smaller vascular bundle, shape of caps formed by the pericyclic fibers: (0)

higher than wide; (1) wider than high.

65. Leaf sheath, bigger vascular bundle, pericyclic fibers: (0) in caps; (1) rounded all

bundle.

66. Leaf sheath, cells of brachiform parenchyma: (0) with short arms; (1) with long arms.

67. Leaf sheath, brachiform parenchyma, raphides: (0) absent; (1) present.

68. Leaf sheath, water storage parenchyma: (0) absent; (1) present.

60 Capítulo I Oliveira, F.M.C.

Leaf anatomy- the blade

69. Leaf blade, adaxial surface, contour: (0) smooth to lightly wavy; (1) furrowed.

70. Leaf blade, abaxial surface, contour: (0) smooth to lightly wavy; (1) furrowed.

71. Leaf blade, shape of abaxial epidermal cells: (0) square; (1) elongated.

72. Leaf blade, adaxial epidermal cells, thickening in leaf cross section: (0) periclinal wall

strongly thick; (1) periclinal wall slightly thick.

73. Leaf blade, abaxial epidermal cells, thickening in leaf cross section: (0) periclinal wall

strongly thick; (1) periclinal wall slightly thick.

74. Leaf blade, wing’s cells of trichome: (0) elongated; (1) rounded.

75. Leaf blade, position of stomata in relation to others epidermal cells: (0) at same level;

(1) in a depression.

76. Leaf blade, subestomatic camara occlusion: (0) absent; (1) present.

77. Leaf blade, cell wall thickening of adaxial mechanical hypodermal cells: (0) absent; (1)

present.

78. Leaf blade, cell wall thickening of abaxial mechanical hypodermal cells: (0) absent; (1)

present.

79. Leaf blade, cell wall thickening of adaxial mechanical hypodermal cells: (0) strongly

thick; (1) slightly thick.

80. Leaf blade, cell wall thickening of abaxial mechanical hypodermal cells: (0) strongly

thick; (1) slightly thick.

81. Leaf blade, number of layers of adaxial mechanical hypodermal cells: (0) one; (1) more

than one;

82. Leaf blade, number of layers of abaxial mechanical hypodermal cells: (0) one; (1) more

than one;

61 Capítulo I Oliveira, F.M.C.

83. Leaf blade, smaller vascular bundle, projection of fibers to adaxial and abaxial sides:

(0) absent; (1) present.

84. Leaf blade, smaller vascular bundle, shape of caps formed by the pericyclic fibers: (0)

higher than wide; (1) wider than high.

85. Leaf blade, cells of brachiform parenchyma: (0) with short arms; (1) with long arms.

86. Leaf blade, brachiform parenchyma, raphides: (0) absent; (1) present.

87. Leaf blade, water storage parenchyma: (0) absent; (1) present.

62 Capítulo I Oliveira, F.M.C.

Appendix III: Characteres and their state of character

63 Capítulo I Oliveira, F.M.C.

Table 3a: Character and their states of character. 1: Leaf, apex, pungent projection: (0) absent, (1) present. 2: Leaf, spines, orientation: (0) one orientation, antrose; (1) one orientation, retrose; (2) two orientations, retrose and antrose at same leaf. 3: Leaf, spines, color: (0) brown; (1) green; 4: Leaf, transition between leaf sheath and leaf blade: (0) abrupt; (1) tenuous. 5: Leaf, foliar texture: (0) thin to moderate thin; (1) sub-rigid to leathery. 6: Leaf, concolor leaf at anthesis: (0) absent; (1) present. 7: Leaf, sheath with purple or brown coloration: (0) absent; (1) present. 8: Leaf, apex: (0) acuminate; (1) acute; (2) rounded. 9: Leaf, length: of the spines: (0) 0,5-1mm; (1)1,1-5mm; (2) bigger than 5mm. 10: Peduncle, length: (0) exceeding the leaf sheath lenght; (1) smaller than leaf sheath length; 11: Peduncle, indumenta: (0) absent; (1) present; 12: Peduncle bracts, morphology: (0) equal; (1) dimorphic;. 13: Peduncle bracts, shape: (0) oval; (1) oblong; (2) elliptic; (3) lanceolate; 14: Peduncle bracts, apex: (0) attenuate; (1) acute; (2) apiculate; 15: Peduncle bracts, apex projection: (0) absent; (1) present. 16: Peduncle bracts, margin: (0) entire; (1) spinose; 17: Peduncle bracts, disposition: (0) lax; (1) imbricate; 18: Peduncle bracts, indumenta: (0) absent; (1) present. 19: Inflorescence, type: (0) simple; (1) compound. 20: Inflorescence, indumenta: (0) absent; (1) present. 21: Inflorescence, length proportion comparing with the leaf sheath: (0) smaller than leaf sheath; (1) bigger than leaf sheath. 22: Inflorescence, primary bracts: (0) absent; (1) present. 23: Inflorescence, primary bracts, shape: (0) oval; (1) oblong; (2) elliptic; (3) lanceolate. 24: Inflorescence, primary bracts, apex: (0) atenuate; (1) acute, (2) obtuse. 25: Inflorescence, primary bracts, apex projection: (0) absent; (1) present. 26: Inflorescence, primary bracts, indumenta: (0) absent; (1) present. 27: Inflorescence, primary bracts, margin: (0) entire, (1) spinose. 28: Floral bracts, morphology: (0) carinate; (1) ecarinate. 29: Floral bracts, apex: (0) acute, (1) acuminate; (2) mucronate; (3) obtuse. 30: Floral bracts, hyaline sheath: (0) absent; (1) present.

64 Capítulo I Oliveira, F.M.C.

Species/ 1 2 3 4 5 6 7 8 9 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 characters 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 Wittrockia 1 0 0 0 1 0 0 0 1 1 0 0 0 2 0 1 1 1 1 0 0 1 0 2 0 1 1 0/ 0 0 superba 1 Aechmea 1 0 0 1 1 0 1 1 1 0 1 0 0/ 1 1 0/ 0 0 1 1 0 1 0 1 0 0 0 1 3 0 distichantha 3 1 Canistropsis 0 0 1 0 0 0 1 1 0 0 1 1 0 0 0 1 1 0 1 0 0 1 0 1 0 0 1 1 0 0 microps Canistropsis 0 0 1 0 0 0 1 0 0 0 1 1 3 0 0 1 1 0 1 0 0 1 3 1 0 0 1 0 0 0 billbergioides Edmundoa 1 0 0 0 1 0 1 1/ 0 0 1 0 0 1 0 1 ? 0 1 1 0 1 0 1 0 1 1 0/ 3 0 lindenii 2 1 Neoregelia 1 1 0 0 1 1 1 0 1 1 0 0 ? ? ? ? ? ? 0 0 0 0 - - - - - 1 1 0 binotii Neoregelia 1 2 0 0 1 1 1 1 2 1 0 0 ? ? ? ? ? 0 1 0 0 1 2 1 1 0 0 1 1 0 eleutheropetal a Quesnelia 1 0 0 1 1 0 1 1 1 0 1 0 0 1 1 1 1 1 0 0 0 0 - - - - - 1 0/ 0 arvensis 3 Neoregelia 0 0 0 0 1 1 1 2 0 1 1 0 2 2 1 0 1 0 0 0 0 0 - - - - - 1 2 1 laevis Edmundoa 1 0 1 0 1 0 1 1 0 0 1 1 2 2 1 1 1 1 1 1 0 1 1 1 1 1 1 1 0 0 perplexa Quesnelia 1 0 0 1 1 0 1 1 1 0 1 0 0 1 1 0/ 1 1 0 1 0 0 - - - - - 1 0/ 0 quesneliana 1 3 Aechmea 1 0 1 1 0 0 0 1 0 0 0 0 3 1 0 0 1 0 0 0 1 0 - - - - - 1 1 0 turbinocalyx Edmundoa 1 0 1 0 1 0 1 1 0 0 1 1 1/ 2 1 0 1 1 1 1 0 1 1 1 1 1 0 0 0/ 0 ambígua 2 3 Wittockia 1 0 0 1 1 0 0 0 1 0 0 1 ? ? 1 1 ? 0 1 0 0 1 0 1 1 0 1 0 1 0 cyathiformis Nidularium 1 0 1 0 0 0 1 ? 0 0/ 0 1 ? ? ? ? ? ? 1 0 0 1 3 0 0 0 1 1 0 1 procerum 1

65 Capítulo I Oliveira, F.M.C.

Table 3b: Character and their states of character: 31: Floral bracts, shape: (0) oval; (1) triangular; (2) oblong; (3) lanceolate. 32: Floral bracts, margin: (0) entire; (1) spinose; 33: Floral bract, apex projection: (0) absent; (1) present. 34: Floral bract, indumenta: (0) absent; (1) present. 35: Flowers: (0) sessile; (1) pedicellate. 36: Sepal, symmetry: (0) asymmetric; (1) symmetric. 37: Sepal, concrescence: (0) absent; (1) present. 38: Sepal, morphology: (0) carinate; (1) ecarinate. 39: Sepal, apex morphology: (0) acute to sub-acute; (1) acuminate; (2) rounded; (3) obtuse to sub- obtuse. 40: Sepal, apex: (0) apiculate; (1) attenuate; 41: Sepals, indumenta: (0) absent; (1) present. 42: Sepal, shape: (0) oval; (1) oboval; (2) elliptic. 43: Petal, concrescence: (0) absent (1) present. 44: Petal, apex: (0) acute to rounded; (1) acuminate; (2) sub-acute; (3) retuse. 45: Petal during the anthesis: (0) erect; (1) sub-erect; 46: Petal appendages: (0) absent; (1) present. 47: Petal, longitudinal callosities: (0) absent; (1) present. 48: Petal, shape: (0) linear (1) spatulated; (2) lanceolate; (3) oblong; (4) oboval. 49: Petal, concrescence: (0) until1/3 of its length; (1) more than1/3 of its length. 50: Filament: (0) free; (1) one serie adnate to petal. 51: Ovary, shape: (0) oblong; (1) elliptic; (2) trigonal; (3) oval; (4) clavate. 52: Leaf sheath, adaxial surface, contour: (0) smooth to lightly wavy; (1) furrowed. 53: Leaf sheath, abaxial surface, contour: (0) smooth to lightly wavy; (1) furrowed. 54: Leaf sheath, shape of adaxial epidermal cells: (0) square; (1) elongated. 55: Leaf sheath, shape of abaxial epidermal cells: (0) square; (1) elongated. 56: Leaf sheath, adaxial epidermal cells, thickening in leaf cross section: (0) periclinal wall strongly thick; (1) periclinal wall slightly thick; 57: Leaf sheath, abaxial epidermal cells, thickening in leaf cross section: (0) periclinal wall strongly thick; (1) periclinal wall slightly thick. 58: Leaf sheath, cell wall thickening of adaxial mechanical hypodermal cells: (0) absent; (1) present. 59: Leaf sheath, cell wall thickening of abaxial mechanical hypodermal cells: (0) absent; (1) present. 60: Leaf sheath, cell wall thickening of adaxial mechanical hypodermal cells: (0) strongly thick; (1) slightly thick

66 Capítulo I Oliveira, F.M.C.

Species/ 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 characters 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 Wittrockia 0 0 1 0 1 0 0 ? 0 0 0/ - 1 1 1 1 1 0 0 1 ? 0 0 0 0 0 0 1 1 0 superba 1 Aechmea ? 0 0 1 0 0 0 1 3 1 1 - 0 3 0 1 0 1 - 0 0 0 0 1 0 0 0 0 0 - distichantha Canistropsis 0 0/ 0 0 1 0 0 1 0 0 0 0 1 0 1 0 1 2 1 ? 1 0 0 1 1 0 1 0 0 - microps 1 Canistropsis 0 0/ 0 0 1 0 0 0 0 0 0 1 1 0 1 0 1 2 1 ? 1 1 1 0 0 0 1 1 1 0 billbergioides 1 Edmundoa 2 0 0 1 1 0 0 1 0 0 1 - 1 0 1 1 1 ? - 1 1 0 0 0 0 0 0 1 0 1 lindenii Neoregelia 3 0 0 0 1 0 0 1 1 0 0 - 1 1 0 0 0 3 1 ? ? 1 1 0 0 0 0 0 0 - binotii Neoregelia 0 0 0 0 1 1 1 1 0 0 0 1 0 ? 0 1 0 ? ? ? ? 0 1 0 0 1 1 1 1 0 eleutheropetal a Quesnelia 2 1 0 1 0 0 0 1 2 0 0 - 0 0 1 1 0 3 - 1 2 0 0 1 1 1 1 1 1 1 arvensis Neoregelia 2 0 0 0 1 1 ? ? ? ? 0 ? ? ? 0 0 0 ? ? 1 ? 0 0 1 1 1 0 0 0 - laevis Edmundoa 0/ 0 1 0 0 0 0 1 1 1 0 2 0 0 0 0 1 0 - 1 1 0 0 0 0 0 0 0 0 - perplexa 2 Quesnelia 2 0 0/ 0 0 0 0 1 3/ 1 0 - 0 0 1 1 0 3/ - ? 2 0 0 0 0 0 0 0 1 - quesneliana 1 2 4 Aechmea 0 0 0 0 1 0 1 1 ? ? 1 ? 0 ? ? 1 0 ? ? 1 ? 0 0 0 1 1 1 0 0 - turbinocalyx Edmundoa 0 0 1 1 0 0 0 1 1 ? 0 - 0 0 1 0 1 0 - 0 3 0 0 0 0 1 1 0 0 - ambígua Wittockia 1 1 0 0 0 0 1 ? 1 1 0 - 0 2 1 1 0 4 - ? 4 0 0 0 0 0 0 0 0 - cyathiformis Nidularium 0 1 0 1 1 1 0 1 0 0 0 2 1 ? 0 0 1 - 0 ? 1 0 0 1 1 0 0 0 1 - procerum

67 Capítulo I Oliveira, F.M.C.

Table 3c: Character and their states of character: 61: Leaf sheath, cell wall thickening of abaxial mechanical hypodermal cells: (0) strongly thick; (1) slightly thick. 62: Leaf sheath, number of layers of adaxial mechanical hypodermal cells: (0) one; (1) more than one. 63: Leaf sheath, number of layers of abaxial mechanical hypodermal cells: (0) one; (1) more than one. 64: Leaf sheath, bigger vascular bundle, pericyclic fibers: (0) in caps; (1) rounded all bundle. 65: Leaf sheath, smaller vascular bundle, shape of caps formed by the pericyclic fibers: (0) higher than wide; (1) wider than high. 66: Leaf sheath, cells of brachiform parenchyma: (0) with short arms; (1) with long arms. 67: Leaf sheath, brachiform parenchyma, raphides: (0) absent; (1) present. 68: Leaf sheath, water storage parenchyma: (0) absent; (1) present. 69: Leaf blade, adaxial surface, contour: (0) smooth to lightly wavy; (1) furrowed. 70: Leaf blade, abaxial surface, contour: (0) smooth to lightly wavy; (1) furrowed. 71: Leaf blade, shape of abaxial epidermal cells: (0) square; (1) elongated. 72: Leaf blade, adaxial epidermal cells, thickening in leaf cross section: (0) periclinal wall strongly thick; (1) periclinal wall slightly thick. 73: Leaf blade, abaxial epidermal cells, thickening in leaf cross section: (0) periclinal wall strongly thick; (1) periclinal wall slightly thick. 74: Leaf blade, wing’s cells of trichome: (0) elongated; (1) rounded;.75: Leaf blade, position of stomata in relation to others epidermal cells: (0) at same level; (1) in a depression. 76: Leaf blade, subestomatic camara occlusion: (0) absent; (1) present. 77: Leaf blade, cell wall thickening of adaxial mechanical hypodermal cells: (0) absent; (1) present. 78: Leaf blade, cell wall thickening of abaxial mechanical hypodermal cells: (0) absent; (1) present. 79: Leaf blade, cell wall thickening of adaxial mechanical hypodermal cells: (0) strongly thick; (1) slightly thick. 80: Leaf blade, cell wall thickening of abaxial mechanical hypodermal cells: (0) strongly thick; (1) slightly thick. 81: Leaf blade, number of layers of adaxial mechanical hypodermal cells: (0) one; (1) more than one. 82: Leaf blade, number of layers of abaxial mechanical hypodermal cells: (0) one; (1) more than one; 83: Leaf blade, smaller vascular bundle, projection of fibers to adaxial and abaxial sides: (0) absent; (1) present. 84: Leaf blade, smaller vascular bundle, shape of caps formed by the pericyclic fibers: (0) higher than wide; (1) wider than high. 85: Leaf blade, cells of brachiform parenchyma: (0) with short arms; (1) with long arms. 86: Leaf blade, brachiform parenchyma, raphides: (0) absent; (1) present. 87: Leaf blade, water storage parenchyma: (0) absent; (1) present.

68 Capítulo I Oliveira, F.M.C.

Species/ 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 characters 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 Wittrockia 0 1 1 1 0 1 1 1 0 1 1 0 0 0 0 1 1 1 0 0 1 1 1 0 1 0 1 superba Aechmea - 0 0 1 0 0 0 0 1 1 1 0 0 1 1 1 1 0 1 0 0 0 1 0 0 0 1 distichantha Canistropsis - 0 0 1 1 1 0 0 0 1 1 0 1 0 0 0 0 0 - - 0 0 1 0 0 1 1 microps Canistropsis 0 0 0 1 0 1 0 1 0 0 0 1 0 0 0 1 1 1 1 1 0 0 0 0 0 0 1 billbergioides Edmundoa - 0 0 0 0 1 1 0 0 1 1 1 1 0 0 1 1 1 0 0 1 1 0 0 0 1 1 lindenii Neoregelia - 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 1 1 1 0 0 0 0 0 1 1 binotii Neoregelia 0 0 1 0 1 0 0 0 0 1 1 1 0 0 0 1 1 1 0 0 0 0 0 1 0 0 1 eleutheropetal a Quesnelia 1 0 0 1 1 0 0 1 0 1 1 1 1 1 0 0 0 0 - - 0 0 1 0 1 0 1 arvensis Neoregelia - 0 0 1 0 0 0 0 0 1 1 1 0 0 0 1 0 0 - - 0 0 1 0 1 0 1 laevis Edmundoa - 0 0 0 0 1 0 0 0 1 1 1 0 0 0 0 1 1 0 0 1 1 0 0 0 0 0 perplexa Quesnelia 1 0 0 1 0 0 0 0 0 1 0 0 0 1 0 1 1 1 1 1 0 0 0 1 1 0 1 quesneliana Aechmea - 0 0 1 0 1 0 0 0 1 1 0 0 1 0 1 1 1 1 1 0 0 1 0 0 0 1 turbinocalyx Edmundoa - 0 0 0 1 1 1 0 0 0 1 1 0 0 0 1 0 1 - 0 0 0 0 0 0 0 0 ambígua Wittockia - 0 0 0 1 1 1 1 0 1 0 1 0 0 0 1 1 1 1 1 1 1 1 0 1 0 1 cyathiformis Nidularium 1 0 0 1 0 1 0 0 0 1 0 0 0 0 0 1 0 1 1 1 0 0 0 0 0 0 1 procerum

69 Capítulo I Oliveira, F.M.C.

References

Adams, W.W.; Martin, C.E. 1986. Morphological changes accompanying the transition from juvenile (atmospheric) to adult (tank) forms in the Mexican epiphyte Tillandsia deppeana (Bromeliaceae). Am. J. Bot. 73(8), 1207-1214.

Aguirre-Santoro, J.; Michelangeli, F. A.; Stevenson, D. W. 2016. Molecular phylogenetics of the Ronnbergia Alliance (Bromeliaceae, Bromelioideae) and its insights into their morphological evolution. Mol. Phylogen. Evol. 100,1-20.

Akaike, H. 1973. Information theory and an extension of the maximum likelihood principle. in Petrov, B. N. and Csaki, F. (Eds.), Proceedings of the second international symposium on information theory. Akademiai Kiado, Budapest, Pp. 267–281:

Almeida, V.R.; Costa, A.F.; Mantovani, A.; Gonçalves-Esteves, V.; Arruda, R.C.O. & Forzza, R.C. 2009. Morphological Phylogenetics of Quesnelia (Bromeliaceae, Bromelioideae). Syst. Bot. 34, 660-672.

APG IV. 2016. An update of the Angiosperm phylogeny group classification for the orders and families of flowering plants: APG IV. Bot. J. of Linn.Soc., 181, 1-20.

Ayoama, E. M.; Sajo, M.G. 2003. Estrutura foliar de Aechmea Ruiz & Pav. subgênero Lamprococcus (Beer) Baker e espécies relacionadas (Bromeliaceae). Rev. Bras. Bot. 26 (4), 461-473.

Benzing, D. H. 1970. Foliar permeability and the adsorption of minerals and organic nitrogen by certain tank bromeliads. Bot. Gaz. 131, 23– 31.

Benzing, D. H. 1976. Bromeliad trichomes: structure, function and ecological significance. Selbyana. 4 (1), 330 – 348.

Benzing, D. H.; Givnish, T. J.; Bermudes, D. 1976. Absorptive trichomes in Brocchinia reducta (Bromeliaceae) and their evolutionary and systematic significance. Selbyana. 10 (1), 81– 91.

Benzing, D. H.; Seeman, J.; Renfrow, A. 1978. The foliar epidermis in Tillandsoideae (Bromeliaceae) and its role in habitat selection. Am. J. Bot. 65 (3), 359–365.

70 Capítulo I Oliveira, F.M.C.

Benzing, D.H. 2000. Bromeliaceae: profile of an adaptative radiation. New York: Cambridge University Press.

Brown, G. K.; Gilmartin, A. J. 1989. Stigma types in Bromeliaceae- a systematic survey. Syst. Bot. 14, 110-132.

Brown, G.K.; Terry, R.G. 1992. Petal appendages in Bromeliaceae. Am. J. Bot. 79 (9), 1051- 1071.

Butcher, D., Gouda, E.J. cont. updated. The new bromeliad taxon list. . University Botanic Gardens, Utrecht (accessed 06.01.16).

Crayan, D.M.; Winter, K.; Smith, A.C. 2004. Multiple origins of crassulacean acid metabolism in the epiphytic habit in the Neotropical family Bromeliaceae. PNAS. 101 (10), 3703-3708.

Doege, S.J.; Korth, K.L. 2003. The role of natural calcium oxalate crystals in plant defense against chewing insects. Inquiry. 4, 88-94.

Edgard, R.C. 2004. Muscle: multiple sequence alignment with high accuracy and high throughput. Nuc. Ac. Resear. 32 (5), 1792-1797.

Evans, T. M.; Jabaily, R. S.; Faria, A. P. G.; Sousa, L. O. F.; Wendt, T.; Brown, G. K. 2015. Phylogenetic relationships in Bromeliaceae subfamily Bromelioideae based on chloroplast DNA sequence data. Syst. Bot. 40 (1), 116-128.

Fahn, A. 1974. Plant anatomy. Oxford: Pergamon Press, 611p.

Fahn, A.; Cutler, D. 1992. Xerophytes. Gebruder Borntraeger, Berlin.

Faria, A.P.G.; Vieira, A.C.M.; Wendt, T. 2012. Leaf anatomy and its contributions to the systematics of Aechmea subgenus Macrocordion (de Vriese) Baker (Bromeliaceae). Ann. Brazilian Acad. Sci. 84(4), 961-971.

Franceschi, V.R.; Horner, H.T. 1980. Calcium oxalate in plants. Bot. Rev. 46 (4), 361-427.

Franceschi, V.R.; Nakata, P.A. 2005. Calcium oxalate in Plants: formation and function. Annu. Rev. Plant Bio. 56, 41-71.

71 Capítulo I Oliveira, F.M.C.

Freschi, L.; Takahashi, C.A.; Cambui, C.A.; Semprebom, T.R.; Cruz, A.B.; Mioto, P.T.; Vervieux, L.M.; Calvente, A.; Latansio-Aidar, S.B.; Aidar, M.P.M.; Mercier, H. 2010. Specific leaf areas of the tank bromeliad Gusmania monostachia perform distinct functions in response to water storage. Journal of Plant Physiol. 167, 526-533.

Givnish, T.J.; Millan,K.C.; Berry, P.E.; Systma, K.J. 2007. Phylogeny, adaptive radiation, and historical biogeography of Bromeliaceae inferred from ndhF sequence data. Aliso, 23: 3-26.

Givnish, T.J.; Barfuss, M.H.J.; Van Ee, Benjamin; Riina, R.; Schulte, K.; Horres, R.; Gonsiska, P.A.; Jabaily, R.S.; Crayan, D.M.; Smith, J.A.C.; Winter, K.; Brown, G.K.; Evans, T.M.; Holst, B.K.; Luther, H.; Till, W.; Zizka, G.; Berry, P.E.; Systma, K.J. 2014. Adaptative radiation, correlated and contingent evolution, and net species diversification in Bromeliaceae. Mol. Phylogenet. Evol., 71, 55-78.

Gomes-da-Silva, J.; Vargens, F.A.C.; Arruda, R.C.O. & Costa, A.F. 2012. A morphological cladistic analysis of the Vriesea corcovadensis group (Bromeliaceae: Tillandsioideae), with anatomical descriptions: new evidence of the non-monophyly of the genus. Syst. Bot. 37, 641- 654.

Heller, S.; Leme, E. M.; Schulte, K.; Benko-Iseppon, A. M.; Zizka, G. 2015. Elucidating phylogenetic relationships in the Aechmea Alliance: AFLP analysis of Portea and the Gravisia Complex (Bromeliaceae, Bromelioideae). Syst. Bot. 40 (3), 716-725.

Hornung-Leoni, C.;Sosa, V. 2008. Morphological phylogenetics of Puya subgenus Puya (Bromeliaceae). Bot. J. Linn. Soc. 156, 93-110.

72 Capítulo I Oliveira, F.M.C.

Johansen, D.A. 1940. Plant microtechnique. McGraw-Hill, New York.

Kowalski, V.K.; Pereira, P.P.D.A.; Oliveira, F.M.C.; Costa, M.E.; Tardivo, R.C. 2016. Are the wing’s cells alive? Study case in Vriesia trichomes. Rodriguésia. 67 (2), 427-435.

Kraus, J.E.; Arduin, M. 1997. Manual básico de métodos em morfologia vegetal. Edur, Seropédica.

Krauss, B.H. 1949. Anatomy of the vegetative organs of the pineapple Ananas comosus (L.) Merr.: II The leaf. Bot. Gaz. 110 (3), 333-404.

Leme, E.M.C. 1997. Canistrum- Bromélias na Mata Atlântica. Rio de Janeiro: Salamandra.

Leme, E.M.C. 1998. Canistropsis - Bromélias na Mata Atlântica. Rio de Janeiro: Salamandra.

Leme, E. M. C. 2000. Nidularium- Bromélias na Mata Atlântica. Rio de Janeiro: Sextante.

Luttge, U.; Stimmel, K.H.; Smith, J.A.C.; Griffiths, H. 1986. Comparative ecophysiology of CAM and C3 Bromeliads. 2. Field-measurements of gas-exchange of CAM bromeliads in the humid tropics. Plant Cell Environ. 9, 377–383.

Males, J. 2016. Think tank: water relations of Bromeliaceae in their evolutionary context. Bot. J. Linn. Soc. 181, 415-440.

Maddison, W. P.; Maddison, D. R. 2015. Mesquite: a modular system for evolutionary analysis. Version 3.04. Https://mequiteproject.org

Miller, M.A., Pfeiffer, W., Schwartz, T., 2011. The CIPRES science gateway: a community resource for phylogenetic analyses. In: Presented at the Proceedings of the 2011 TeraGrid Conference: Extreme Digital Discovery. ACM, p. 41.

Monteiro, R.F.; Forzza, R.C.; Mantovani, A. 2011. Leaf structure of Bromelia and its significance for the evolution of Bromelioideae (Bromeliaceae). Plant Sys Evol. 293, 53-64.

Nakata, P.A. 2003. Advances in our understanding of calcium oxalate formation and function in plants. Plant Sci. 164, 901-909.

Nakata, P.A. 2012. Plant calcium oxalate Crystal formation, function, and its impact on human health. Front. Biol. 7 (3), 254-266.

73 Capítulo I Oliveira, F.M.C.

Prychid, C.J.; Rudall, P.J. 1999. Calcium oxalate crystals in Monocotyledons: a review of their structure and systematics. Ann. Bot.. 84, 725-739.

Proença, S.L.; Sajo, M.G. 2007. Anatomia foliar de bromélias ocorrentes em áreas de cerrado do Estado de São Paulo, Brasil. Acta Bot. Bras. 21 (3), 675-673.

Ramírez, I. M. 1994. Notes on Neoregelia subgenus Hylaeaicum (Bromeliaceae: Bromelioideae). Selbyana 15 (2), 82-84.

Ramírez, I. 2000. Neoregelia subgenus Hylaeaicum. In: Benzing, D.H. 2000. Bromeliaceae: profile of an adaptative radiation. New York: Cambridge University Press.

Ronquist, F., Teslenko M., Van DerMark P., Ayres D. L., Darling A., H¨ohna S., Larget B., Liu L., Suchard M. A., Huelsenbeck J.. 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542.

Santos-Silva, F.; Saraiva, D.P.; Monteiro, R.F.; Pita, P.; Mantovani, A.; Forzza, R.C. 2013. Invasion of the South American dry diagonal: What can leaf anatomy of Pitcairnoideae (Bromeliaceae) tell us about it? Flora. 208, 508-521.

Saraiva, D. P.; Mantovani, A.; Forzza, R.C. 2015. Insights into the Evolution of Pitcairnia (Pitcairnoideae- Bromeliaceae), based in morphological evidence. Syst. Botany. 40 (3), 726- 736.

Scatena, V.L.; Segecin, S. 2005. Anatomia foliar de Tillandsia L. (Bromeliaceae) dos Campos Gerais, Paraná, Brasil. Rev. Bras. Bot. 28 (3), 635-649.

Schulte, K.; Zizka, G. 2008. Muti locus plastid phylogeny of Bromelioideae (Bromeliaceae) and the taxonomic utility of petal appendages and pollen charactes. Candollea 63 (2), 209-225.

Schulte, K., Barfuss, M.H.J., Zizka, G., 2009. Phylogeny of Bromelioideae (Bromeliaceae) inferred from nuclear and plastid DNA loci reveals the evolution of the tank habit within the subfamily. Mol. Phylogen. Evol. 51, 327–339.

74 Capítulo I Oliveira, F.M.C.

Smith L.B.; Downs R.J. 1974. Pitcairnioideae (Bromeliaceae). Flora Neotropica Monograph 14: 1-662.

Smith L.B.; Downs R.J. 1977. Tillandsioideae (Bromeliaceae). Flora Neotropica Monograph 14: 663-1492.

Smith L.B.; Downs R.J. 1979. Bromelioideae (Bromeliaceae). Flora Neotropica Monograph 14: 1493-2142.

Silveira, M. 1989. O preparo de amostras biológicas para microscopia eletrônica de varredura. In: W. de Souza (ed.). Manual sobre Técnicas Básicas em Microscopia Eletrônica de Varredura. Sociedade Brasileira de Microscopia Eletrônica,v. 1, pp. 71-82.

Silvestro, D.; Zizka, G.; Schulte, K. 2014. Disentangling the effects of key innovations on the diversification of Bromelioideae (Bromeliaceae). Evolution 68 (1), 163-175.

Sousa, L. O. F.; Wendt, T.; Brown, G. K.; Tuthill, D. E.; Evans, T. M. 2007. Monophyly and phylogenetic relationships in Lymania (Bromeliaceae: Bromelioideae) based on morphology and chloroplast DNA sequences. Syst. Bot.32 (2), 264-270.

Sousa, G.M.; Estelita, M.E.M.; Wanderley, M.G.L. 2005. Anatomia foliar de espécies brasileiras de Aechmea subg. Chevaliera (Gaudich. ex. Beer) Baker, Bromelioideae- Bromeliaceae. Rev. Bras. Bot. 28 (3), 603-613.

Strhel, T. 1983. Forma, distribuição e flexibilidade dos tricomas foliares usados nas filogenias de Bromélias. Iheringia- Série Botânica. 31,105-119.

Tomlinson, P. 1969. Commelinales- Zingiberales. In: Metcalf, CR. (ed.) Anatomy of the Monocotyledons: III. Claredon Press, Oxford. Pp, 193-294.

Versieux, L.M.; Elbl, P.M.; Wanderley, M.G.L.; Menezes, N.L. 2010. Alcantarea (Bromeliaceae) leaf anatomical characterization and it systematics implications. Nord. J. Bot. 28, 385-397.

Wanderley, M. G. L.; Martins, S. E.; Proença, S. L.; Moreira, B. A. 2007. Canistrum. In: Wanderley, M.G.L; Shepherd, G. J; Melhem, T.S; Giulietti, A.M. Flora Fanerogâmica do Estado de São Paulo. São Paulo: FAPESP.

75 Capítulo I Oliveira, F.M.C.

Yu, Y.; Harris, A. J.; He, X. J. 2012. RASP (reconstruct ancestral state in phylogenies), v. 2.1b. Available at http://mnh.scu.edu.cn/soft/blog/RASP.

Capítulo II

Floral anatomy of Bromeliaceae with special reference on androecium and gynoecium

Fernanda Maria Cordeiro de Oliveira1,2 e Gladys Flavia de Albuquerque Melo-de-Pinna1

¹Universidade de São Paulo, Instituto de Biociências, Departamento de Botânica, Laboratório de

Anatomia Vegetal, Rua do Matão 321 Travessa 14, 05508-090 São Paulo, SP, Brasil.

2Corresponding author: [email protected]

Running title: Floral anatomy of Bromeliaceae

Submetido ao periódico Botanical Journal of Linnean Society

Capítulo II Oliveira, F.M.C.

Abstract

Bromeliaceae is a family with predominant neotropical distribution and is included in Poales

Order. Its flowers, trimerous, pentacyclic, showy and colorfull are being studied for a long time, particularly about the septal nectaries. In this study, we prioritized to describe and discuss the anatomy of androecium and gynoecium from species belonging to three out of eight

Bromeliaceae subfamilies. Flower buds in pre-anthesis were collected, fixed and processed by usual technics in vegetal anatomy. We found some characters that probably has importance in pollination such as conical epidermis presents on anthers, filaments and stigma. The vascularization of filaments and style is discussed under a phylogenetic perspective and a new synapomorphie for Pitcairnoideae is proposed. Secretory structures of gynoecia- septal nectary, obturator and transmitting tissue- are comparatively described, giving data for future studies on this group evolution.

Key-words: Poales, floral anatomy, vascularization, secretory structures.

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Resumo

Bromeliaceae é uma família com distribuição predominantemente neotropical e encontra-se inserida na Ordem Poales. Suas flores, trímeras, pentacíclicas, vistosas e coloridas são alvo de estudos há tempos, principalmente em relação aos seus nectários septais. Neste estudo, priorizou-se descrever e discutir a anatomia dos androceu e gineceu de espécies pertencentes a três das oito subfamílias de Bromeliaceae. Para tanto, botões florais em pré-antese foram coletados, fixados e processados segundo técnicas usuais em anatomia vegetal. Alguns caracteres, tais como a presença de epiderme cônica nos filetes, anteras, estiletes e estigmas provavelmente tem papel na polinização do grupo. A vascularização dos filetes e estiletes é discutida sob perspectiva filogenética, sendo que uma nova sinapomorfia para Pitcairnoideae é proposta. As estruturas secretoras do gineceu- nectário septal, tecido obturador e tecido transmissor- são descritas de forma comparativa, fornecendo dados para futuros estudos de evolução no grupo.

Palavras chave: Poales, anatomia floral, vascularização, estruturas secretoras.

80 Capítulo II Oliveira, F.M.C.

1. Introduction

The family Bromeliaceae has an almost exclusively neotropical distribution, with the exception of the species Pitcairnia feliciana (A. Chev.) Harms & Mild that occurs in West

Africa (Smith, 1974, Jacques-Félix, 2000). The family has ca. 3,400 species distributed among

56 genera (Butcher & Gouda, continuously updated). This family along with Typhaceae and

Rapateaceae occupy a basal position in Poales (APG IV, 2016), and was for a long time subdivided into three subfamilies: Bromelioideae, Pitcairnioideae s. l. and Tillandsioideae. The three subfamilies could be differentiated by the foliar margin, ovary position, and fruit and seed types (Smith & Downs, 1974, 1977, 1979). Nevertheless through advances in the area of molecular systematics it is now known that Pitcairnioideae s. l. does not represent a monophyletic group, and thus Bromeliaceae was reorganized into eight subfamilies:

Bromelioideae, Tillandsioideae, Pitcairnioideae s. s., Navioideae, Puyoideae, Brocchinioideae,

Hechtioideae and Lidmanioideae (Givnish et al., 2007; Givnish et al., 2011).

The flowers in Bromeliaceae are trimeric, pentacyclic, usually showy, colorful and attractive to pollinators especially due to the presence of nectar in the septal nectaries (Smith &

Downs, 1974; Sajo et al., 2004a). According to Sajo et al. (2004a) the presence of septal nectaries and its evolution in relation to the group's epigyny is one of the features that make this family so singular within the Poales. The presence of petal appendages in some genera within this family were also the subject to many studies in Bromeliaceae, especially because of its taxonomic importance for the group. These appendages, commonly used for genera delimitation, are the result of a growth in the adaxial portion of the petals, are not vascularized, and are the last structures to be formed in the flower (Brown & Terry, 1992; Sajo et al., 2004a;

Oliveira et al., 2016).

81 Capítulo II Oliveira, F.M.C.

The floral anatomy of Bromeliaceae has been studied aiming to detect characters with taxonomic importance, mainly regarding ovary and ovule anatomy (Novikoff & Odintsova,

2008; Nogueira et al., 2015; Kuhn et al., 2016). Ovule development has been described for distinct species within the family, being the presence of chalazal and micropylar appendages one of the most striking traits (Sajo et al., 2004b; Fagundes & Mariath, 2014; Mendes et al.,

2014). Regarding the ovary little is known about the anatomical structure of the septal nectaries and the composition of its secretion (Fiordi & Palandri, 1982; Bernadello et al., 1991; Sajo et al., 2004a). However, studies that describe the obturator tissue are scarce, and little is yet known about the origin of the characteristics of its secretion (Sajo et al., 2004a; Nogueira et al., 2015;

Oliveira et al., 2016).

Aiming to better understand the anatomy of both the androecium and gynoecium, we analyzed floral buds of 16 species belonging to three of the eight subfamilies of Bromeliaceae.

We described characters that can be useful for taxonomy in this family, and discuss them under an ecological and phylogenetic perspective.

2. Material and methods

We analyzed flower buds in pre-anthesis of 16 species, comprehending 10 genera and three subfamilies of Bromeliaceae. A full description of the testimony material and sampling areas are present on table I.

The sampled inflorescences were fixed in FAA 50 (Formaldehyde, Acetic Acid and

Ethanol 50˚GL) as described in Johansen (1940), and posteriorly stored in 70% ethanol. Flower buds were then dehydrated in an ethanol/butanol series and embedded in Paraplast® (Ruzin,

1999). We obtained serial transversal and longitudinal sections between 8 and 12µm thick using a rotary microtome. The waxed slides were stained using Toluidine O (Sakai, 1973) for 30

82 Capítulo II Oliveira, F.M.C.

minutes. The excess dye was washed with water and the slides were dried in room temperature.

After dry the slides were de-waxed using xylol and mounted in Entellan® (Ruzin, 1999).

For scanning electron microscope (SEM) examination, filaments, anthers, stigmas and ovaries were dehydrated in an ethanol series. The samples were then critical-point-dried and coated with gold (Silveira, 1989). The analysis was done using a Zeiss DMS-940 Scanning

Electron Microscope.

3. Results

The main anatomical features of both the androecium and the gynoecium are summarized in table II.

Androecium - the studied species show two series of stamens, with the first

(antisepalous) adnate to the petals in the basal region, and the second (antipetalous) free (Fig.

1A-C). They form a corolla-stamen tube in Nidularium longiscapum, Neoregelia spectabilis,

Crypthantus bahianus, and Dyckia tuberosa Dyckia tuberosa (Fig. 1D). The upper portion of the filaments is free (Fig. 1 E-F), and these filaments can be round, triangular or flat (Fig. 1).

In some species flat and round filaments occur simultaneously in the same flower (table II).

When callus or petal appendages are present the antisepalous filaments are embedded between these structures and the petals themselves, as it can be seen in N. amazonicum (Fig. 1 B).

The epidermis of the filaments is uniseriated and do not show cell wall thickening, but there can be a prominent cuticle deposition in the apical portion, as in Neo. spectabilis, N. longiflorum, N. amazonicum, Q. testudo and V. platynema (Fig. 2). The mesophyl is homogeneous, being possible to see idioblasts with raphides in A. gamosepala, A. cylindrata,

C. bahianus, Encholirium sp., P. flammea and Q. testudo (Fig. 2 A-F). Aerenchyma in the antisepalous filaments is only seen in V. platynema. Regarding vascularization, a single collateral bundle occupies the central portion of the filaments (Fig. 2 A-C). However in

83 Capítulo II Oliveira, F.M.C.

Encholirium sp. there are three vascular traces (Fig. 2D) and in N. procerum, P. flammea, V. inflata and D. tuberosa (Fig. 2B, E-F) there are two vascular traces.

The anthers are dithecous and tetrasporangiate (Fig. 3). The epidermis is uniseriated, papillose, composed by conic-shaped cells without cell wall thickening. The cuticle deposition emphasizes the conic shape of epidermal cells (Fig. 4 arrows). In frontal view, the cuticle is striated (Fig. 5). One to four cell layers with ring wall thickening compose the endothecium.

The middle layer and the tapetum are not observed in pre-anthesis. Idioblasts containing raphides are present in the anthers of A. cylindrata, A. gamosepala, A. ornata, D. tuberosa,

Encholirium sp., and V. inflata. Anther vascularization in the connective region is done by a concentric vascular bundle located between the two theca (Fig. 3).

Gynoecium: The gynoecium in species from the Bromelioideae subfamily is characterized by an inferior ovary that is adnate to the hypanthium (Fig. 6 A-B). The external epidermis of the hypanthium is uniseriated with each cell containing a silica crystal. The epidermal cells also show cell wall thickening in their anticlinal and periclinal walls in A. nudicaulis, A. cylindrata, and A. gamosepala (Fig. 6 C). Stomata are present in A. nudicaulis and A. cylindrata, and trichomes can be found in A. ornata, A. cylindrata and Q. testudo (Fig.

6 D). The hypoderm has cells with thickened walls only in A. nudicaulis and Q. testudo. The mesophyll is homogeneous (Fig. 6 E-F) with an aerenchyma formed by braciform cells present only in N. procerum and N. longiscapum (Fig. 6 F). Idioblasts containing raphides are present in the wall of the hypanthium, and the vascularization is done by the presence of the sepals, petals, stamens and dorsal carpelar bundle complexes.

Species from the Pitcairnioideae and Tillandsioideae show superior ovaries (Table II,

Fig. 7 A-C). The ovary external epidermis is uniseriated (Fig. 7 D-F), and the cells show no wall thickening. In D. tuberosa the epidermal cells are periclinally elongated and stomata are present (Fig. 7 D). The ovarian mesophyll is homogeneous and Encholirium sp. an aerenchyma

84 Capítulo II Oliveira, F.M.C.

is present (Fig. 7 E); vascularization is performed by the presence of the dorsal and ventral carpelar bundles.

All studied species show trilocular and tricarpelar ovaries with axial placentation (Fig.

6 A-B and 7 A-B). The ovules are anatropous, bitegumented and its vascularization (as well as for the placenta) is done by the ventral bundles (Fig. 8 A-F). Ovules of D. tuberosa (Fig. 8 B),

Encholirium sp., N. amazonicum, P. flammea (Fig. 8 C) and V. platynema (Fig. 8 F) show chalazal appendages. There is obturator tissue in the placenta, which is constituted by a layer of secretive cells with prominent nucleus (Fig. 8 G-H). Sometimes it is possible to note the presence of secretion in the ovary's locule that comes from the obturator tissue. The cells in this obturator tissue are short or elongated, and vary among the studied species (table II). The ovary of species of the Bromelioideae subfamily also shows a septal interlocular nectary, that is characterized by having cells with heavily stained content and a prominent nucleus that highlight its secretive activity (Fig. 9 A-D). For the species of the other two studied subfamilies, in which the ovaries are all superior, the nectaries are located below the locules, and are therefore named infraloculars (Fig. 9 E-G). The only exception is P. flammea, in which the most developed portion of the septal nectary lies below the locules, but still has part of the nectary in the septal region (Fig. 7 C). In this species the nectariferous tissue forms labyrinth

In the style region the external epidermis is uniseriated and in some species this epidermis can be papillous (Table II, Fig 10). The internal epidermis is differentiated into transmitting tissue, which cells show prominent nucleus and dense cytoplasm that characterize its secretive function. The mesophyll is homogeneous and in some cases idioblasts with raphides are present (Fig. 10). Vascularization is performed only by the dorsal carpelar bundles for most species (Fig. 10 A-B, E), but for Encholirium sp (Fig. 10 D), D. tuberosa (Fig. 10 C) and P. flammea the vascularization is performed by both the dorsal and ventral carpelar bundles).

85 Capítulo II Oliveira, F.M.C.

Stigmas are of the conduplicated-spiralling type for most studied species (Fig. 11 G-

H). Only in V. platynema and V. inflata the stigmas are from the simple-erect type (Fig. 11 K).

They are anatomically very similar, with a papillous abaxial epidermis. The mesophyll is homogeneous in all studied species, and vascularization is performed by the dorsal carpelar bundles (Fig. 11 A-E). Only in D. tuberosa, Encholirium sp. and P. flammea the vascularization is performed by both the ventral and dorsal carpelar bundles (Fig. 11 F).

4. Discussion

4.1 General characteristics

In the studied species, the epidermis in the apical portion of the filaments, styles and anthers is conical. Nevertheless this shape, which is observed in both transversal and longitudinal sections, is caused by cuticle deposition, and not by the thickening of the cell wall or by its shape itself. These traits have already been reported in petals of Aechmea distichantha

Lem., and Canistropsis billbergioides (Schult. & Schult. f.) Leme by Oliveira et al. (2016), in stigmas of species of Bromelioideae and Tillandsioideae by Souza et al. (2016), and in anthers of species of the genus Dyckia by Carvalho et al. (2016). Within the order Poales an epidermis with conical aspect was observed in the anthers of Rapataceae (Venturelli & Bouman, 1988,

Oriani & Scatena, 2013, Ferrari & Oriani, 2016), and Mayacaceae (Carvalho et al., 2009).

The ecological implications for the presence of a conical epidermis with cuticle deposition in the petals have been discussed by several authors. According to Koch et al. (2008) and Whitney et al. (2011) this feature could enhance the adherence of pollinator insects to the sepals during flower visitation, and also to avoid the accumulation of residuals in the surface.

Oriani & Scatena (2013) also observed ornamented cuticle in the epidermis of the petals, anthers, and styles in Rapataceae. The authors discuss that the presence of a thick cuticle could be an adaptation against the excess of light by increasing solar radiation reflection. In relation

86 Capítulo II Oliveira, F.M.C.

to the cuticle ornamentation, the same authors infer that it can be related to pollinator attraction.

We believe that the presence of a thick, ornamented cuticle in the filaments, anthers, styles, and stigmas in Bromeliaceae is also related to pollinator attraction, once when the cuticle is present in the filaments and styles it occurs in their upper position, and the anthers and stigmas are exposed during anthesis.

The presence of idioblasts containing raphides in vegetative organs of Bromeliaceae is well documented (Krauss, 1949, Tomlinson, 1969); in flowers, they were documented in the anthers by Sajo et al. (2005) and in the remaining whorls by Oliveira et al. (2016). In the studied species the idioblasts with raphides are present both in the androecium and in the gynoecium.

The presence of raphides is usually associated with a demand to neutralize the great amount of oxalic acid that is produced (Brighina et al., 1984), in addition to making the plant less edible to herbivores (Mauseth, 1988).

When studying the flowers of species belonging to the Nidularioid complex

(Canistropsis billbergioides, Canistrum auratiacum, Edmundoa lindenii, Nidularium inocentii,

Neoregelia johanis and Wittrockia superba), Nogueira et al. (2015) reported the presence of aerenchyma in the ovary walls. The authors associated this presence to a condition of hypoxia to which the ovaries are submitted once these structures are in direct contact with water. This contact is due to either the inflorescences being submerged in the water accumulated in the central tank or because the primary bracts and floral bracts accumulate water. The authors also highlight that the presence of aerenchyma in the ovary walls could be considered a synapomorphy for the Nidularioid clade.

Species belonging to the Nidularioid complex were also analyzed in this study (N. amazonicum, N. procerum and Neo spectabilis). Between the three species Neo. spectabilis is the only species which inflorescence is submerged in the central tank. The other two show an elongated floral peduncle and well developed primary bracts, being able to accumulate water

87 Capítulo II Oliveira, F.M.C.

from the rain. Thus, this trait might not necessarily be related to hypoxia, since the species that has submerged inflorescence does not show such feature. However, it could represent a synapomorphy for the Nidularioid complex, corroborating Nogueira et al.'s (2015) hypothesis.

Nevertheless, studies with a larger number of species are necessary. In the remaining species the aerenchyma is present in the walls of the ovary in Encholirium sp., and in the antisepalous filaments of V. platynema. Those two species show well-developed floral pedicel, meaning that their inflorescences are not submerged and their floral bracts do not accumulate water.

Ovules in Bromeliaceae are anatropous, bitegumented, and can display the so called chalazal appendages (Sajo et al., 2004b; Palací et al., 2004; Fagundes & Mariath, 2014; Mendes et al., 2014; Nogueira et al., 2015; Kuhn et al., 2016). This structure is formed by a growth in the dermal and subdermal layers of the chalazal region, and the final product is a whole or fimbriated protuberance (Palací et al., 2004; Sajo et al., 2004b; Fagundes & Mariath, 2014;

Mendes et al., 2014). These appendages are present in the ovules of several monocotyledon families such as Tofieldiaceae, and Nartheaceae (Remizowa et al., 2006). In Poales, the families

Bromeliaceae, Rapateaceae, and Juncaceae show chalazal appendages in their ovules

(Venturelli & Bouman 1988; Sajo et al., 2004b; Oriani et al., 2012).

According to Smith & Downs (1977) and Palací et al. (2004), the presence of chalazal appendages is related to the development of feathery seeds in Tillandsioideae. In Catopsis, for instance, Palací et al. (2004) observed that the chalazal appendages of the ovules are fimbriated and grow along the development of the seeds generating the feathery appendages of these seeds that help in wind dispersal. For species in the genus Tillandsia, the appendages are whole. The authors discuss that the feathery appendages of the seeds in this genus originate from the growth of both the internal and external ovule teguments. Hence the authors propose that the feathery appendages of the seeds of Tillandsioideae are not homologous despite having the same purpose of aiding on seed dispersal through wind.

88 Capítulo II Oliveira, F.M.C.

In the studied species of Vriesea, only V. platynema show a well-developed, whole chalazal appendage. It is also possible to note that the internal and external teguments show a more pronounced growth when compared to the remaining studied species. The feathery appendages of the seeds of Vriesea species are most likely derived from the growth of the teguments as related by Palací et al. (2004) for species of Tillandsia. However to confirm such hypothesis ontogenetic studies of the seeds of Vriesea species are needed.

The chalazal appendages also seem to be related to the development of winged seeds in some Pitcarnioideae (Varadarajan & Gilmartin 1988). Specifically in Dyckia the whole, asymmetric-shaped chalazal appendages seem to be related to the asymmetrical growth typical from the seed's wings (Dorneles et al. 2014). In Dyckia tuberosa, the chalazal appendage is whole and shows a pronounced growth, resembling the morphology of the future seed.

However, as for Vriesea seeds, ontogenetic studies to confirm this hypothesis are lacking.

4.2 Vascularization

In Bromeliaceae filaments that are vascularized by more than one vascular bundle are present in Pitcarnioideae (Dickia tuberosa, D. racinae, Encholirium sp., Encholirium subsecundum and P. flammea), in Tillandsioideae (Vriesea oligantha, V. inflata) and in

Bromelioideae (Nidularium procerum) (Arrais, 1989; Carvalho et al., 2016). Data about the remaining subfamilies are not available in the literature.

Stamen vascularization is in general formed by a single vascular bundle in the majority of Angiosperms (Eames, 1931). However, filaments vascularized by more than one bundle

(generally three bundles) occur in stamens of Magnoliaceae, Lauraceae and Musaceae (Eames,

1931; Wilson, 1942; Puri, 1951).

89 Capítulo II Oliveira, F.M.C.

According to Eames (1931) stamens that are vascularized by three vascular bundles likely represent a plesiomorphic condition among the Angiosperms. Thus, stamens vascularized by a single vascular trace probably had an ancestor that was vascularized by three traces.

Puri (1951) arguments that in some cases the androecium structure becomes more complex due to adnation, conation, or loss of parts. Eames (1931) also highlights that when there is fusion between whorls, this fusion is first noted morphologically, with the internal structure often unmodified. In those cases floral vascularization can provide information for a better interpretation of those structures (Puri, 1951; Carlquist, 1969).

In Velloziaceae, for instance, Menezes (1979) noted that the filaments of Vellozia declinans have three bundles. The author interpreted such character as a reduction in the number of stamens in this species in relation to its ancestor. Hence the filaments with three vascular bundles would be a result of stamen fusion, indicating that this species ancestor likely had a greater number of stamens.

Among the studied species Encholirium sp has all filaments - both the antisepalous and antipetalous - vascularized by three bundles. In Dyckia tuberosa, P.flammea, V. inflata and

Nidularium procerum the filaments are vascularized by two vascular bundles. As in Poales, trimerous pentacyclic flowers are plesiomorphic (Remizowa et al., 2010), and thus the presence of more than one vascular bundle in the filaments probably do not indicate whorl fusion, as proposed by Menezes (1979) in Velloziaceae. Due to the absence of data about floral vascularization in the basal subfamilies of Bromeliaceae it is not possible to state that this is a plesiomorphic character. What we can state based on data both from the literature and from the studied species is that this character was originated several times in Bromeliaceae since they occur in species of the Bromelioideae, Tillandsioideae and Pitcairnioideae subfamilies.

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In the gynoecium of the studied species both dorsal and ventral carpelar bundles are responsible for the vascularization. When the ovary is inferior - as in species belonging to the

Bromelioideae subfamily - the ventral bundles are responsible for the vascularization of the placenta and the septal nectaries. On the other hand in species of Pitcairnioideae and

Tillandsioideae, which flowers have superior ovary, the ventral bundles vascularize only the placenta once the nectary is located below the ovary's locules. In species of Pitcairnioideae the ventral bundles vascularize the gynoecium as a whole, including the style and the stigma.

The style and stigma vascularization by three vascular bundles, being one dorsal and two ventral, is also considered a plesiomorphic character among Angiosperms (Eames, 1931;

Wilson, 1942; Puri, 1951). For that matter, the condition of a single trace could have been originated by the fusion of the two lateral traces with the median trace, or still by the loss of the lateral traces (Wilson, 1942). In Bromeliaceae, styles and stigmas that are vascularized by the dorsal and ventral bundles are only present in Pitcairnioideae, and might represent a synapomorphy for this subfamily. However it is not possible to state that this character is plesiomorphic for the family due to the absence of floral vascularization data for the remaining subfamilies of Bromeliaceae.

4.3 Secretory structures

One of the most striking features of flowers in Bromeliaceae is the presence of septal nectaries (Sajo et al., 2004a). Those nectaries, also known as gynopleural nectaries (Remizowa et al., 2010), are found only in flowers of Monocotyledons and were lost several times throughout the evolutionary history of the group (Fahn, 1952; Rao, 1975; Rudall, 2002;

Bernadello, 2007). In the order Poales these structures were only registered in Bromeliaceae and Rapateaceae (Sajo et al., 2004a), maintaining its basal position within this order (Linder &

Rudall, 2005). In the remaining lineages of Poales this structure was lost, and this loss is likely related to the wind pollination in this group (Rudall, 2002). Thus, pollination by animals that

91 Capítulo II Oliveira, F.M.C.

are attracted by the nectar produced in the flowers is likely a plesiomorphic condition in Poales

(Givnish et al., 2010).

According to Schmid (1985) the septal nectary is a secretive tissue that is located in a chamber formed in the septum of the ovary due to the absence of post-genital intercarpelar fusion in the septum region. Remizowa et al. (2010) state that in a syncarpic gynoecium with septal nectary the carpel primordia are initiated individually. The ventral portion of adjacent primordia is joined in a relatively late stage of the development. The nectaries are developed in the external wall of those primordia before the fusion of the carpels. Thus after the fusion the nectaries are internalized in the septa region or in the basal region of the carpels (below the locules). In flowers with inferior ovary the intercalary growth occurs since the base of the primordium, including the region of the nectariferous tissue proliferation. In this case the nectaries are found in the region of the ovary septa. In flowers with superior ovary the nectariferous tissue starts to proliferate in the basal portion of the primordia, and the region of the carpel with intercalary growth is located above the proliferation of the nectariferous tissue, and thus the nectaries are located below the locules of the ovary - formed by the folding of the carpel and by the intercalary growth.

In the studied species of the subfamily Bromelioideae, which have flowers with inferior ovary, the nectaries are truly septal, since they occur in the septa of the ovary. In the

Pitcairnioideae and Tillandsioideae subfamilies we opted to name them infralocular nectaries, since they occur below the locules and not between them (in the septa region).

According to Remizowa et al. (2010) the septal nectaries evolved towards their internalization, which means that infralocular nectaries would be considered plesiomorphic in relation to the septal interlocular nectaries. This hypothesis suggest that through the internalization of the nectaries their secretive surface would be expanded, producing more nectar when compared to infralocular nectaries. However, Sajo et al. (2004a), when analyzing

92 Capítulo II Oliveira, F.M.C.

species from five of the eight subfamilies of Bromeliaceae, propose that the condition of epigynous flowers (and consequently with septal interlocular nectaries) would be plesiomorphic - with several reversions to the hypogynous condition - or that epigynous flowers would be originated more than once in the group. When analyzing flower buds of species of

Bromelioideae, Pitcairnioideae and Tillandsioideae we verify that with the exception of species of Bromelioideae the remaining species have infralocular nectaries, corroborating the results of

Sajo et al. (2004a).

Regarding their classification, nectaries are structured - i.e. they are morphologically recognizable - and show a specialized anatomical structure: they have vascular bundles that are responsible for supplying water and nutrients, parenchyma where the nectar is stored, and epidermal cells through which the nectar is excreted (Fahn, 1979). The region where the nectaries are active varies between the studied species. In the species with superior ovaries, the nectaries are active below the locules even when only a small portion of the nectaries is present in the locule region as in P. flammea. In the flowers with inferior ovaries, the nectaries can be active in the region of ovule insertion or even below this region.

The obturator tissue has been thoroughly studied especially for its importance to reproduction, being responsible for chemically and/or physically directing the pollen tube towards the micropyle and for the nutrition of the pollen tube (Tilton & Horner Jr., 1980;

Herrero, 2000; Vardar et al., 2012). When the pollen tube is chemically directed, the obturator is referred to as being secretive. Its cells generally show prominent, heavily stained nuclei that are indicative of its secretive activity. Sometimes it is possible to note the presence of secretion in the locules of the ovaries. The chemical composition of the exudate can vary, showing polysaccharides, proteins and lipids (Singh & Walles, 1992; Hudák et al., 1993; Herrero, 2000;

Oliveira et al., 2016). When the obturator directs the pollen tube mechanically, this tissue resembles trichomes (Tilton & Horner Jr., 1980; Herrero, 2000). Its origin can be placentary as

93 Capítulo II Oliveira, F.M.C.

seen in the studied species, or even funicular. In some cases it has mixed origin (Tilton & Horner

Jr., 1980).

In Poales this tissue is quite diverse regarding either its origin or its shape. In

Cyperaceae it is formed by trichomes with funicular origin (Reynders et al., 2012), in Juncaceae and Rapateaceae by trichomes with placentary origin (Oriani & Scatena, 2012; Oriani &

Scatena, 2013). In Mayacaceae the obturator is secretive, and originated from the placenta

(Oriani et al., 2012). In Bromeliaceae the obturator tissue has a palisade shape with evident and heavily stained nuclei, and secretive activity. They can be present in a single or in multiple cell layers (Fagundes & Mariath, 2010; Oliveira et al., 2016). In the studied species the obturator tissue has a single layer of cells in palisade with evident nuclei. In all species the tissue has placentary origin similarly to what was observed for the remaining species of the family by

Fagundes & Mariath (2010) and Oliveira et al. (2016).

In the style region, the internal epidermis has cells with prominent nucleus and dense cytoplasmatic content that highlights its secretive nature. This tissue is named transmitting tissue, stimulates through its secretion the growth and directing of the pollen tube (McCormick

& Yang, 2005). There are two types of transmitting tissue: the first type is formed by secretive epidermal cells in which the pollen tube grows in the stylar channel - the style is hollow; in the second type the transmitting tissue has several layers of cells underneath the epidermis. In this type the pollen tube goes through those cells until it reaches the ovary locule - the style is solid

(Went & Willemse, 1984; Erbar, 2003). In monocotyledons prevails the hollow style in which the cells from the stylar channel generally have dense cytoplasm and are rich in organells (Went

& Willense, 1984; Sajo & Rudall, 2012). According to this classification, Bromeliaceae has the type I, hollow, sometimes with secretion in the stylar channel, as observed in the current study.

In Poales hollow styles with secretive epidermis were recorded in Xyridaceae (Remizowa et

94 Capítulo II Oliveira, F.M.C.

al., 2012), Mayacaceae (Oriani & Scatena, 2012), Juncaceae (Oriani et al., 2012) and

Rapateaceae (Oriani & Scatena, 2013), and are likely plesiomorphic in the order.

5. Conclusions

The results obtained in this work reveal anatomical characters of both the androecium and gynoecium that are useful for the systematics of the family, especially regarding the gynoecium vascularization. It is also noteworthy the necessity for future works to approach the floral anatomy in Bromeliaceae by analyzing all eight subfamilies in order to better understand the evolution of this group.,

6. Acknowledgements

The authors thank Shyguek N.A.Miyamoto, Mathias E. Engels, Vanessa K. Kowalski and Rafael B. Louzada for providing the plant material for this research. The authors also acknowledge the National Counsel of Technological and Scientific Development (CNPq - process number 140115/2013-7 and 308070/2012-7) for the funding.

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Table 1: Studied species, grouped by subfamily, and their respective vouchers. Subfamily Specie Voucher Occurence Bromelioideae Aechmea bromeliifolia S. N.A Miyamoto e Ponta Grossa- (Rudge) Baker A.C. Azevedo 150 PR (HUPG) Aechmea gamosepala S.N.A. Miyamoto, M. Matinhos-PR Wittm. Engels & Kowalski, V.K. 95 (HUPG) Aechmea cylindrata Lindm. S.N.A. Miyamoto & Quatro Barras- F.M.C. de Oliveira 61 PR (HUPG) Aechmea nudicaulis (L.) S.N.A. Miyamoto, A.C. Paranaguá- PR Griseb. Azevedo, B.N.S. Lima, G. Migliorini & M. Santana 44 (HUPG) Aechmea ornata Baker S.N.A. Miyamoto & Guaratuba- PR V.K. Kowalski, 115 (HUPG) Cryptanthus bahianus R.B. Louzada s/n (SP) Recife - PE L.B.Sm. Nidularium amazonicum M.E.Engels s/n (UPCB) Antonina-PR (Baker) Linden & E. B.N.S. Lima, Morren ex Lindm. V.K.Kowalski, S.N.A. Miyamoto, 50 Nidularium longiscapum F.M.C de Oliveira, 54 São Paulo-PR B.A. Moreira & Wand. (HUPG) Neoregelia spectabilis (T. F.M.C. de Oliveira 55 Ponta Grossa- Moore) L.B.Sm. (HUPG) PR Nidularium procerum Lima, B.N.S.; Koza, Morretes- PR Lindm. V.K.; Miyamoto, S.N.A. 15 (HUPG) Quesnelia testudo Lindm. F.M.C. de Oliveira, Guaraqueçaba- S.N.A. Miyamoto, M.E. PR Engels & V.K. Kowalski, 38 (HUPG) Pitcairnoideae Dyckia tuberosa (Vell.) F.M.C de Oliveira, 60 São Paulo -SP Mez (HUPG) Encholirium sp. F. M. C. de Oliveira, 61 São Paulo -SP (HUPG) Pitcairnia flammea Lindl. Engels, M.E. 310 Jaguariaíva- PR (HUPG) Tillandsioideae Vriesea inflata (Wawra) M.P.M. Martínez et al. Morretes-PR Wawra 218 (HUPG) Vriesea platynema Gaudich. V. K. Kowalski & R. Balsa Nova- Kowlaski 22 (HUPG) PR

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Table II: Anatomical characters of the androecium and gynoecium of the studied species of Bromeliaceae. Character 1: Presence of aerenchyma in the filaments. Character 2: Presence of ornamented cuticle in the anthers. Character 3: Number of vascular bundles in the filaments. Character 4: Flattened filaments. Character 5: Round or triangular filaments. Character 6: Superior ovary. Character 7: Inferior ovary. Character 8: Aerenchyma in the ovary/hypanthium walls. Character 9: Ornamented cuticle in the style and stigma. Character 10: Stomata in the walls of the ovary/hypanthium. Character 11: Obturator with short walls. Character 12: Obturator with long walls. Character 13: Ventral and dorsal carpelar bundles in the style and stigma. Character 14: Ovules with chalazal appendages.

Species/ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 characters Bromelioideae Aechmea - - 1 - + - + - - - - + - - bromeliifolia A. cylindrata - - 1 + - - + - + + + - - - A. gamosepala - + 1 + - - + - - - + - - - A. nudicaulis - + 1 - + - + - - + - + - - A. ornata - + 1 - + - + - + - + - - - Cryptanthus - - 1 + - - + - - - + - - - bahianus Neoregelia - - 1 - + - + - - - + - - - spectabilis Nidularium - + 1 + - - + - + - + - - + amazonicum N. longiscapum - - 1 - + - + + + - + - - - N. procerum - + 2 + - - + + - - + - - - Quesnelia - + 1 + - - + - + - + - - - testudo Pitcairnoideae Dyckia - - 2 + - + - + + + + - + + tuberosa Encholirium sp. - + 3 + - + - - - - + - + + Pitcairnia - + 2 + - + - - - - + - + + flammea Tillandsioideae Vriesia inflata - - 2 - + + - + - - + - - - V. platynema + + 1 + - + - - - - - + - +

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Figure 1: Transversal sections of the flower buds. A: Nidularium longiscapum B. A. Moreira & Wand. B: Nidularium amazonicum (Baker) Linden & E. Morren ex Lindm. C: Nidularium procerum Lindm. D: Cryptanthus bahianus L. B. Sm. E: Vriesea inflata (Wawra) Wawra. F: Quesnelia testudo Lindm. An = Anther, Fi = Filament, Ov = Ovary, Pe = Petal, Pe-Fi = Petal- Stamen Tube, Se = Sepal, St = Style

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Figure 2: A-G: Transversal sections of the flower buds highlighting the filaments. H-I: Scanning Electron Microscopy (SEM) of the apical portion of the filaments. A: Nidularium amazonicum (Baker) Linden & E. Morren ex Lindm. B: Pitcairnia flammea Lindl. C: Vriesea inflata (Wawra) Wawra. D: Encholirium sp. E: Nidularium procerum Lindm. F: Dyckia tuberosa (Vell.) Mez. G: Nidularium amazonicum (Baker) Linden & E. Morren ex Lindm. H: N. amazonicum. I: Quesnelia testudo Lindm. Thick arrows indicate vascular bundles. Thin arrows indicate cuticle deposition in the epidermal cells.

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Figure 3: Transversal sections of the flower buds highlighting the anthers. A: Aechmea ornata Baker. B: Quesnelia testudo Lindm.. C: Nidularium procerum Lindm. D: Nidularium longiscapum B.A. Moreira & Wand. E: Encholirium sp. F: Cryptanthus bahianus L.B.Sm. Ar = Aerenchyma. Arrows indicate the endotecium, which cells show wall thickening.

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Figure 4: Transversal sections of the flower buds. Details of the anthers. A: Dyckia tuberosa (Vell.) Mez. B: Encholirium sp. C: Nidularium longiscapum B.A. Moreira & Wand. D: Quesnelia testudo Lindm. Arrows indicate epidermal walls with cuticle deposition.

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Figure 5: Scanning Electron Microscopy (SEM) of the anthers in the studied species. A: Dyckia tuberosa (Vell.) Mez. B: Encholirium sp. C: Neoregelia spectabilis (T. Moore) L.B.Sm. D: Nidularium amazonicum (Baker) Linden & E. Morren ex Lindm. E: Aechmea nudicaulis (L.) Griseb. F: Vriesea platynema Gaudich.

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Figure 6: Transversal sections of the flower buds of the sudied species from the Bromelioideae subfamily, in the ovary region. A: Nidularium procerum Lindm. B: Nidularium amazonicum (Baker) Linden & E. Morren ex Lindm. C: Aechmea nudicaulis (L.) Griseb. D: Aechmea ornata Baker. E: Nidularium procerum Lindm. F: Neoregelia spectabilis (T. Moore) L.B.Sm. Ar = Aerenchyma. Hi = Hypanthium. Lo = Ovary locule. Pl = Placenta, Sn = Septal nectary. Arrows indicate epidermis with cells with thickened walls and with a silica crystal.

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Figure 7: Transversal sections of the flower buds of the studied species from the Pitcairnioideae and Tillandsioideae subfamilies, in the ovary region. A: Vriesea inflata (Wawra) Wawra. B: Dyckia tuberosa (Vell.) Mez. C: Pitcairnia flammea Lindl. D: Dyckia tuberosa (Vell.) Mez. E e F: Encholirium sp. Fi = Filament. Lo = Locule. Ov = Ovary. Pe = Petal. Se = Sepal. Arrows indicate vascular bundles of the filaments.

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Figure 8: Transversal sections of the ovaries, highlighting the ovules. A: Aechmea gamosepala Wittm. B: Dyckia tuberosa (Vell.) Mez. C: Pitcairnia flammea Lindl. D: Nidularium procerum Lindm. E-F: Scanning Electron Microscopy (SEM) of the ovules. E: Nidularium amazonicum (Baker) Linden & E. Morren ex Lindm. F: Vriesea platynema Gaudich. G-H: Transversal sections of the ovaries highlighting the obturator tissue. G: Vriesea platynema Gaudich. H: Aechmea ornata Baker. Arrows indicate the chalazal appendages of the ovules

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Figure 9: Transversal sections of the flower buds, highlighting the septal and infralocular nectaries. A: Quesnelia testudo Lindm. B: Neoregelia spectabilis (T. Moore) L.B.Sm. C e D: Nidularium amazonicum (Baker) Linden & E. Morren ex Lindm. E: Longitudinal section of the flower bud of Vriesea platynema Gaudich. F: Dyckia tuberosa (Vell.) Mez. G: Vriesea inflata (Wawra) Wawra.

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Figure 10: Transversal sections of the flower buds, highlighting the styles. A: Nidularium longiscapum B.A. Moreira & Wand. B: Nidularium amazonicum (Baker) Linden & E. Morren ex Lindm. C: Dyckia tuberosa (Vell.) Mez. D: Encholirium sp. E: Quesnelia testudo Lindm. F: Detail of the epidermis of the style in N. longiscapum. Thick arrows indicate vascular bundles of the style. Thin arrows indicate cuticle deposition in the epidermal cells of the style

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Figure 11: Transversal sections of the flower buds, highlighting the stigmas. A: Quesnelia testudo Lindm. B: Nidularium procerum Lindm. C: Nidularium longiscapum B.A. Moreira & Wand. D: Aechmea ornata Baker. E: N. longiscapum. F: Dyckia tuberosa (Vell.) Mez. G-K: Scanning Electron Microscopy (SEM) of the stigma in the studied species. G: Aechmea gamosepala Wittm. H: Dyckia tuberosa (Vell.) Mez. I: Nidularium procerum Lindm. J: D. tuberosa. K: Vriesea platynema Gaudich. Thick arrows indicate vascular bundles of the stigma.

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References

APG IV. 2016. An update of the Angiosperm phylogeny group classification for the orders and families of flowering plants: APG IV. Botanical Journal of Linnean Society 181: 1-20.

Arrais MG. 1989. Aspectos anatômicos de espécies de Bromeliaceae da Serra do Cipó- MG, com especial referência à vascularização floral. Master Dissertation. Universidade de São Paulo, São Paulo.

Bernadello LM, Galetto L, Juliani HR. 1991 Floral nectar, nectary structure and polinators in some Argentinean Bromeliaceae. Annals of Botany 67: 401-411.

Bernadello G. 2007. A systematic survey of floral nectaries. In: Nicolson, S.W.; Nepi, M.; Pacini, E. (Eds.). Nectaries and Nectar. Springer

Brighina L, Fiordi ACF, Palandri MR. 1984. Structural characteristics of mesophyll in some Tillandsia species. Phytomorphology 34:191-200.

Brown GK, Terry RG. 1992. Petal appendages in Bromeliaceae. Am. J. Bot. 79 (9): 1051-1071.

Butcher D., Gouda, EJ. cont. updated. The new bromeliad taxon list. . University Botanic Gardens, Utrecht (accessed 13.01.17).

Carvalho MLS, Nakamura AT, SajoMG. 2009. Floral anatomy of Neotropical species of Mayacaceae. Flora 204: 220-227.

Carvalho JDT, Oliveira, JMS, Freitas CC, Martins MS. 2016. Stamen morphoanatomy of Dyckia Schult.f. (Bromeliaceae, Pitcairnoideae) species: new data for taxonomic use. Acta Botânica Brasilica doi: 10.1590/0102-33062016abb0112.

Carlquist S. 1969. Toward acceptable evolutionary interpretations of floral anatomy. Phytomorphology 19 (4): 332-362.

Dorneles MP, Oliveira JMS, Canto-Dorow TS. 2014. Dyckia racinae L.B.Sm. (Bromeliaceae): morphological description emphasizing the reproductive structures. Iheringia 69 (2): 397-404.

Eames AJ. 1931. The vascular anatomy of the flower with refutation of the theory of carpel polymorphism. American Journal of Botany 28 (3): 147-188.

123 Capítulo II Oliveira, F.M.C.

Erbar C. 2003 Polen tube transmitting tissue: place to competition of male gametophytes. International Journal of Plant Science 164 (S): S265-S277.

Fagundes NF, Mariath JEA. 2010. Morphoanatomy and ontogeny of fruit in Bromeliaceae species. Acta Botânica Brasílica 24 (3): 765-779.

Fagundes NF, Mariath JEA. 2014. Ovule ontogeny in Billbergia nutans in the evolutionary context of Bromeliaceae (Poales). Plant Systematics and Evolution 300: 1323-1336.

Fahn A. 1952. On the structure of floral nectaries. Botanical Gazette 465-470.

Fahn A. 1979. Secretory tissues in plants. Academic Press, London.

Ferrari RC, Oriani A. 2016. Floral anatomy and development of Saxofridericia aculeate (Rapateaceae) and its taxonomic and phylogenetic significance. Plant Systematics and Evolution. DOI 10.1007/s00606-016-1361-z.

Fiordi AC, Palandri MR. 1982. Anatomic and ultrastructural study of septal nectary in some Tillandsia (Bromeliaceae) species. Caryologia 35 (4): 477-489.

Givnish TJ, Millan KC, Berry PE, Systma KJ. 2007. Phylogeny, adaptive radiation, and historical biogeography of Bromeliaceae inferred from ndhF sequence data. Aliso 23: 3-26.

Givnish TJ, Ames M, McNeal JR, McKain MR, Steele PR, de Pamphilis CW, Graham SW, Pires JC, Stevenson DW, Zomlefer WB, Briggs BG, Duvall MR, Moore MJ, Heaney JM, Soltis DE, Soltis PS, Thiele K, Leebens-Mack JH. 2010. Assembling the tree of the monocotyledons: plastome sequence phylogeny and evolution of Poales. Annals of Missouri Botanical Garden 97: 584–616

Givnish TJ, Barfuss MHJ, Van EEB, Riina R, Schulte K, Horres R, Gonsiska PA, Jabaily RS, Crayan M, Smith AC, Winter K, Brown GK, Evans TM, Holst BK, Luther H, Till W, Zizka G, Berry PE, Systma KJ. 2011. Phylogeny, adaptive radiation, and historical biogeography in Bromeliaceae: insights from an eight-locus plastid phylogeny. American Journal of Botany 98 (5): 1-24.

Herrero M. 2000. Changes in the Ovary Related to Pollen Tube Guidance. Annals of Botany 85: 79-85.

124 Capítulo II Oliveira, F.M.C.

Hudák J, Walles B, Vennigerholz F. 1993. The transmitting tissue in Brugmansia suaveolens L.: ultrastructure of the stylar transmitting tissue. Annals of Botany 71:177–186

Jacques-Félix H. 2000. The discovery of a Bromeliad in Africa: Pitcairnia Feliciana. Selbyana 21 (1,2): 118-124.

Johansen DA 1940. Plant microtechnique. McGraw-Hill, New York.

Koch K, Bhushan B, Barthlott W. 2008. Diversity of Structure, morphology and wetting of plants surfaces. Soft Matter 4: 1943-1963.

Krauss B H. 1949. Anatomy of the vegetative organs of the pineapple, Ananas comosus (L.) Merr. Botanical Gazette 110: 333-404.

Kuhn SA, Nogueira FM, Fagundes NF, Mariath JEA. 2016. Morphoanatomy of the ovary and the ovule in Bromeliaceae subfamily Tillandsoideae and its systematic relevance. Botanical Journal of Linnean Society 181: 343-361.

Linder HP, Rudall PJ. 2005. Evolutionary history of Poales. Annual Review of Ecology Evololution and Systematics 36:107–124

Mauseth JD. 1988. Plant anatomy. Benjamin/ Cummings. Menlo Park, California.

McCormick S, Yang H. 2005. Is there more than one way to attract a pollen tube? Trends on Plant Science 10 (6): 260-261.

Mendes SP, Mastroberti AA, Mariath, JEA, Vieira RC, de Toni KV. 2014. Ovule and female gametophyte development in the Bromeliaceae: an embryological study of Pitcairnia encholirioides. Botany . 92 (12): 883-894.

Menezes NL. 1979. Evolution in Velloziaceae, with special reference to androecial characters. In: Brickell, C.D.; Cutler, D.F.; Gregory, M. (Eds.) Petaloid Monocotyledons: Hoorticultural and Botanical Research (Linnean Society Symposium Series): 117-138.

Nogueira FN, Fagundes NF, Kuhn SA, Fregonezi JN, Mariath JEA. 2015. Ovary and ovule anatomy in the Nidularioid Complex and its taxonomic utility. Botanical Journal of Linnean Society 177: 66-77.

125 Capítulo II Oliveira, F.M.C.

Novikoff AV, Odintsova A. 2008. Some aspects of comparative gynocium morphology in three bromeliad species. Wulfenia 15:13-24.

Oliveira FMC, Souza AM, Correa BBR, Maeda TM, Melo-de-Pinna GFA .2016. Anatomia floral de Aechmea distichantha Lem. e Canistropsis billbergioides (Schult. & Schult.f) Leme (Bromeliaceae). Hoehnea 43 (2): 183-193.

Oriani A, Scatena VL. 2012. Floral anatomy of xyrids (Poales): contribution to their reproductive biology, taxonomy, and phylogeny. International Journal of Plant Science 173 (3): 767-779.

Oriani A, Stutzel T, Scatena VL. 2012. Contributions to the floral anatomy of Juncaceae (Poales- Monocotyledons). Flora 207: 334-340.

Oriani A, Scatena VL. 2013. The taxonomic value of floral characters in Rapateaceae (Poales- Monocotyledons). Plant Systematics and Evolution 299: 291-303.

Palací CA, Brown GK, Tuthill DE. 2004. The seeds of Catopsis (Bromeliaceae, Tillandoideae). Systematic Botany 29 (3): 518-527.

Puri V. 1951. The role of floral anatomy in the solution of morphological problems. Botanical Review 22 (7): 471-553.

Rao VS. 1975. Septal glands: their form, structure and function. In: Ram, H.Y.M.; ShaH, J.J.; Shah, C.K. (eds). Form, Structure and function in plants. Sarita Prakashan: Nauchandi Meerut India.

Reynders M, Vrijdaghs A, Larridon I, Huygh W, Leroux O, Muasya AM, Goetghebeur P. 2012. Gynoecial anatomy and development in Cyperioideae (Cyperaceae, Poales): congenital fusion of carpels facilitates evolutionary modifications in pistil structure. Plant Ecology and Evolution 145: (1): 96-125.

Remizowa MV, Sokoloff DD, Rudall P. 2006. Evolution of the monocot gynoecium: evidence from comparative morphology and development in Tofieldia, Japonolirium, Petrosavia and Narthecium. Plant Systematics and Evolution 258: 183-209.

Remizowa MV, Sokoloff DD, Rudall P. 2010. Evolutionary history of the monocot flower. Annals of the Missouri Botanical Garden 97 (4): 617-645.

126 Capítulo II Oliveira, F.M.C.

Remizowa, MV, Kuznetsov, AN, Kuznetsova, SP, Rudall, PJ, Nuraliev, M S, Sokoloff, DD. 2012. Flower development and vasculature in Xyris grandis (Xyridaceae, Poales); a case study for examining petal diversity in monocot flowers with a double perianth. Botanical Journal of Linnean Society 170: 93-111.

Rudall P. 2002. Homologies of inferior ovaries and septal nectaries in monocotyledons. International Journal of Plant Science 163 (2): 261-276.

Ruzin SE. 1999. Plant Microtechnique and Microscopy. Oxford University Press, New York.

Sajo MG, Rudall PJ, Prychid CJ. 2004a. Floral anatomy of Bromeliaceae, with particular reference to the evolution of epigyny and septal nectaries in commelinid monocots. Plant Systematics and Evolution 247: 215-231.

Sajo MG, Prychid CJ, Rudall PJ. 2004b. Structure and development of the ovule in Bromeliaceae. Kew Bulletin 59: 261-267.

Sajo MG, Furness CA, Prychid CJ, Rudall PJ. 2005. Microsporogenesis and anther development in Bromeliaceae. Grana 44:65-74.

Sajo MG, Rudall P. 2012. Morphological evolution in the graminid clade: comparative floral anatomy of the grass relatives Flagellariaceae and Joinvilleaceae. Botanical Journal on Linnean Society 170: 393-404.

Sakai WS. 1973. Simple method for diferential staining of paraffin embedded plant material using Tolidine Blue O. Stain technology 48 (5): 247-249.

Schmid R. 1985. Functional interpretations of the morphology and anatomy of septal nectaries. Act Bototanica Neerland 34 (1): 125-128.

Silveira M. 1989. O preparo de amostras biológicas para microscopia eletrônica de varredura. In: W. de Souza (ed.). Manual sobre Técnicas Básicas em Microscopia Eletrônica de Varredura. Sociedade Brasileira de Microscopia Eletrônica,v. 1, pp. 71-82.

Singh S,Walles B. 1992. The ovarian transmitting tissue. International Journal of Plant Sciences 153: 205-211.

127 Capítulo II Oliveira, F.M.C.

Smith LB. 1974. Introduction. In: Pitcairnioideae (Bromeliaceae). Flora Neotropica. New York: Hafner Press, 14 (1): 1-3.

Smith LB,Downs RJ. 1974. Pitcairnioideae (Bromeliaceae). Flora Neotropica.New York, Hafner Press, 14 (1): 1-658.

Smith LB, Downs RJ. 1977. Tillandsioideae (Bromeliaceae). Flora Neotropica. New York: Hafner Press,14 (2): 663-1492.

Smith LB, Downs RJ. 1979. Bromelioideae (Bromeliaceae).Flora Neotropica.New York, Hafner Press, 14 (3):1493-2142.

Souza EH, Carmello-Guerreiro SM, Souza FVD, Rossi ML, Martinelli AP. 2016. Stigma structure and receptivity in Bromeliaceae. Scientia Horticulturae 203: 118-125.

Tilton VR, Horner Jr, HT. 1980. Stigma, style, and obturator of Ornithogalum caudatum (Liliaceae) and their function in the reproductive process. American Journal of Botany 67: 1113-1131.

Tomlinson PB. 1969. Comelinales - Zingiberales.In: C.R. Metcalfe (ed.).Anatomy of the monocotyledons: III. Claredon Press, Oxford, pp.193-294.

Varadarajan GS, Gilmartin AJ. 1988. Seed morphology of the subfamily Pitcairnoideae (Bromeliaceae) and its systematic implications. American Journal of Botany 75 (6): 808-818.

Vardar F, Ismailoglu I, Ünal M. 2012. Embryological and cytological features of Gagea bohemica (Liliaceae). Turkish Journal of Botany 36: 462-472.

Venturelli M, Bouman F. 1988. Development of ovule and seed in Rapateaceae. Botanical Journal of Linnean Society 97: 267-294.

Went, JL, Willemse, MTM. 1984. Fertilization. In: Embryology of Angiosperms, ed. B.M. Johri, pp. 273-317. Berlin: Springer-Verlag.

Whitney HM, Bennet V, Dorling M, Sandbach L, Prince D, Chittka L, Glover B. 2011. Why do so many petals have conical epidermal cells? Annals of Botany 108: 609-616.

Wilson CL 1942. The telome theory and the origin of the stamen. American Journal of Botany.29: 759-764.

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Capítulo III

New record of colleters in Monocots: Bromeliaceae (Poales)

Fernanda Maria Cordeiro de Oliveira 1,2e Gladys Flavia de Albuquerque Melo-de-Pinna1

1 Universidade de São Paulo, Instituto de Biociências, Departamento de Botânica, Laboratório

de Anatomia vegetal, Rua do Matão 277, 05508-090, São Paulo, SP, Brasil

2Corresponding author: [email protected]

A ser submetido ao periódico Botany

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Abstract Tillandsia L. is the largest genus within the subfamily Tillandsoideae, and it comprises species with filiform or linear leaves that have a circular form when observed in cross-section (terete leaves). It is currently known that terete leaves can be formed as result of a diffuse growth or peripheral activity of the marginal meristem. Studies on foliar development are important not only as a way to identify the growth regions related to the leaf formation, but they also allow for the observation of structures responsible for the protection of this region, such as the colleters. Colleters are secretory structures present in growth regions, such as the shoot apical meristem. These structures are often reported for Eudicot species, have been attributed with taxonomic values for some families. Among Monocots, colleters are still scarcely studied, and they have been described only for Orchidaceae and Rapateaceae. In the present study we report data on foliar ontogenesis of three Tillandsia species. In order to do so, vegetative shoot apices were sampled, fixed and processed according to usual plant anatomy techniques. Histochemical tests were carried out in order to characterize the main classes of compounds in the exudate of the glandular trichomes. Only T. tricholepis was observed to have glandular trichomes, which were observed in the adaxial surface of the foliar sheath close to the shoot apical meristem. The secretion produced by these trichomes was observed to be a mixed one, composed of lipophilic, mucilaginous and protein compounds. The localization of these trichomes and the composition of their secretion indicate the relationship between these colleters and the protection of the shoot apical meristem. This is the first record of colleters in Bromeliaceae. This data can be useful for future studies on the colleters of Monocotyledons.

Key-words: anatomy, histochemisty, trichomes, foliar ontogenesis, apical meristem protection.

131 Capítulo III Oliveira, F.M.C.

Resumo Tillandsia L., maior gênero da subfamília Tillandsoideae, possui espécies cujas folhas são filiformes ou lineares, e em secções transversais possuem forma terete. Hoje sabe-se que as folhas teretes podem ser resultado de um crescimento difuso ou ainda da atividade periférica do meristema marginal. Estudos de desenvolvimento foliar são importantes não apenas para identificar as regiões de crescimento envolvidas na formação da folha, mas também para o registro de estruturas responsáveis pela proteção desta região, como os coléteres. Coléteres são estruturas secretoras que ocorrem em regiões ainda em crescimento, como por exemplo o meristema apical caulinar. Estas estruturas são muito reportadas para as eudicotiledôneas, possuindo valor taxonômico em algumas famílias. Nas monocotiledôneas ainda são pouco estudados, sendo descritos apenas para Orchidaceae e Rapateaceae. Neste estudo, são relatados dados da ontogênese foliar de três espécies de Tillandsia. Para tanto, ápices vegetativos foram coletados, fixados e processados segundo técnicas usuais em anatomia vegetal. Testes histoquímicos foram realizados para a caracterização das principais classes de compostos presentes no exudado dos tricomas glandulares. Apenas T. tricholepis apresenta tricomas glandulares na face adaxial da bainha foliar, em regiões próximas ao meristema apical caulinar.

A secreção produzida por estes tricomas é mista, contendo compostos lipofílicos, mucilagens e proteínas. A localização destes tricomas e a composição de sua secreção indicam o envolvimento destes coléteres com a proteção do meristema apical caulinar. Este é o primeiro registro de coléteres em Bromeliaceae e nossos dados podem ser úteis para futuros estudos de coléteres nas monocotiledôneas.

Palavras chave: anatomia, histoquímica, tricomas, ontogênese foliar, proteção do meristema apical.

132 Capítulo III Oliveira, F.M.C.

1. Introduction

Bromeliaceae is nearly exclusively a neotropical family, the only exception being

Pitcairnia flammea (A. Chev.) Harms & Mild., which occurs in the western region of the

African continent (Smith 1974, Jacques-Félix 2000). The family comprises ca. 3400 species grouped in 58 genera and currently subdivided in eight subfamilies: Bromelioideae,

Tillandsioideae, Pitcairnioideae s. s., Navioideae, Puyoideae, Brocchinioideae, Hechtioideae and Lindmanioideae (Givnish et al. 2007, Givnish et al. 2012, Butcher and Gouda, continuously updated).

The subfamily Tillandsoideae is characterized for comprising species that have entire foliar blades, superior ovary, capsule fruits and seeds with appendices (Till 2000). The most representative genus within the subfamily is Tillandsia L., which groups ca. 550 species, ca. 70 of them occurring in Brazil (Butcher and Gouda, continuously updated; BFG 2015). Most species within this genus inhabits dry environments, and their leaves usually have highly specialized peltate trichomes that assure water absorption (Benzing 1976, Benzing 2000).

The genus Tillandsia also contains species that have linear of filiform leaves, which are circular when observed in cross-sections, i.e. terete (Scatena and Segecin 2005). Terete leaves occur in many Angiosperm families, and according to Esau (1960) their morphology results from a reduced or even absent activity of marginal growth during the leaf development.

Recent works on foliar development have shown that many terete leaves may also result from diffuse growth, such as it occurs in Didiereaceae (Hernandes-Lopes et al. 2016), or from a peripheral activity of the marginal meristem, such as in succulent leaves of Anacampserotaceae,

Orchidaceae, Aizoaceae and Asteraceae (Melo-de-Pinna et al. 2016).

Studies on foliar development allow not only the description of growth regions involved during leaf formation, but they also allow the observation of specialized structures

133 Capítulo III Oliveira, F.M.C.

that protect the meristematic region, such as colleters. Colleters are glandular trichomes or complex secretory structures found close to growth regions. The mucilaginous and/or lipophilic secretion they release helps protecting the meristematic region against desiccation (Fahn 1979,

Fahn 1982, Cardoso-Gustavson et al. 2014). These structures are widely described among

Eudicot species, and have been attributed with taxonomic value for some families, such as

Rubiaceae and Apocynaceae (Lersten 1974, Thomas 1991, Thomas and Dave 1991). On the other hand, scarce observations of colleters have been made among Monocotyledons, and authors have only reported these structures to occur in Orchidaceae (Mayer et al. 2011,

Cardoso-Gustavson et al. 2014) and Rapateaceae (Oriani and Scatena 2012, Ferrari and Oriani

2016).

Based on the analysis of the vegetative shoot apical meristem region of Tillandsia species the present study reports data regarding foliar ontogenesis and describes for the first time the presence of colleters in Bromeliaceae.

2. Materials and methods

2.1 Plant material

Three species of the genus Tillandsia were selected: Tillandsia recurvata (L.) L.,

Tillandsia tricholepis Baker and Tillandsia usneoides (L.) L., all of which have filiform leaves.

Part of the sampled material was deposited at the Herbarium of the State University of Ponta

Grossa (HUPG).

2.2 Light microscopy

Shoot apices were fixed in a 50% solution of formaldehyde-ethanol-acetic acid (FAA

50), as described by Johansen (1940) for the preservation of hydrophilic compounds. For the preservation of liposoluble compounds, shoot apices were fixed in neutral buffered formalin

134 Capítulo III Oliveira, F.M.C.

(NBF) as described by Lillie (1965). The material was dehydrated in ethanol/butanol series and embedded in paraplast (Ruzin 1999). Seriated transversal and longitudinal sections (6 to 10 µm thick) were obtained in a rotatory microtome.

Sections were deparaffinized by using a xylol solution, stained with 1% aqueous Astra blue and 1% Safranin in a 50% ethanol solution (modified from Kraus and Arduin, 1997) and finally mounted in Canada balsam.

A variety of histochemical tests was performed. Ruthenium red (Gregory and Baas

1989), tannic acid x ferric chloride (Pizzolato 1977) and methylene blue (Johansen 1940) were applied for the detection of pectin. Starch black B (Fisher 1968) was applied for the detection proteins. Nile blue sulfate (Cain 1947) was applied for the detection of acid and neutral lipids.

Copper acetate/ Rubeanic acid (Ganter and Jollés 1969) was applied for the detection fatty acids. The PAS reaction (McManus 1948) was applied for the detection of polysaccharides.

Sudan IV (Pearse 1985) and Sudan black B (Pearse 1985) were applied for the detection of total lipids. Finally, ferric chloride (Johansen 1940) was applied for the detection of phenolic compounds.

2.3. Scanning Electron Microscopy (SEM)

In order to perform SEM analysis shoot apices were carefully dissected and dehydrated in ascending ethanol series. Samples were then submitted to critical point drying with CO2 and metalized with gold (Silveira 1989). The analysis was carried out using a Zeiss DMS- 940

Scanning Electron Microscope.

3. Results Seriated cross-sections of the vegetative shoot apical region show that Tillandsia recurvata and T. usneoides have an alternate phyllotaxis (Fig. 1), while T. tricolepsis has a spiral phyllotaxis (Fig. 2).

135 Capítulo III Oliveira, F.M.C.

During foliar ontogenesis, differences regarding the activity of the marginal growth region were observed among the species. In T. recurvata a marked growth of the foliar sheath results in leaf margins that overlap each other (Fig.1A-B). Despite also having an alternate phyllotaxis, T. usneiodes does not show overlapping in the margins of the foliar sheath (Fig.

1C-D).

The species T. tricholepis, which has a spiral phyllotaxis (Fig. 2A-B) shows an equitant vernation pattern only in distalmost regions of the shoot apical meristem (Fig. 2C).

Cross-section of the regions where leaf primordia are individualized show no glandular trichomes in neither one of the three analyzed species (Fig. 1 e 2A-B). Glandular trichomes were only observed at the adaxial surface of the adjacent leaf sheaths of T. tricolepsis (Fig. 2A-

B; Fig. 3A-B). When seen in frontal view these multicellular trichomes are observed to be palmate (Fig. 3C) or digitate (Fig. 3D-E) ones. There are no distinction between the cell of the peduncle and those of the secretory head. These trichomes were additionally observed to occur close to the axillary buds (Fig. 3F).

The results of the histochemical tests carried out for the species T. tricholepis in order to characterize the main compounds of the trichome’s secretion are summarized in Table I. The secretion produced by the trichomes showed a positive reaction when tested for lipophilic and hydrophilic compounds. Among the former, the following tests had positive results: Sudan IV

(Fig. 4A), Sudan black (Fig. 4B-C), Nile blue sulfate (Fig. 4D-E) and copper acetate/Rubeanic acid (Fig. 4F-G), thus indicating the presence of neutral lipids and fatty acids. Among the hydrophilic compounds, the following tests had positive results: PAS reaction (Fig. 5A-B),

Ruthenium red (Fig. 5C) and methylene blue (Fig. 5D-E), thus indicating the presence of acid mucilage. The secretion did not react positively when tested with ferric chlorite, thus indicating the absence of phenolic compounds (Fig. 5F). The secretion react positively with Starvh Black

B, indicating the presence of proteins (Fig. 5G-H).

136 Capítulo III Oliveira, F.M.C.

4. Discussion

The concept of colleter is a confuse one, and it may vary according to the morphology and the localization of the colleter, altough they usually occur in developing organs (Fahn 1979,

Fahn 1982, Cardoso-Gustavson et al. 2014). According to Fahn (1979) colleters are structures

– usually trichomes – that occur close to the shoot apical meristem (SAM) and leaf primordia.Such trichomes secrete a viscous (“sticky”) substance that is usually lipophilic or mucilaginous. A mixture of both lipophilic and mucilaginous substances can also be secreted by these trichomes.

The current concept of colleter is related to the chemical nature of the exudate and the position where this structure occurs, as it corroborates the structure’s function in the plant.

Therefore, collecters can be defined as structures – emergences, appendices or trichomes – that secrete lipophilic and/or mucilaginous substances, and occur close to meristematic regions protecting them from desiccation (Meyer et al. 2011; Meyer et al. 2013). The exudate produced by the colleters can also protect the meristems against the attack of pathogens; it can act as a lubricant; and it can promote symbiotic association with bacteria (Lersten 1975, Thomas 1991,

Miguel et al. 2006, Meyer et al. 2011).

Glandular trichomes have been reported to occur among the Bromeliaceae only at the foliar sheath of Navia fontoides (Tomlinson 1969) and Ananas comosus (Krauss 1949), although the secretion content hasn’t been described. The trichomes reported here for T. tricholepis produce a mixed secretion, composed of lipophilic, mucilaginous, and protein substances. This mixed composition of the colleters’ secretion has already been described for species in other Eudicot families, such as Apocynaceae (Thomas and Dave 1989), Rubiaceae

(Barreiro and Machado 2007) and Caryocaceae (Paiva and Machado, 2006). Among the

Monocots, it has been described for Orchidaceae (Meyer et al. 2011).

137 Capítulo III Oliveira, F.M.C.

The mucilage secreted by the colleters has the important role of avoiding desiccation of meristematic regions due to the mucilage’s capacity of retaining water (Fahn 1979). On the other hand, the lipophilic portion of the secretion probably acts a barrier against microorganism, as it is proposed to happen for the species Oncidium flexuosum (Orchidaceae, Mayer et al.

2011).

The glandular trichomes of T. tricholepis are observed at the adaxial surface of the foliar sheath that is closer to the SAM and the axillary buds. In the foliar sheaths that are further away from the SAM no trichomes were observed. The occurrence of these trichomes exclusively close to meristematic regions is probably related to lubrication and protection of these meristems against desiccation, given that Tillandsia species are epiphytes and/or rockery plants, and unlike most Bromeliaceae species, do not form water tanks at the base of its leaves.

Therefore, without the secretion produced by the glandular trichomes the SAM would be exposed to solar radiation and subject to desiccation. Thus, these trichomes fit the current functional definition of colleter, which makes this the first report of these structures for the

Bromeliaceae family.

Colleters are commonly located at the adaxial surface of stipules, bracts and sepals, as observed for T. tricholepis, in which they occur at the adaxial surface of the foliar sheath. This localization is related to the structure’s function, considering that the adaxial surface of an organ is usually closer to developing structures, such as the SAM (Paiva 2009, Cardoso-Gustavson et al. 2014).

Usually, colleters do not have pores in their cuticle, so the secretion is liberated through a rupture that results from the inner pressure caused by the exudate accumulated in the subcuticular space (Mohan and Inamdar 1986). However, no ruptures were observed in the cuticle of Bromeliaceae’s colleters. The secretion is then most likely to be liberated by micro

138 Capítulo III Oliveira, F.M.C.

pores on the cuticle, as it is proposed for the colleters of the species Oncidium flexuosum

(Orchidaceae) (Meyer et al. 2011).

Colleters are estimated to occur in approximately 60 Eudicot families (Thomas 1991) and to have taxonomic value for Rubiaceae (Lersten 1974, Thomas 1991, Miguel et al. 2016) and Apocynaceae (Thomas and Dave 1991, Thomas 1991; Rio et al. 2005). Among the

Monocotyledons, Cardoso-Gustavson et al. (2014) highlight that colleters are still little studied, which makes it difficult to infer their ecological role, as well as their evolutionary history.

Among the Poales, colleters were previously reported only for Rapateaceae as glandular trichomes that occur during floral development (Oriani and Scatena 2012, Ferrari and Oriani

2016). It is possible that these structures also occur in other families within species of this order, for which foliar and/or floral developmental studies are still scarce or absent.

5. Conclusion

We describe for the first time the presence of colleters in a Bromeliaceae species.

These structures are glandular trichomes present at the adaxial surface of the foliar sheath, in regions that are close to the shoot apical meristem. Histochemical tests revealed a mixed composition of the exudate with lipophilic, mucilaginous and protein substances.

Complementary studies dealing with a larger number of species are necessary in order to assign a possible evolutionary approach to this finding.

6. Acknowledgements

The authors thank D. Demarco for helping with histochemical tests interpretation. The authors also acknowledge the National Counsel of Technological and Scientific Development

(CNPq - process number 140115/2013-7 and 308070/2012-7) for the funding.

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Table 1: Results of histochemical characterization of the main secretory products of Tillandsia tricholepis Baker colleters.

Class of compound Reagent Reaction Total polysaccharides PAS reagent + Mucilage Rutenium red + Methylene Blue + Fatty acid Copper acetate/rubeanic acid + Acid and neutral lipids Nile blue sulfate + (acid) Total lipids Sudan IV + Sudan Black + Phenolic compounds Ferric chloride - Proteins Starch Black B +

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Figure 1: Cross section of shoot apical region of Tillandsia recurvata (L.) L. (A-B) and T. usneoides (L.) L. (C-D). A: Leaf sheath of T. recurvata’s primordia showing the presence of overlapping in margin (arrow). B: Detail of leaf sheath primordia. C: Leaf sheath of T. usneoides primordia. D: Detail of a mature leaf sheath, showing the absence of overlapping in the margins (arrow). Asterisks indicate marginal growth region in leaf sheath primordia.

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Figure 2: Cross section of shoot apical region of Tillandsia tricholepis Baker. A: Leaf blade of primordia. B: Detail of primordia leaf blade. C: Leaf sheath, showing glandular trichomes in adaxial surface (arrows). D: Detail of glandular trichomes, present in adaxial surface of leaf sheath (arrows).

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Figure 3: Colleters on vegetative apex of Tillandsia tricholepis Baker. A-B: Longitudinal sections of shoot apical region. Black arrows indicate colleters. C-F: Scanning Electron Microscopy (SEM) of glandular trichomes present in leaf sheath of T. tricholepis. C: Palmate trichome. D-E: Digitiform trichomes. F: Colleters next to an axillary bud. White arrow indicate axillary bud.

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Figure 4: Cross sections of shoot apical region of Tillandsia tricholepis Baker. Histochemical characterization of colleter secretion. A: Sudan IV for lipids. B-C: Sudan Black for lipids. D- E: Nile blue sulfate for acid and neutral lipids. F-G: Copper acetate/rubeanic acid for fatty acids.

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Figure 5: Cross sections of shoot apical region of Tillandsia tricholepis Baker. Histochemical characterization of colleter secretion. A-B: PAS reagent for total polysaccharides. Note the abundant secretion toward the trichome (A). C: Rutenium red for acid mucilage. D-E: Methylene Blue for acid mucilage. F: Ferric chloride for phenolic compounds. G-H: Starch Black B for proteins.

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References

Barreiro, D. P. and Machado, S. R. 2000. Coléteres dendróides em Alibertia sessilis (Vell.) K. Schum. Rev. Bras. Bot. 30 (3): 387-399.

Benzing, D. H. 1976. Bromeliad trichomes: structure, function and ecological significance. Selbyana. 4 (1), 330 – 348.

Benzing, D.H. 2000. Bromeliaceae: profile of an adaptative radiation. New York: Cambridge University Press.

BFG. 2015. Growing knowledge: an overview of Seed Plant diversity in Brazil. Rodriguésia, 66 (4): 1085-1113. DOI: 10.1590/2175-7860201566411

Butcher, D. and Gouda, E.J. 2017. The new bromeliad taxon list. . University Botanic Gardens, Utrecht (accessed 15.January 17).

Cain A.J. 1947. The use of Nile Blue in the examination of lipids. Quarterly Journal of Microscopical Sci. 88:383-392.

Cardoso-Gustavson, P.; Campbel, L.M.; Mazzoni-Viveiros, S.C. and Barros, F. 2014. Floral colleters in Pleurothallidinae (Epidendroideae: Orchidaceae). Am. J. Bot, 101 (4): 587-597.

Esau, K. 1960. Anatomy of seed plants. Wiley & Sons, New York

Fahn, A. 1979. Secretory tissues in plants. London: Academic Press.

Fahn, A. 1982. Plant Anatomy. Oxford: Pergamon Press.

Ferrari, R. C. and Oriani, A. 2016. Floral anatomy and development of Saxofridericia aculeata (Rapateaceae) and its taxonomic and phylogenetic significance. Plant Syatematic and Evolution, DOI 10.1007/s00606-016-1361-z

Fisher, D.B. 1968. Protein Staining of Ribboned Epon Sections for Light Microscopy. Histochemie 16: 92-96.

Gantner , P. and Jolles, P. 1969 . Histologie normale et pathologique, vols. I, II. Gauthier- Villars, Paris, France.

153 Capítulo III Oliveira, F.M.C.

Givnish, T.J.; Millan,K.C.; Berry, P.E. and Systma, K.J. 2007. Phylogeny, adaptive radiation, and historical biogeography of Bromeliaceae inferred from ndhF sequence data. Aliso. 23: 3- 26. Givnish, T.J.; Barfuss, M.H.J.; Van EE, B.; Riina, R.; Schulte, K.; Horres, R.; Gonsiska, P.A.; Jabaily, R.S.; Crayan, D.M.; Smith, A.C.; Winter, K.; Brown, G.K.; Evans, T.M.; Holst, B.K.; Luther, H.; Till, W.; Zizka, G.; Berry, P.E. and Systma, K.J. 2011. Phylogeny, adaptive radiation, and historical biogeography in Bromeliaceae: insights from an eight-locus plastid phylogeny. Am. J. Bot.. 98 (5): 1-24. Gregory M. and Baas, P. 1989. A survey of mucilage cells in vegetative organs of the dicotyledons. Israel J. Bot. 38:125-174. Hernandes-Lopes, J.; Oliveira-Neto, M.A. and Melo-de-Pinna, G.F.A. 2016. Different ways to build succulent leaves in Portulacineae (Caryophyllales). Int. J. Plant Sci. 177 (2): 198-208.

Jacques-Félix, H. 2000. The discovery of a Bromeliad in Africa: Pitcairnia feliciana. Selbyana 21 (1,2): 118-124.

Johansen, D.A. 1940. Plant microtechnique. McGraw-Hill, New York.

Kraus, J.E. and Arduin, M. 1997. Manual básico de métodos em morfologia vegetal. Edur, Seropédica.

Krauss, B.H. 1949. Anatomy of the vegetative organs of the pineapple Ananas comosus (L.) Merr.: II The leaf. Bot. Gaz. 110 (3): 333-404.

Lersten, N.R. 1974. Morphology and distribution of colleters and crystals in relation to the taxonomy and bacterial leaf nodules in Psychotria (Rubiaceae). Am.J. Bot. 61: 973–981.

Lersten, N.R. 1975. Colleter types in Rubiaceae, especially in relation to the bacterial leaf nodule symbiosis. Bot. J. Linn. Soc.. 71: 311-319.

Lillie, R. D. 1965. Histopathologic technic and pratical histochemistry. 3ª Ed. McGraw Hill, New York.

Mayer, J.L.S.; Cardoso-Gustavson, P. and Appezzato-Da-Gloria, B. 2011. Colleters in monocots: New record for Orchidaceae. Flora. 206: 185-190.

Mayer, J.L.S.; Carmelo-Guerreiro, S.M. and Mazzafera, P. 2013. A functional role for the colleters of coffee flowers. Ann. Bot. DOI: 5:10.1093/aobpla/plt029.

154 Capítulo III Oliveira, F.M.C.

McManus, J.F.A. 1948. Histological and Histochemical Uses of Periodic Acid. Stain. Technology 23: 99-108.

Melo-de-Pinna, G.F.A.; Hernandes-Lopes, J.; Ogura, A.S.; Santos, L.K.; Silva, D.C. and Haevermans, T. 2016. Growth patterns and diferent arrangements of vascular tissues in succulent leaves. Int. J. Plant Sci. 177 (8): 643-660.

Mohan, J. and Iinamdar, J. 1986. Ultrastructure and secretion of extrafloral nectaries of Plumeria rubra L. Annals of Botany 57 (3), pp 389-401. Miguel, E.C.; Gomes, V.M., Oliveira, M.A. and Cunha, M.D. 2006. Colleters in Bathysa nicholsonii K. Schum. (Rubiaceae): Ultrastructure, secretion protein composition and antifungal activity. Plant Bio. 8: 715-722. Miguel, E.C.; Da cunha, M.; Miguel, T.B.A. and Barros, C.F. 2016. Ontogenisis secretion ans senescense of Tocoyena bullata (Vell.) Mart. (Rubiaceae) colleters. Plan. Bio. 18: 851-858. Oriani, A. and Scatena, V.L. 2012. The taxonomic value of floral characters in Rapateaceae (Poales- Monocotyledons). Plant Systematics and Evolution, DOI 10.1007/s00606-012-0721- 6 Paiva, E.A.S. 2009. Occurrence, structure and functional aspects of Copaifera langsdorfii Desf. (Fabaceae, Caesalpinioideae). Comptes Rendus Biologies 332: 1078-1084. Paiva, E. A. and Machado, S. R., 2006. Colleters in Caryocar brasiliense (Caryocaraceae): ontogenesis, ultrastructure and secretion. Braz. J. Bio. 66 (1), pp. 301-308.

Pearse, A.G.E. 1985. Histochemistry: theoretical and applied. Little, Brown & Company, Boston.

Pizzolato, T. D. and Lillie, R. D. 1973. Mayer’s tannic acid-ferric chloride stain for mucins. J. Histochem. Cytochem. 2 (1): 56-64.

Rio, M.C.S.; Kinoshita, L.S. and Castro, M.M. 2005. Anatomia foliar como subsídio para a taxonomia de espécies de Fosteronia G.Mey. (Apocynaceae) dos cerrados paulistas. Rev. Bras. Bot. 28 (4): 713-726.

Ruzin, S.E. 1999. Plant Microtechnique and Microscopy. Oxford University Press, New York.

Scatena, V.L. and Segecin, S. 2005. Anatomia foliar de Tillandsia L. (Bromeliaceae) dos Campos Gerais, Paraná, Brasil. Rev. Bras. Bot. 28 (3): 635-649.

155 Capítulo III Oliveira, F.M.C.

Smith, L.B. 1974. Introduction. In: Pitcairnioideae (Bromeliaceae). Flora Neotropica. New York: Hafner Press, 14 (1): 1-3.

Thomas, V. 1991. Structural, functional and phylogenetic aspects of the colleter. Ann. Bot. 68: 287- 305.

Thomas, V.and Dave, Y. 1989.Histochemistry and senescence of colleters of Allamanda cathartica (Apocynaceae). Ann.Bot.64: 201–203.

Thomas, V.and Dave, Y. 1991. Comparative and phylogenetic significance of the colleters in the family Apocynaceae. Feddes Repertorium, 102 (3-4): 177-182.

Till, W. 2000. Tillandsoideae. In: Benzing, D.H. Bromeliaceae: Profile of an adaptative radiation. Cambridge University Press. Cambridge, 690p.

Tomlinson, P. 1969. Commelinales- Zingiberales. In: Metcalf, CR. (ed.) Anatomy of the Monocotyledons: III. Claredon Press, Oxford. Pp: 193-294.

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Conclusões Gerais

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Conclusões Gerais

A proposta deste trabalho foi estabelecer caracteres anatômicos que fossem úteis para a sistemática em Bromeliaceae, bem como discuti-los sob uma perspectiva filogenética.

No primeiro capítulo, o objetivo principal foi estabelecer sinapomorfias morfológicas e anatômicas para o clado Nidularióide. Com as reconstruções de estado de carácter, pudemos concluir que caracteres morfológicos frequentemente utilizados na delimitação dos gêneros pertencentes ao Complexo Nidularióide, tais como o tipo de inflorescência, presença de flores pediceladas/sésseis, a presença de apêndices nas pétalas, e a presença de calosidades nas pétalas são homoplásticos, e não deveriam ser utilizados na circunscrição destes gêneros. A maioria dos caracteres anatômicos analisados também representam homoplasias, no entanto, foi possível estabelecer duas novas sinapomorfias para o clado Nidularióide: (1) presença de tricomas peltados absorventes cujas células da ala são alongadas e (2) presença de espessamento na parede periclinal interna das células epidérmicas adaxiais da lâmina foliar.

Neste estudo, percebemos que trabalhos que envolvem evolução de caracteres em

Bromelioideae ainda são escassos, principalmente os que envolvem a análise de caracteres anatômicos. Ainda, demostramos que a anatomia foliar em Bromelioideae pode fornecer subsídios para futuros estudos de evolução neste grupo, propondo novas sinapomorfias para este grupo que é morfologicamente tão diverso e de difícil delimitação genérica.

No segundo capítulo, ao estudar a anatomia floral de espécies pertencentes à três das oito subfamílias de Bromeliaceae, pudemos descrever caracteres que podem ser úteis na sistemática da família. Propusemos uma nova sinapomorfia anatômica para

Pitcairnoideae, baseando-se no padrão de vascularização do gineceu. Também

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encontramos caracteres que possuem significância ecológica, tais como a presença de epiderme cônica, cujo formato é dado pela deposição da cutícula. Acreditamos que, pela ocorrência nas porções apicais dos filetes e estiletes, bem como nas anteras e estigmas, esta característica esteja envolvida com a atração de polinizadores. As estruturas secretoras também foram analisadas, estando presentes os nectários septais/ infraloculares, obturador e tecido transmissor. Em relação aos nectários, optamos por chamar de septais apenas os presentes em Bromelioideae, cujos ovários são ínferos e estes ocorrem verdadeiramente nos septos. Já para os nectários de Pitcairnoideae e Tillandsoideae, optamos por chamar de nectários infraloculares, uma vez que a porção secretora ocorre abaixo dos lóculos dos ovários.

Os caracteres descritos neste capítulo mostram a necessidade de estudos posteriores, envolvendo as oito subfamílias de Bromeliaceae, a fim de melhor entender a evolução floral neste grupo.

No terceiro capítulo, ao analisarmos três espécies do gênero Tillandsia L.

(Tillandsoideae), descrevemos pela primeira vez, a presença de coléteres em Bromeliaceae.

Estes coléteres são tricomas, que ocorrem na face adaxial da bainha foliar, próximos ao meristema apical caulinar. Possuem secreção de composição mista, i.e. secretam substâncias lipofílicas e hidrofílicas. Visto que a espécie em que estes coléteres ocorrem é epífita e não forma um tanque central com a sobreposição das bainhas foliares, esta secreção provavelmente tem relação com a proteção do meristema apical caulinar contra o dessecamento.

Acreditamos que estas estruturas ocorram em outros membros da família e que, devido à ausência de estudos de desenvolvimento foliar e floral, ainda não haviam sido documentados para Bromeliaceae, e possuem poucos registros na Monocotiledôneas em

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geral. Assim, reinforçamos a necessidade de futuros estudos envolvendo o desenvolvimento foliar em Bromeliaceae.

Ao analisar os resultados dos três capítulos conjuntamente, percebe-se que os caracteres anatômicos têm grande potencial para o uso na sistemática de Bromeliaceae, no entanto estes são subutilizados. Assim, acreditamos que os caracteres descritos nesta tese possuem relevância na sistemática do grupo e podem ser aplicados em futuros estudos de evolução em Bromeliaceae.

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