FISIOLOGIA DA GERMINAÇÃO DAS CIPSELAS HETEROMÓRFICAS EM Bidens L

UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” unesp INSTITUTO DE BIOCIÊNCIAS – RIO CLARO

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS (BIOLOGIA VEGETAL)

FISIOLOGIA DA GERMINAÇÃO DAS CIPSELAS HETEROMÓRFICAS EM Bidens L.

PAULO ROBERTO DE MOURA SOUZA FILHO

Dissertação/tese apresentada ao

Instituto de Biociências do Câmpus

de Rio Claro, Universidade

Estadual Paulista, como parte dos

requisitos para obtenção do título de Doutor em Ciências Biológicas (Biologia Vegetal) .

Setembro - 2014

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS (BIOLOGIA VEGETAL)

FISIOLOGIA DA GERMINAÇÃO DAS CIPSELAS HETEROMÓRFICAS EM Bidens L.

PAULO ROBERTO DE MOURA SOUZA FILHO

Tese apresentada ao Instituto de Biociências do Câmpus de Rio Claro, Universidade Estadual Paulista, como parte dos requisitos para obtenção do título de Doutor em Biologia Vegetal.

Setembro - 2014 581.1 Souza Filho, Paulo Roberto de Moura S729f Fisiologia da germinação das cipselas heteromórficas em Bidens L. / Paulo Roberto de Moura Souza Filho. - Rio Claro, 2014 105 f. : il., figs., gráfs., tabs., quadros

Tese (doutorado) - Universidade Estadual Paulista, Instituto de Biociências de Rio Claro Orientador: Massanori Takaki

1. Fisiologia vegetal. 2. Biologia reprodutiva. 3. Atraso da germinação. 4. Heterocarpia. 5. Mecanismos hormonais. I. Título.

Ficha Catalográfica elaborada pela STATI - Biblioteca da UNESP Campus de Rio Claro/SP

DEDICATÓRIA

Dedico esse trabalho a uma pessoa simples cuja baixa escolaridade não foi limitação para me ensinar, por meio de exemplos e vivências, valores que carrego e carregarei pela vida. Dedico a José de Souza Sobrinho (in memoriam) essa tese.

AGRADECIMENTOS

Primeiramente agradeço a minha família, pelo esforço de ter me fornecido uma boa educação e pelo apoio em todos os momentos, inclusive nos difíceis. Entre eles: Paulo R.M. Souza; Izabel C.B.M Souza; Luiz J.B.M. Souza e Eduardo A.B.M. Souza.

Ao professor Dr. Massanori Takaki pela orientação e amizade.

Ao CNPq pelo apoio financeiro.

À UNESP, assim como o programa de pós-graduação em Biologia Vegetal pela oportunidade de aprendizado e aperfeiçoamento.

À professora Dra. Alessandra Coan por apoiar, disponibilizando as instalações do Laboratório de Anatomia Vegetal, IB, UNESP, Rio Claro, para realização do trabalho e auxiliando com seu conhecimento na área.

Ao professor Dr. Massuo J. Kato e à Dra. Lydia F. Yamaguchi ambos do IQ, USP, São Paulo, por apoiar o trabalho realizando análises químicas do material botânico e auxilio conceitual.

À CAPES/Fulbright por ter fornecido bolsa de doutorado-sandwiche.

À professora Dra. Kathleen Donohue pela orientação e apoio, assim como membros do seu laboratório pela amizade.

À companheira Fernanda D. Santos por apoio e compreensão.

Aos colegas de laboratório Henrique H. Tozzi, Bruna Locardi, Camilla Kissmann, Lorena Egídio, Clara Machado e Sarah Bianchessi, pela amizade e apoio.

Aos integrantes da República Nemelés: Laura K. Honda, Thales H. D. Leandro, Igor Otero, Rafael H. Sugohara, Rebeca M.S. Alves, Felipe C. Nominatto, Claudia Kanda, Danilo J.L. Sousa, Raíssa Fonseca, Alexandre H. Takara e Pedro H. Mainardi

Aos amigos da pós-graduação: Diego E. Escobar, Luís F. Daibes, Gabriela Camargo, Mayra Eichemberg, Natália Costa, Odair Almeida, Shirley Martins.

Aos docentes da pós-graduação: Victor J.M. Cardoso, Alessandra Fidelis, Vera L. Scatena.

Aos amigos do departamento: Célia M. Hebling, Ari R. Pesce, Daniela O. Dinato, Sílvia R. Bettani.

Aos amigos do dia-a-dia, entre eles Tatiane C. Basconi e outros não mencionados que foram cruciais para alcançar esse objetivo.

SUMÁRIO

SUMÁRIO ...... 3 RESUMO ...... 5 ABSTRACT ...... 6 INTRODUÇÃO ...... 7 BIDENS HETEROMORPHISM: ROLE OF PERICARP ON SEED GERMINATION ...... 12 ABSTRACT ...... 13 INTRODUCTION ...... 13 MATERIALS AND METHODS ...... 16 Plant information and seed collection ...... 16 Cypsela feature measurements ...... 17 Germination assays ...... 18 Cypsela anatomical analysis ...... 18 Chemical composition of the pericarp ...... 19 RESULTS AND DISCUSSION ...... 19 Cypsela feature measurements ...... 19 Cypsela germination ...... 24 Pericarp anatomy...... 25 Chemical composition of cypsela parts ...... 30 SUPPLEMENTARY MATERIAL ...... 35 GERMINATION CONSTRAINTS OF DICARPIC CYPSELAE OF Bidens pilosa ...... 45 ABSTRACT ...... 46 INTRODUCTION ...... 46 MATERIALS AND METHODS ...... 49 Cypsela collection ...... 49 Germination variation among populations ...... 50 Variation of sensitivity to abscisic acid ...... 50 Pericarp influence in seed germination ...... 50 Gibberellic acid effects on germination ...... 51 Sensitivity to exogenous gibberellic acid ...... 51 Statistical analysis ...... 52 RESULTS AND DISCUSSION ...... 54 Variation of germination among populations ...... 54 Variation of sensitivity to abscisic acid (ABA) ...... 55 Pericarp influence on seed germination ...... 58 Response to germination hormonal promoter ...... 61 CONCLUSIONS ...... 64 AMONG-POPULATION DICARPY VARIATION IN Bidens bipinnata L...... 66 ABSTRACT ...... 67

INTRODUCTION ...... 68 MATERIALS AND METHODS ...... 70 Study species ...... 70 Plant material and micro-environmental data collection ...... 71 Laboratory germination assays ...... 71 Field germination assays ...... 72 Hormonal assays ...... 72 Statistical analysis ...... 73 RESULTS AND DISCUSSION ...... 75 Lab germination assays ...... 79 Field germination assay ...... 82 Hormonal assays ...... 84 SUPPLEMENTARY MATERIAL ...... 87 CONSIDERAÇÕES FINAIS ...... 91 REFERÊNCIAS GERAIS: ...... 93

RESUMO Bidens é um gênero de Asteraceae que apresenta cipselas heteromórficas que diferem no comportamento germinativo. No gênero, o padrão encontrado é que as cipselas periféricas tem uma menor taxa de germinação que as centrais. Os objetivos foram investigar quais são os fatores intrínsecos que estão relacionadas aos distintos comportamentos germinativos, e como essa estratégia varia na natureza. Focamos inicialmente no fruto (ou pericarpo) de oito espécies de Bidens para verificar se existe um padrão na estrutura e na composição química dos diferentes morfotipos e relacionar aos processos germinativos (Capítulo 1). Verificamos que os pericarpos dos tipos diferem anatomicamente, porém não há relação com a diferença na germinação. Por outro lado, a composição dos compostos secundários dos frutos apresenta diferença entre os tipos, e foi relacionado com a diferença fisiológica. Posteriormente, para verificar de que modo o controle hormonal e as restrições do pericarpo estão relacionadas com a diferença na germinação, nós utilizamos Bidens pilosa como espécie dicárpica modelo (Capítulo 2). Como resultado, os tipos não diferiram na resposta ao ácido abscísico (ABA), hormônio responsável pela dormência: ambos tipos têm sensibilidades semelhantes e não catabolizam ABA. Quando as cipselas foram tratadas com ácido giberélico exógeno (GA3), hormônio promotor da germinação, o tipo central respondeu a menores concentrações. Contudo, quando síntese de GA foi bloqueada, ambos os tipos apresentaram sensibilidades semelhantes ao GA3 aplicado. Ainda foi testada as influências física e química do fruto. Quando foram removidos os frutos, as sementes periféricas isoladas germinaram mais rápido que as cipselas intactas, contudo a diferença na germinação foi mantida entre os tipos. Quando as cipselas intactas foram tratadas com extrato dos frutos, apenas o tipo central sofreu inibição do extrato do fruto periférico. Discutimos a possível natureza e mecanismo de inibição da substância que ainda é desconhecida. Por fim, focamos na variação da dicarpia: morfologia e comportamento, ao longo de populações de B. bipinnata (Capítulo 3). A quantidade de cipselas periféricas variam em função da disponibilidade de luz na população, e o tamanho de ambos os tipos variam em função da luz e da presença de NH4 e K no solo. A taxas de germinação em laboratório de ambos os tipos variaram de acordo com a fertilidade do solo e com a disponibilidade de luz, apesar de que a proporção final de germinação do tipo periférico variou mais do que o central. O experimento realizado em campo apresentou resultados semelhantes aos obtidos em laboratório. Assim podemos concluir que a natureza do heteromorfismo em Bidens varia de acordo com as condições ambientais e está relacionada com componentes químicos encontrados nos frutos.

Palavras-chave: Atraso da germinação, Heterocarpia, Mecanismos hormonais. 6

ABSTRACT

Bidens is an Asteraceae genus that presents heteromorphic cypsela which differ in the germination behavior. For the genus, the pattern is that the peripheral cypselae have lower germination rate than the central ones. The objectives were to investigate which are the intrinsic diaspora factors that are related to different germination behaviors, and how that strategy varies in the nature. We initially focused in the fruit (or pericarp) from eight Bidens species to verify if there is a pattern in it structure and chemical composition on the different morphotypes and relate to its germination processes (Chapter 1). We verified that the type pericarps differ anatomically, but it are not related to germination difference. On the other hand, the secondary compounds composition of the fruits showed difference between the types, thus it was related to the physiological difference. Then, to verify by which way the hormonal control and pericarp constraints differ in the dicarpic species germination, we used Bidens pilosa as dicarpic model species (Chapter 2). As results, the types did not differ in the response to abscisic acid (ABA), hormone that induces dormancy: both types showed similar sensitivity and did not catabolize ABA. When the cypselae were treated with exogenous gibberellic acid (GA3), germination promoter hormone, the central type answer to low concentration. However, when endogenous GA synthesis was blocked, both types presented similar sensitivity to added GA3. Also were tested the physical and chemical influence from the fruit. When fruits were removed, the isolated peripheral seeds germinated faster than intact cypsela, but the final germination percentage difference were kept between the types. When the intact cypsela were treated with fruit extracts, just the central types suffered inhibition from peripheral fruit extract. We discuss the possible nature and inhibition mechanisms of the compound, that still unknown. At last, we focused on the dicarpic variation: morphology and behavior, across populations of B. bipinnata (Chapter 3). The amount of peripheral cypsela varied in relation to light availability to population, and both type sizes varied in relation to light and to presence of NH4 and K in soil. The laboratory germination rates of both types varied in relation to the soil fertility and to the light availability, despite of the final germination proportion of the peripheral types varied more than central ones. The field experiment presented similar results to the obtained in the laboratory. Thus we conclude that the nature of Bidens hetermorphism varies according to the environmental conditions and that it is related to chemical components found in the fruits.

Key-words: Heterocarpy, Hormonal mechanisms, Germination delay.

INTRODUÇÃO

O heteromorfismo somático é uma estratégia vegetal no qual uma planta produz diásporos com características morfo-fisiológicas diferentes. Tamanho, cor, presença de estruturas acessórias, todas são características que estão relacionadas a variação morfológica dos diásporos. Por outro lado, fatores associados a germinação das sementes estão relacionados a variação na resposta fisiológica, entre as características estão: grau de dormência, resposta a fatores ambientais específicos, velocidade de germinação, longevidade, entre outros (VENABLE 1985, IMBERT 2002, MATILLA et al. 2005, BASKIN et al. 2013). Essa estratégia reprodutiva inicialmente denominada de polimorfismo somático (HARPER, 1977), passou a ser conhecida como heteromorfismo somático (VENABLE 1985). Posteriormente devida a natureza do diásporo e sua disposição sofreu algumas especificações (VAN DER PJIL 1982, MANDÁK 1997, BASKIN, et al. 2013): heteroespermia, quando se trata de sementes no mesmo fruto e sofrem variações; heterocarpia quando uma mesma planta produz frutos diferentes, normalmente frutos secos; anficarpia quando há a produção de frutos em regiões diferentes da planta, diásporos diferentes são produzidos tanto acima como abaixo do solo (MANDÁK 1997). De modo geral, as espécies heteromórficas são de porte herbáceo, com algumas arbustivas, e sua maioria tem ciclos de vida curta como anuais ou bianuais. Os diásporos heteromórficos apresentam a vantagem por se estabelecerem em microambientes diferentes, espacial ou temporalmente (VENABLE 1985, IMBERT 2002, BASKIN et al. 2013). Espécies com essa estratégia são frequentemente encontradas em locais semiáridos, desérticos e costeiros, que são ambientes naturais que sofrem grandes variações ao longo do tempo (VAN DER PJIL 1982, SNYDER 2011). Ainda, algumas espécies heteromórficas, se adaptaram a ambientes artificiais variáveis, como plantações e ambientes urbanos (IMBERT 2002). Essa variação nos diásporos implica em um maior potencial competitivo, já que os diásporos produzidos podem se estabelecer em uma maior variedade de microambientes (VERNABLE and LEVIN 1985). Os diásporos heteromórficos adquirem características específicas o que vão facilitar sua dispersão e seu estabelecimento em determinadas situações. De modo geral, os tipos de diásporos seguem duas estratégias distintas (com possível a presença de intermediários): (1) mantedor e (2) colonizador (BAKER e O’DOWD 1982). De modo geral, o tipo mantedor tem baixa dispersão espacial, associado a sua maior massa ou ausência de estruturas acessórias, sendo este “responsável” por garantir a permanência local da população. Ainda o tipo mantedor é normalmente associado a um maior atraso na germinação para muitas espécies (IMBERT 2002, BASKIN et al. 2013). Enquanto o tipo 8 colonizador, com maior capacidade de dispersão espacial, tem a estratégia de se estabelecer em novos locais (BAKER e O’DOWD 1982, VENABLE 1985). Baskin et al. (2013) baseado em classificações anteriores (VENABLE 1985) desenvolveu um modelo de classificação para as espécies heteromórficas usando o grau de dispersão e dormência dos diásporos. Por exemplo, Garhadiolus papposus, uma espécie trimorfica apresenta o seguinte padrão H/H(CA)–I/I(IA)–L/L(PA), ou seja: tipo central (Central Achene) tem alta dispersão (High dispersal) e alta germinação (High germination); tipo intermediário (Intermediate Achene) tem estratégias intermediárias (Intermediate dispersal/germination); e o tipo periférico (Peripheral Achene) apresenta baixa dispersão (Low dispersal) e baixa germinação (Low germination) (BASKIN et al. 2014). Desse modo as espécies apresentam estratégias mistas. Existem ainda casos específicos nos quais os tipos apresentam a dispersões especificas a determinados vetores, por exemplo Picris echioides, apresentam cipselas preferencialmente anemocóricas e outros ectozoocóricas (SONRESEN 1978). Assim a produção dos diásporos heteromórficos podem variar, e esta ocorre em detrimento a determinações impostas pelo ambiente. A produção dos diferentes tipos podem apresentar um comportamento de bet-hedging (tradução livre: cercando uma aposta) para produção dos diásporos (VENABLE 1985). Essa hipótese relaciona a imprevisibilidade do ambiente com a variação no fenótipo dos descendentes de terminada planta. Assim, a variação na produção dos tipos de diásporos assegura que ao menos um grupo de descendentes sobreviva para o próximo evento reprodutivo (CREAN e MARSHALL 2009). Outra teoria é a dispersão informada (informed dispersal) que relaciona o heteromorfismo a uma resposta da planta mãe. A planta mãe influenciada por sinais ambientais, altera o fenótipo dos descendentes para se “adequarem” às novas condições ambientais (MARTORELL e MARTÍNEZ-LÓPEZ 2014). Em ambas as teorias, as plantas alteram o número e a qualidade dos diásporos para aumentar seu sucesso reprodutivo. Assim, o que causa o heteromorfismo é uma característica somática, ou seja, determinada pela planta mãe e seus tecidos. Em teoria, as sementes heteromórficas são geneticamente semelhantes, o que irá variar são os tecidos maternos, ou seja, os integumentos e os ovários pré ou pós a antese (HARPER 1977, GIBSON 2001, GIBSON e TOMLINSON 2002). Contudo existem algumas controvérsias a serem levadas em consideração, nos quais as sementes heteromórficas apresentam expressões de genes diferentes (SOLIMAN 2003). Entre as famílias que apresentam o maior número das espécies heteromórficas estão as Asteraceae (IMBERT 2002). Isso se deve, em parte, ao fato de que os frutos são formados por flores diferentes presentes no mesmo capítulo. Elas apresentam heterocarpia, nos quais o tipo mais comum é o dicarpia, ou seja, produção de dois tipos de 9 frutos, chegando a policarpia, produção de mais de quatro tipos, por exemplo Calendula micrantha que apresenta seis morfotipos (SOLIMAN 2003). De modo geral a heterocarpia é decorrente da variação nos tipos florais, ou seja, o tipo periférico é originado da flor do raio, enquanto o central é originado da flor do disco (IMBERT 2002). Contudo para algumas espécies heterocárpicas um dos tipos florais é estéril ou exclusivamente masculino, e a diferenciação do tipo de cipsela é decorrente, aparentemente, a um tipo floral. Heterosperma pinnatum apresenta cipsela oriundas das flores do disco hermafroditas, enquanto as flores do raio são estéreis (VENABLE et al. 1987). Em Calendula micrantha e C. arvensis as flores liguladas do raio formam cipselas, sendo as do disco sem gineceu (GARDOCKI et al. 2000, RUIZ DE CLAVIJO 2005, respectivamente). Assim, para essas espécies, a variação no número das cipselas não fica restrito ao número dos tipos florais. Por exemplo, em Hypochoeris glabra o número de frutos periféricos é menos variável, enquanto o número do tipo central varia de acordo com a densidade de plantas nas populações das plantas mãe (BAKER e O’DOWD 1982). Em Asteraceae, o fruto, ou pericarpo, é a estrutura que protege a única semente, e é tida como o principal responsável pelo heteromorfismo: ou pela variação na forma dos diásporos, ou pela restrição germinativa. A variação na forma irá influenciar no processo de dispersão. A presença de estruturas acessórias como pappus ou expansões no fruto podem favorecer as cipselas a se deslocarem para maiores distancias (VAN DER PIJL 1982). Em Crepis sancta as cipselas centrais possuem pappus plumosos o que favorece a dispersão pelo vento, sendo ausente nas cipselas periféricas (IMBERT, et al. 1996). Em Heterosperma pinnatum as cipselas centrais são compridas e apresentam pappus com aristas que se prendem a pelos de animais, diferentemente das cipselas periféricas que não apresentam essa estrutura, mas apresentam maior dispersão secundária (VENABLE et al. 1987). Em Anacyclus, as cipselas periféricas apresentam expansões no fruto o que auxilia na dispersão anemocórica (TORICES et al. 2013). Em Synedrella nodiflora as cipselas centrais possuem pappus com aristas, e as cipselas periféricas, expansões de aerênquima no pericarpo, provavelmente associado a dispersão pela água (SOUZA-FILHO e TAKAKI 2011). É também atribuído ao pericarpo o atraso na germinação das sementes. Em algumas espécies as cipselas atuam como resistência mecânica contra a germinação, enquanto em outras é atribuída a ela a presença de inibidores químicos. A cipsela, além de desempenhar proteção, causa resistência mecânica contra a germinação. Essa característica é bem observada em algumas espécies heterocárpicas, na qual existe uma variação na espessura e constituição das cipselas. Em Senecio jacobaea o mesocarpo, camada de células intermediárias do pericarpo, possui células mais espessas entre as “costelas” (ribs) o que 10 fornecem maior resistência mecânica. Em Heterotheca latifolia o pericarpo da cipsela periférica, além de mais espesso, apresenta fibras circundando completamente o embrião, diferente do central que apresenta feixes (VENABLE e LEVIN 1985). Em Catanache lutea a presença de estruturas impermeáveis nos diásporos subterrâneos dificulta a entrada de água o que torna lenta a germinação em relação aos frutos aéreos (CLAVIJO e JIMÉNEZ 1998). Em Anthemis chrysantha a ausência de espaço intercelular no mesocarpo torna difícil a difusão de água para embebição, além da resistência mecânica (AGUADO et al. 2011). Essas limitações são aliviadas com a remoção do fruto, e deixando a semente livre a germinação entre os tipos se iguala. O fruto também pode conter substâncias químicas que inibem a germinação das sementes. Em periantos de aquênios escuros de Salsola komarovii (Chenopodiaceae) foi encontrado maior grau de ácido abscísico, hormônio inibidor da germinação (TAKENO e YAMAGUCH 1991). Já para Asteraceae os efeitos dos inibidores químicos foram constatados, mas não identificados. Em Artotis fastuosa (dicarpica), Dimorphotheca sinuate (monocarpica) e D. polyptera (dicarpica) os extratos aquosos dos frutos inibiram a germinação das sementes, confirmada pelo efeito aditivo do aumento de concentração. O efeito da substância química foi observado mesmo com sementes isoladas (BENEKE et al. 1993). Em Bidens pilosa, inibidores extraídos com etanol de frutos periféricos foram capazes de reduzir a germinação dos frutos centrais (FORSYTH e BROWN 1982). Compostos secundários estão cada vez mais adquirindo importância na biologia de sementes (JIA et al. 2012). Trabalhos focando nos efeitos aleloquímicos dos frutos estão mostrando que a resposta da semente pode sim ser afetada por substâncias antes discriminadas, por não possuir um caráter hormonal. Trabalhos recentes com Arabidopsis thaliana (Brassicaceae), planta modelo para trabalhos moleculares, vem mostrando que compostos presentes no tegumento das sementes são capazes de estimular a produção de hormônios inibidores da germinação. Compostos como proantocianidinas, ou taninos condensados, além de dar coloração ao tegumento das sementes, são responsáveis por uma maior dormência das sementes (JIA et al. 2012). O gênero Bidens apresenta um grande número de espécies, sendo algumas delas consideradas daninhas e/ou invasoras. Esse gênero apresenta-se distribuído por muitos países, porém o mesmo é originário das Américas (MITICH 1994, HOLM et al. 1997, KIM et al. 1999). Algumas espécies do gênero são heterocárpicas, variando de dicárpicas a policárpicas. E seguem padrões de heteromórficos semelhantes entre si, com uma exceção conhecida (DAKSHINI e AGGARWAL 1974, FORSYTH e BROWN 1982, BROWN e MITCHELL 1984, ROCHA 1996, AMARAL e TAKAKI 1998, BRÄNDEL 2004a). Ainda o padrão comum ao gênero difere do modelo encontrado para outras heteromórficas 11

(IMBERT 2002, MATILLA 2005, BASKIN et al. 2013). Assim espécies desse gênero apresentam características interessantes a serem analisadas e melhor compreendidas. Desse modo, este gênero foi escolhido para investigação do heteromorfismo. Procuramos focar na verificação de quais são os fatores que geram o heteromorfismo entre algumas espécies do gênero Bidens (Capítulo 1). Depois focando em uma espécie como modelo, B. pilosa, verificamos como os tipos de cipselas respondem aos estímulos hormonais e qual o papel do pericarpo na germinação. A resposta foi relacionada a um modelo do mecanismo proposto para a germinação dos tipos (Capítulo 2). Ainda foi verificada a variação da dicarpia morfológica (variação na forma) e fisiológica (variação germinativa) em outra espécie modelo, B. bipinnata, ao longo de diferentes populações e quais fatores ambientais estão associados as variações encontradas (Capítulo 3).

CAPÍTULO 1

BIDENS HETEROMORPHISM: ROLE OF PERICARP ON SEED GERMINATION

Bidens Heteromorphism: the role of pericarp in seed germination Paulo Roberto de Moura Souza Filho1, Sarah Bayod Bianchessi1,Lydia Yamaguchi2, Massuo Kato Jorge2; Alessandra Coan1, Massanori Takaki1

Departamento de Botânica, Universidade Estadual Paulista, Rio Claro, São Paulo, 13506-125, Brazil. Instituto de Química, Universidade de São Paulo, São Paulo, 05508-000, Brazil.

ABSTRACT

Diaspore heteromorphism is a common reproductive strategy on Asteraceae. Bidens is an important genus which have some species with such strategy. The dicarpy is more frequent among them, however what leads to physiological behavior variance is unknown. We investigate the variation on pericarp anatomy and secondary compounds composition (HPLC/MS) of eight species aiming to find the pattern by which their germination mechanism varies. We classified the heterocarpic species by it morpho-physiological features in dicarpic (B. alba; B. aristosa; B. bipinnata; B. pilosa; B. subalternans; B. frondosa), polycarpic (B. gardneri) and monocarpic (Bidens sp.). The transversal slides analysis did showed some difference on the cypsela type’s structures but it is not related to germination barrier due to single cell layer between the ribs. The mesocarp, the most developed layer, presented similar layer numbers, cell compositions and cell-wall structures. However, the pericarp chemical composition varied by cluster analysis for B. aristosa, B. bipinnata, B. pilosa and B. subalternans. Focusing on the identified relevant secondary compounds on fruits: nitrogen compounds (sphingamine) for the central cypselas and flavonols (quercetin and kaempferol) for the peripheral ones. Despite still have some unidentified compounds, we assumed that there is an inhibitor on peripheral cypselas due to literature. We also discussed how the flavonoids may affect the germination of the peripheral types via reactive oxygen species scavenging. To sum, we did not find anatomical clues to a physical constraint difference. But the chemical composition variance among the cypsela types indicate to us that the dicarpic germination difference may occurs via chemical inhibition.

Key-words: Chemical inhibition, Heterocarpy, Physical constraints,

INTRODUCTION

Diaspore heteromorphism is a strategy in which a single plant produces two or more types of seeds in its fruits (heterospermy) or different kinds of fruits (heterocarpy) (MANDÁK 14

1997; IMBERT 2002). Since it increases offspring spread in time, via the degree of dormancy, or in space, due to different dispersal structures, it is known as mixed strategy (MCEVOY 1984). In the one-seeded fruits, known as cypsela (or achenes), of heterocarpic Asteraceae, type differentiation, which causes morpho-physiological variations, occurs in the ovary (IMBERT 2002, BASKIN et al. 2013). In most Asteraceae, morphologically distinct florets have different functions and their different ovaries develop into mature cypsela types. In Hemizonia increscens (TANOWITZ et al. 1987) and Galinsoga ciliata (KUCEWICZ et al. 2010), ligulate ray flowers produce large, peripheral cypselae with a limited spatial dispersal. However, in a few species, morphologically distinct fruits originate from a single floret type. In Hetherosperma pinnatum, the dicarpic cypselae originate from disk florets, while ray florets are sterile (VENABLE et al. 1987). In Calendula micrantha, the ligulate ray florets produce six cypsela types (GARDOCKI et al. 2000) and they may be related to embryo gene expression (SOLIMAN 2003). In heterocarpic Anacyclus clavatus, A. valentinus, and A. homogamos (sterile ray florets), the winged cypselae are bigger and more peripheral in the capitulum than the unwinged ones, but their size gradation does not depend on floret type (TORICES et al. 2013). Although differential development occurs after fertilization, during cypsela ontogeny, the causes of this morphological differentiation are unknown. It is hypothesized that cypsela size inside the head varies due to source-sink and/or architectural effects. In the first case, the variation would be induced by the level of accumulated resources: the first fertilized ones receive more maternal resources. On the other hand, the architectural effect hypothesis states that cypsela size is determined by intrinsic factors, e.g. genetic organ identity, that determine cypsela features. In monocarpic Tragopogon porrifolius, both mechanisms generate the variation in cypsela sizes. However, size is not related to the cypsela germination behavior or fitness. The causes of the variation in dicarpic cypselae originating from a single floret type are still unknown (TORICES and MENDEZ 2010). Variations in pericarp structure can cause differences in germination behavior. Highly lignified fiber layers in the pericarps of ray cypselae of Heterotheca latifolia cause a mechanical resistance, which is released when seeds are excised, while thin-layered disk cypselae have a short delay of germination (VENABLE and LEVIN 1985). In Anthemis chrysantha, both the thick cell walls and the number of cell layers of the peripheral pericarp type inhibit embryo growth and cause a delay of germination. Thick pericarps diminish water uptake and induce a physical resistance to embryo growth (AGUADO et al. 2011). In Senecio jacobaea, the pericarps of ray cypselae are thicker than those of the central type and their cells are thick-walled between achene coat ribs. When these pericarps were 15 partially removed (e.g. proximal nick, distal nick, and split), cypselae germinated according to removal damage, thus there is no inhibition through water uptake and/or gas exchanges (MCEVOY 1984). Thus, clues in the pericarp anatomy indicates the physical constraints to germination, such as: (1) more cell layers, (2) thicker cell walls and/or (3) more sclerenchyma cells. In addition, fruits may contain chemical compounds that inhibit embryo growth and maintain seed dormancy. This is the case with the pericarps of ray achene of Dimorphotheca polyptera and Artotis fastuosa whose chemical compounds inhibited the germination of cypselae and excised embryo (BENEKE et al. 1993). When testing the effects of surrounding tissues on Bidens cernua embryo, Hogue (1976) concluded that seed coats and cypsela pericarps presented some germination inhibitors. Their nature can be (1) hormonal, acting directly on the embryo or live surrounding tissues, or (2) secondary, upregulating seed hormones. In Salsola komarovii (Chenopodiaceae), high amounts of abscisic acid were found in the dicarpic perianths of the dormant type (TAKENO and YAMAGUCH 1991). In wild seeds of Arabidopsis thaliana, testae contained proanthocyanidins, which induce ABA de novo synthesis, thus maintaining dormancy. Transparent testa mutants germinate faster than wild seeds (JIA et al. 2012). Proanthocyanidins, also known as condensed tannins, are colorless flavonoids that, after oxidation, become brown and act as antioxidant and protect against microbial pathogens, insect pests, and larger herbivores (DIXON et al. 2005; NONOGAKI 2006). Other flavonoids may act as seed germination inhibitors (POURCEL et al. 2007). Bidens is one of the largest genera of Asteraceae. It comprises circa 340 species (FUNK 2009). Cypsela heteromorphism was reported for some species and the common pattern is dicarpy, despite a cypsela size gradient in the capitulum (BROWN and MITCHELL 1984; AMARAL and TAKAKI 1998). However, at least one known species presents a continuous variation, whose size is directly related to germination (B. gardneri [SASSAKI et al. 1999]). Its dicarpic pattern follows the H/L - L/H strategy (see also BASKIN et al. 2013), in which central cypselae have a wide spatial dispersal and shallow dormancy, while peripheral ones have a narrow dispersal and deep dormancy (DAKSHINI and AGGAWAL 1974, BROWN and MITCHELL 1984, CORKIDI et al. 1991, ROCHA 1996, AMARAL- BAROLI and TAKAKI 2001). Yet B. frondosa is a known exception, which presents an H/L - L/H strategy, i.e. the central with High dispersal / Low germination and the peripheral with Low dispersal / High germination (BRÄNDEL 2004a). Despite seed biology studies, there are no reports of heteromorphism in other Bidens species as B. cernua (HOUGUE 1976, BRÄNDEL 2004b), B. polylepis (BASKIN et al. 1995), B. laevis (LECK et al. 1994), B. alba (RAMIREZ et al. 2012), B. tripartita (BRÄNDEL 2004b), and B. sulphurea (BORGHETTI 16

1998). The absence of morphological dicarpy does not necessarily mean that the species do not have heteromorphic diaspores, since it may present a cryptic heteromorphism, which is difficult to identify just by it morphs. Germination tests should thus be carried out to verify behavioral heterocarpy (BASKIN et al. 2013, LEVERETT and JOLLS 2014). Our objective is to verify the variations of the heterocarpic cypselae of some Bidens species external and internal pericarp morphology and chemical composition and correlate to it germination behavior. For that we, firstly distinguish the species different types, by measuring it external morphology (size and structure mass) and testing it germination. Then we analyze its anatomy structure of the types. Finally we compare the chemical composition and according to their secondary compound profile.

MATERIALS AND METHODS

Plant information and seed collection

Bidens alba (L.) DC. It bears capitula with white, sterile ray florets. The population was found near a highway roadside in Recife, PE, Brazil (8°4'39.43"S; 34°58'35.23"W). In this dicarpic species, tetragonal, peripheral cypselae present hairs on the pericarp surface, while, on the trigonal, central ones, hairs appear around pappies (Figure 1). It is part of the B. pilosa complex and shares some features with B. pilosa (BALLARD 1986). No reports of dicarpy were found in the literature. Bidens aristosa (Michx.) Britton is a weed with yellow, sterile ray florets. It is very common around Duke Forest, Durham, NC, U.S.A. (36°0'10.82"N; 78°57'7.53"W). Its dicarpic cypselae present slight differences: the peripheral ones are short, dark, and ovate, while the central ones are long and oblanceolate (Figure 1). No reports of dicarpy were found in the literature. Bidens bipinnata L. is a weed that shares morphological traits with B. subalternans (DUVIGNEAUD 1975). It was collected around Duke Forest, Durham, NC, U.S.A. (35º59’ 20”N; 78º57’24” W). Its dicarpic cypselae have already been reported before, with differential behavior like the Bidens patterns (DAKSHINI and AGGARWAL 1974, BROWN AND MITCHELL 1984). Bidens frondosa L. is a weed that bears yellow, sterile ray florets. It grows in mudflats sites. Its population was found to be sympatric to B. aristosa (36° 0'10.82"N; 78°57'7.53"W). It is a dicarpic species whose central cypselae are longer and lighter colored than the peripheral ones (Figure 1). This species is an exception to the dicarpic behavior pattern because its central cypselae are deeper dormant than the peripheral ones (BRÄNDEL 2004a). 17

Bidens pilosa L. is a worldwide dangerous weed native Tropical America (HOLM et al. 1977, KISSMANN and GROTH 1992, MITICH 1994). Its ray florets are white and sterile or can be absent, as is the case of our collected population, in which the tubule of peripheral, hermaphrodite disk florets suffered a slight modification (personal observation). This population of B. pilosa was found around a sugarcane crop in Rio Claro, SP, Brazil (22°25'32.92"S; 47°30'11.94"W). It is the most studied Bidens species. In its capitulum, cypsela length gradually increases from periphery to center (DE MARINIS 1983). However, cypsela size is not linked to the dicarpic cypsela behavioral differences (ROCHA 1996, FORSYTH and BROWN 1982, AMARAL and TAKAKI 1998). Physiological dicarpy is related to pericarp features: the orange-to-yellowish, peripheral cypselae have ornaments and hairs on their surface, while the dark-colored, central cypselae pericarps are only haired close to the pappi (Figure 1). Bidens subalternans DC. is a worldwide weed. It has an inconspicuous, yellowish, sterile ray floret and may be sympatric to B. pilosa in many sites (22°25'32.92"S; 47°30'11.94"W) (KISSMANN and GROTH 1992; GROMBONE-GUARATINI et al. 2005). The dicarpic cypsela traits differ markedly: the orange-to-brownish peripheral cypselae are haired and ornamented along their surface, while the gray central ones are glabrous (Figure 1). No reports of dicarpy were found in the literature Bidens gardneri Baker is found in Brazilian savannas and it is considered as a weed. It has orange or yellowish sterile ray floret. This population was found in the Reserva Ecológica de Mogi Guaçu, Mogi Guaçu, SP, Brazil (22°18'S; 47°11'W) along a roadside. This species has polycarpic cypselae whose germination behavior varies according to cypsela size. Rondon (2001) divided the types by length: short (5-8 mm), intermediate (9- 10 mm), and long (>11 mm). Short cypselae are located at the periphery, while long ones are found in the capitulum center. Although we considered her classification, for some analyses, we only used long and short cypselae as central and peripheral, respectively (Figure 1). Bidens sp. is a non-identified, rare species. It occurs in shaded places in forest borders. It has no ray ligulate florets, only hermaphrodite tubular ones. This population was found by a trail in the Floresta Estadual Edmundo Navarro de Andrade (FEENA), Rio Claro, SP, Brazil (22°24'51.71"S; 47°32'1.50"W). This species is monomorphic since all cypselae present the same features and show no visible difference.

Cypsela feature measurements

For each species, cypsela types were separated by morphological traits according to the literature information or visual patterns. Fifty cypselae from each type were individually photographed using the Leica M125 Stereo Microscope with Leica DFC280 digital camera. The pictures were measured with software ImageJ (1.42q) to obtain cypsela length and the width and length of the longer pappi. To evaluate mass allocation, the pericarps of 50 fresh cypselae were manually removed using a forceps and a stereoscope. Groups of ten cypselae were oven-dried (105°C during 24h), as replicates, and weighed with an analytical balance (0.01 g) to obtain the dry biomass of each cypsela part. The morphological data were statistical analyzed for the cypsela type difference with the Welsh Two Sample t-test which assumes unequal variance and applies the Welsh degrees of freedom modification. For B. gardneri, which have three levels, we performed one-way ANOVA, and Tukey HSD were carried out. For the mass allocation analysis were performed a mixed one-way ANOVA to verify the variance of the dry mass between the types within the species, then was carried out a Welsh Two Sample t-test to confirm specific differences.

Germination assays

To verify the germination of cypsela types, 25 fresh cypselae were placed in four 90mm Petri dishes lined with two filter paper layers moistened with deionized water and maintained at 25°C under continuous white light, or at 22°C with a 12h photoperiod (for B. bipinnata), during 30 days. Seed germination was checked daily and germinated cypselae were removed. Bidens pilosa, B. gardneri, B. subalternans, B. bipinnata, and B. alba were used for this assay. Due to their low germination rate or low cypsela availability B. frondosa, B. aristosa, and Bidens sp. were excluded. Germinability and mean germination time were statistical analyzed for cypselae type using the individual dishes values as the replicates. We performed Welsh Two Sample t- test. And for B. gardneri one-way ANOVA (three levels), then Tukey HSD were carried out. All the statistical analysis were made using the R software (R CORE TEAM 2013)

Cypsela anatomical analysis

To verify the structure of the pericarp according to types and species, we prepared cross sections of fully developed unripe cypselae. We chose immature cypselae from closed capitula because they are less hard. Cypselae of the monomorphic species and of the two types of the dicarpic and polycarpic ones were fixed in FAA 50 and preserved in 70% ethanol 19 with a few glycerol drops (JENSEN 1962). Cypselae were dehydrated in n-butanol series, followed by infiltration in glycol methacrylate (Leica) as a supporting medium. Historesine blocks were sectioned 6-10µm wide at the cypsela proximal and medial regions with a Reichert-Jung Leica microtome (model 2040). The slides were then stained with Toluidine blue 0.05%, diluted in acetate buffer pH 4.7 (O’BRIEN et al. 1964), and mounted in Entellan®. They were later photographed using the Leica DMLB light microscope with Leica DFC280 digital camera for further analyses. Chemical composition of the pericarp

For the extractions, twenty separated cypsela parts, i.e. seeds and pericarps, were crushed with a mortar and pestle and silica sand was added. They were successively extracted with MeOH (2.5 mL), chloroform (5 mL) and milli-Q water (2.5 mL) before they were centrifuged twice at room temperature for phase separation. The aqueous phase was filtered and concentrated under vacuum to obtain crude MeOH extracts. The samples were analyzed by liquid chromatography and mass spectrophotometry (LC/MS). Shimadzu liquid-chromatography (Kyoto, Japan) was composed by two analytic pumps (model LC-20AD), an automatic injector (SIL-20AHT), a UV/Vis detector (SPD-20A), and a column oven (CTO-20A) controlled by CBM-20A. The mobile phase contained MeOH and 0.2% formic acid diluted in water, the gradient was 5% MeOH at 0 min (for up to 2 min), and then it was linearly raised to 100% MeOH (from 2 to 25 min) and kept at a plateau for 5 min. The column was reverse phase Luna 5u PFP (2) 100A, 150 x 2mm (Phenomenex). The monitored wavelength was between 254 and 280 nm, and the columns oven was set to 40ºC. The mobile phase flux was 200 µl/min directly infused in the mass spectrophotometer. The mass spectrophotometer (Bruker micrOTOF-QII) worked in positive mode with

N2 as nebulizer gas and dried at 4 Bar and 8 L/min. The drying temperature was set to 200ºC; the collision energy and the quadruple energy were set to 12 and 6 eV, respectively. RF1 and RF2 funnels were programmed to 200Vpp and the monitored mass range was 100- 1000kDa. Data were analyzed by XCMS (U.S.A. [SMITH et al. 2006]) for retention time alignment and correction. The data obtained were normalized according to their area and submitted to a multivariate analysis using Unscrambler (v. 10).

RESULTS AND DISCUSSION

Cypsela feature measurements

To the eight Bidens species, the central cypselae type are longer which favors it exposition to dispersal vectors, while the larger, peripheral ones, due to less spatial 20 constraints, are hidden in the open ripe capitulum (Table 1, Figure 1). The awns of the peripheral cypselae are also shorter in most species, except B. alba and B. gardneri. In B. aristosa, it is not sure whether pappi, even on the central type, serve to adhere to vectors, because they seem to be vestigial structures (Figure 1). These morphological traits contribute to the spatial dispersal of the species. Using B. pilosa as a model, De Marinis (1983) observed type arrangement in the capitulum and pointed out size gradient difference in globose, ripe capitula. Rocha (1996) tested dispersal strategies through two field observations: (1) outer cypselae remained longer on the infructescence than central ones; (2) central cypselae have more chances to attach to a vector than the peripheral ones, according to observations made using an artificial dispersal vector. He thus concluded that the central cypselae of B. pilosa are more dispersive than the peripheral ones. In H. pinnatum, the central cypselae remains on the head longer than the peripheral ones, which soon fall close to their mother plant. Also for H. pinnatum some traits were correlated: (1) the number of awned cypselae, (2) the length of both types of cypsela, and (3) the awn length of the central ones. Thus, these traits indicate the importance of the central cypselae for plant dispersal (VENABLE et al. 1995). The high length of the central cypselae of Bidens also exposes their awn, since they stay at a higher level, above the other structures, e.g. in B. frondosa, which favors exposure to animal vectors (CARLQUIST 1966). In most heteromorphic Asteraceae species, central cypselae behave as colonizers and present morphological features that allow farther dispersal (IMBERT 2002, BASKIN et al. 2013). In the heteorcarpic types, the total dry biomass of cypsela types differed for B. alba, B. pilosa, B. subalternans and B. bipinnata (Table 1). The peripheral type for these species was lighter than the central ones, on the contrary of the previously reported (IMBERT 2002). However, for B. frondosa, B. gardneri and B. aristosa, there is no difference between the types. For them, the equal dry mass indicates that the resources allocated to produce each cypsela type were similar. This is uncommon in other heterocarpic species. Usually, central cypselae are lighter than the peripheral ones (IMBERT 2002, MATILLA 2005). Since it favors seed dispersal, this weight difference is important for anemochorous diaspores. On the other hand, in Bidens cypselae, as in other ectozoochoric seeds, the cypsela weight reduction contributes little to seed dispersal distances. The peripheral type, on the contrary for other species, its dark color, reduced weight and size may have led to the wrong idea of peripheral cypsela malformation. The dry mass of the cypsela parts indicates that resources allocated to the fruits types low varies (F1,62=5.467, p=0.023) when compared to the seeds types (F1,62=40.2, p<0.001), despite both are significant different (Figure 2 and Table S1). The total mass variation is more related to seed mass variation among types than to the pericarp. In B. alba, B. 21 bipinnata, B. frondosa, B. gardneri, B. pilosa and B. subalternans the peripheral seeds were lighter than the central ones. Pericarp mass just varied between types in B. gardneri and B. subalternans, whose peripheral pericarps were slightly heavier than the central ones (discussed in the next section). The reduced seed size in peripheral cypselae may indicate low embryo growth potential, thus more resistance from the pericarp compared to the central seeds, which reflects directly on germination behavior. However to the B. frondosa which the central have slow germination this idea is not aplicable (BRÄNDEL 2004). In Senecio jacobaea, ray cypselae are heavier and germinate more slowly than disk ones. The time ray cypselae take to germinate depends on the pericarp mass, which physically impede germination. On the other hand, the heavier the embryo, the faster it germinates (MCEVOY 1984). In Crepis tectorum (ANDRESSON 1990) and Tragopogon pratensis (VAN MÖLKEN et al. 2004), seed size is also directly related to germination rate. 22

Figure 1. Bidens ssp. fruit morphology.

Table 1: Morphologic features of Bidens cypselae. Means of cypsela length and width (mm), pappus length (mm) and dry mass of cypselae (mg). Followed by the results of the Welch T-test between the mean of the two cypsela types within the species, and one-way ANOVA for B. gardneri. Standard error reported in brackets. Species Types Length Width Pappus length Dry mass B. alba Cen. 7.628(0.173) 0.714(0.011) 2.347(0.062) 1.144(0.023) Per. 5.139 (0.088) 0.905(0.012) 2.195(0.058) 0.996(0.022) t=12.827, t=-11.562, t=1.8015, t=4.629, df=72.679, df=96.177, df=97.537, df=7.999, p<0.001 p<0.001 p=0.075 p=0.002 B. pilosa Cen. 7.089 (0.193) 0.794(0.020) 2.580(0.044) 1.494(0.028) Per. 4.721(0.089) 0.998(0.023) 2.080(0.046) 1.318(0.024) t=11.126, t=-6.657, t=7.848, t=4.808, df=68.708, df=96.677, df=97.701, df=7.839, p<0.001 p<0.001 p<0.001 p=0.001 B. subalternans Cen. 8.994 (0.150) 0.681(0.011) 2.192(0.045) 1.996(0.038) Per. 5.15(0.073) 0.91(0.012) 1.47(0.034) 1.774(0.059) t=23.019, t=-14.119, t=12.749, t=3.177, df=71.051, df=96.668, df=90.815, df=6.840, p<0.001 p<0.001 p<0.001 p=0.016 B. bipinnata Cen. 14.454(0.242) 0.886(0.016) 3.454(0.066) 5.669(0.067) Per. 9.954(0.134) 1.126(0.019 2.836(0.074) 4.627(0.112) t=16.285, t=-9.5839, t=6.216, t=7.979, df=76.457, df=95.353, df=96.805, df=6.564, p<0.001 p<0.001 p<0.001 p<0.001 B. frondosa Cen. 7.518(0.092) 1.986(0.043) 3.986(0.073) 3.328(0.099) Per. 5.291(0.077) 2.709(0.029) 2.733(0.052) 3.068(0.082) t=18.540, t=-13.861, t=13.937, t=2.021, df=94.975, df=86.290, df=88.493, df=7.749, p<0.001 p<0.001 p<0.001 p=0.079 B. gardneri Long 11.763(0.126) 0.846(0.013) 1.807(0.039) 5.115(0.039) Inter. 10.27(0.119) 0.933(0.022) 1.802(0.032) - Short 8.459(0.093) 1.05(0.021) 1.85(0.038) 4.818(0.179) t=1.621, F2,147=212.3, F2,147=29.61, F2,147=0.51, df=4.372, p<0.001 p<0.001 p=0.601 p=0.174 B. aristosa Cen. 5.906(0.075) 2.9418(0.056) - 3.564(0.100) Per. 4.849(0.049) 3.400(0.046) - 3.664(0.096) t=11.674, t=-6.345, t=-0.721, df=84.877, df=94.669, df=7.986, p<0.001 p<0.001 p=0.492 Bidens sp. 10.121(0.185) 0.769(0.012) 2.366(0.051) 3.714(0.021)

Figure 2. Dry mass of cypsela tissues according to type in Bidens species. The different letters represents significant difference (p<0.05) in Welch T-test between the two cypsela types within the species. For further details, see Table S1. Error bars= Standard error.

Cypsela germination

A variation in the germination behavior of dicarpic types was observed for the analyzed species (Figure 3 and Table S2). Central cypselae presented higher germination proportion and rate than the peripheral ones. B. subalternans, B. pilosa, and B. bipinnata showed difference in the final germination proportion. B. gardneri presented a variation in the germination proportion of short cypselae as compared to long ones (p<0.001, Tukey HSD) and intermediate cyspelae (p<0.001, Tukey HSD), but no difference between long and intermediate ones (p=0.58, Tukey HSD). However, the germination rates of both types statistically differed. Despite its morphological variation, B. alba presented no significant difference in type germinability, but a difference in germination rate. Brändel (2004) verified that, unlike the other Bidens species, the central type of B. frondosa germinated more slowly than the peripheral one. However, he used after-ripened cypselae, which could have biased this result (BASKIN et al. 2013). Since it is not sure whether morphological dicarpy is correlated to germination behavior in B. aristosa, B. riparia, and Bidens sp., future works need to be carried out. Heterocarpy is characterized by the variation in cypsela morphology (i.e. size, color, dispersal appendices), but also by germination behavior (IMBERT 2002, BASKIN et al. 2014). In some species, behavioral variation may not be detected by morphotype selection because some apparently monocarpic species may present cryptic dicarpy (LEVERETT and JOLLS 2014). The opposite may also happen, a morphological difference may not represent 25 behavioral heteromorphism, as reported for some species (IMBERT et al. 1996, SOUZA- FILHO and TAKAKI 2011).

Figure 3. Germinability (%) and Germination rate (days-1) of the cypsela type assays. Asterisks represent significant difference between the cypsela types means by Welch T-test and one-way ANOVA (just for B. gardneri) = ***<0.001 and **<0.05. The symbols= means and Error bars= Standard error. For further details, see Table S2.

Pericarp anatomy

The overall pattern of the pericarp structures of Bidens is similar in all studied species: three layers, the middle one being more developed (Figures 3 and 4; S1 to S6). The exocarp, external layer, is uniseriate with uniform cube-shaped cells and external cuticle. The mesocarp is divided into three distinct regions: (1) outer mesocarp, with a variable diameter, and thin-walled cells composing one to five layers; (2) middle mesocarp, with thick-walled cells composing one to five layers; (3) inner mesocarp, whose thin-walled, slightly crushed cells have a variable diameter. Endocarp is uniseriate, slightly conspicuous and, in most species, it collapses at maturity (JULIO and OLIVEIRA 2009, JANA and MUKHERJEE 2013). Mesocarp is the most important tissue for seed protection. It consists of pitted palisade parenchyma, subjacent sclerenchyma, and other parenchyma cells. The middle mesocarp has thick-walled, lignified cells (confirmed by histochemical analyses) to protect the embryo. In two species: B. aristosa and B. frondosa (Figures S2 and S4, respectively), the middle mesocarp presented one or two layers displayed homogeneously. However, the other species present a ribbed cypselae pattern that varies between one, in the furrow or striation, and circa five cells thickness, in the ribs. It consists of ribs and furrow regions 26 between the outer (parenchyma) and middle (sclerenchyma) mesocarp layers (TADESSE and CRAWFORD 2014). This anatomical pattern probably allows embryo cell expansion during seed imbibition. The phytomelanin is deposited in the schizogenous cavity (JULIO and OLIVEIRA 2009). This phytomelanin layer is related to the mechanical protection of cypselae (PANDEY and DHAKAL 2001). The distribution of this acellular layer generates some patterns that have an ecological and taxonomic importance (TADESSE and CRAWFORD 2014). Fruit maturation ends in: (1) phytomelanin deposition, (2) induration, and (3) detachment of external layers (exocarp and outer mesocarp), this last step could not occurs in some peripheral cypselae. We believe that the time at which phytomelanin is deposited is related to the different cypsela ontogeny. However, there is no clear pericarp variation between ripped cypsela types. We found some the clues that could indicate germination physical restrictions difference among the cypsela types, but these are not strong enough to proof the pericarp resistance difference. For B. subalternans (Figure 3B and 3F), B. bipinnata (Figure S3B and S3F) and B. pilosa (Figure S5B and S5F) number of cell layers in the ribs varies between central and peripheral types. The peripheral middle mesocarp are thicker than central ones. But we do not atribute an physical restiction to germination due to similar layer thickness in the furrow (Figures 3D and 3H, S3D and S3H and S5D and S5H). In addition, for these species, the extra rib layers explains the higher dry mass/size proportion of peripheral cypselae (see Table 1, Figure 2 and Table S1). But for B. gardneri, which the peripheral fruit is heavier than the central ones, any extra tissue were found. In Catananche lutea, aerial cypselae differ from amphicarpic ones, whose pericarp slows down water uptake (RUIZ DE CLAVIJO 1995). The tricarpic cypselae of Garhadiolus papposus have different degrees of seed dormancy maintained by their pericarp structure, where more sclerenchyma cell layers act as a barrier to embryo growth. Germination occurs when the embryo growth potential is superior to the fruit mechanical constraint (SUN et al. 2009). In dicarpic Anthemis chrysantha, the darker cypselae are more dormant than the lighter, central ones. Seed dormancy is caused by a thick mesocarp with no intracellular spaces, which thus acts as a mechanical constraint slowing down seed imbibition (AGUADO et al. 2011). In the tetraploid varieties of Aster pilosus, the pericarp weight is correlated to germination time since the lighter the pericarp, the faster the germination (PRINZIE and CHMIELEWSKI 1994). Another possible explanation for high mass pericarps of the peripheral cypselae of Bidens can be remaining external layers in the ripe peripheral cypselae. We observed that the exocarp and the outer mesocarp layers keep surrounding the ripened peripheral cypselae. This can be confirmed by the presence of ornaments formed by the enlargement of outer mesocarp cells 27 on the pericarps (see Figure 1). Differently, in the final stage of development of the central cypselae, a schizogenous cavity forms between the middle and outer mesocarp and it detaches these layers (JULIO and OLIVEIRA 2009). This pattern was observed in B. pilosa, B. alba, B. subalternans, and B. bipinnata. The presence of external layers possibly does not affect the germination processes through mechanical inhibition, because these layers are formed by cells with primary cell walls. But it may explain why some authors reported the presence of a thick pericarp in peripheral cypselae (ROCHA 1996). The morphological difference of cypselae in the capitulum is attributed to two hypotheses: (1) resource competition among fruits and (2) architectural effects (TORRICES and MENDEZ 2010). The first one implies that the difference in development time causes variation in the resources allocated to each cypsela type, i.e. peripheral cypselae should be bigger than central ones because their floret anthesis begins before. The second one assumes that cypsela sizes differ according to genetic processes. Both patterns have been reported in monocarpic species as Tragopogon porrifolius (TORRICES and MENDEZ 2010), Aster acuminatus (PITELKA et al. 1983), and Coreopsis lanceolata (BANOVETZ and SCHEINER 1994). They create a continuous size variation within the head (IMBERT 2002), but have no relation with germination behavior. In heteromorphic species, as Bidens, different fruit development disagrees with the resource competition theory. In fact, although peripheral cypselae open and develop first, they are smaller than the central ones. Thus, this differentiation occurs through inherent ontogenetic mechanisms.

A E

B F

200µm

C G

mm ex ts en ts 40µm mm en om

H D

mm ex ex mm en ts en ts co co om om

Figure 3. Immature cypsela transversal slides of Bidens subalternans. Left: central cypsela: A and C proximal region slices, near to the radicle, B and D medial region slices. Right: peripheral cypsela: E and G proximal region slices, F and H medial region slices. Black arrow points to phytomelanin deposition (co = cotiledons; ex = exocarp; om = outer mesocarp; mm = median mesocarp; im = inner mesocarp; en = endocarp; ts = seed testa). 29

E A

F B

200µm

C G om ex ex mm om t mm

D H

ex om om mm en mm

en 40µm

Figure 4. Immature cypsela transversal slides of Bidens gardneri. Left: central cypsela: A and C proximal region slices, near to the radicle, B and D medial region slices. Right: peripheral cypsela: E and G proximal region slices, F and H medial region slices. Black arrow points to phytomelanin deposition (co = cotiledons; ex = exocarp; om = outer mesocarp; mm = median mesocarp; im = inner mesocarp; en = endocarp; ts = seed testa).

Chemical composition of cypsela parts

The PCA generated by the chemical analysis of methanol extracts of pericarps of Bidens showed their composition varies in most species, but in some of them, such variation was not clear (Figure 5-11). In B. aristosa, B. bipinnata, B. pilosa, and B. subalternans, the scores generated by the HPLC loadings show that each cypsela type is clearly grouped apart from the other. In these species, the peripheral type contains some extra compounds that were not found in the central one. Yet in B. alba, B. frondosa, and B. gardneri, the clusters of the sample scores are not clear since all the cypsela type samples grouped together, however for some there is a small tendency of group separation. Thus, despite physiological differences, we did not see any correlation to chemical composition for these three species. Among the most relevant compounds that differentiate cypsela types, we found flavonoids in the peripheral fruits, and nitrogen compounds in the central ones. The peripheral fruits of B. aristosa, B. bipinnata, and B. subalternans contain kaempferol and those of B. pilosa, quercetin. These compounds are important for their antioxidant activity (BORS et al.1990, RICE-EVANS et al. 1996). Kaempferol is highly efficient to decrease the damages of peroxy radicals on linoleic acid (TOREL et al. 1986). Both kaempferol and - quercetin also inhibit the O2 promoted redox reactions of methyl viologen (MeV). They are

- known as potent O2 quenchers in the photosynthetic pathway (LARSON 1988). These reactive oxygen species (ROS) are known to alleviate the dormancy of several species (GRAEBER et al. 2012), including B. pilosa (WHITAKER et al. 2012). They promote germination through endosperm/seed coat degradation or the stimulation of hormone production (EL-MAAROUF-BOUTEAU et al. 2014). MeV is effective to stimulate the - germination of the peripheral cypselae of B. pilosa. It acts as an O2 donor that triggers the germination process. Flavonoids act as scavengers for that radicals and their action may reduce the germination rate in the peripheral cypselae of B. pilosa, B. subalternans, and B. bipinnata. Assays with these antioxidants need to be carried out to prove their biological action. Most of the relevant compounds have not yet been identified. They must be described and tested for their biological activity in seed germination. They may also have another biological meaning for the cypsela type ecology, e.g. increase seed longevity by avoiding oxidative stress (JIA et al. 2012).

REFERENCES For the reference, please check it at page 95. 31

B. alba

Figure 5. Principal Component Analysis biplot of the loadings, summary of the variables (black dots), and scores relative to central (light gray triangles) and peripheral (dark gray squares) replicates of pericarps of B. alba.

B. aristosa

Figure 6. Principal Component Analysis biplot of the loadings, summary of the variables (black dots), and scores relative to the central (light gray triangles) and peripheral (dark gray squares) replicates of pericarps of B. aristosa.

B. bipinnata

Figure 7. Principal Component Analysis biplot of the loadings, summary of the variables (black dots) and scores relative to the central (light gray triangles) and peripheral (dark gray squares) pericarps of B. bipinnata.

B. frondosa

Figure 8. Principal Component Analysis biplot of the loadings, summary of the variables (black dots), and scores relative to the central (light gray triangles) and peripheral (dark gray squares) pericarps of B. frondosa. 33

B. gardneri

Figure 9. Principal Component Analysis biplot of the loadings, summary of the variables (black dots), and scores relative to the central (light gray triangles) and peripheral (dark gray squares) replicates of pericarps of B. gardneri.

B. pilosa

Figure 10. Principal Component Analysis biplot of the loadings, summary of the variables (black dots), and scores relative to the central (light gray triangles) and peripheral (dark gray squares) pericarps of B. pilosa. 34

B. subalternans

Figure 11. Principal Component Analysis biplot of the loadings, summary of the variables (black dots), and scores relative to the central (light gray triangles) and peripheral (dark gray squares) pericarps of B. subalternans.

SUPPLEMENTARY MATERIAL

Table S1: Resource allocation of the Bidens cypselae types. Dry mass means of cypsela seed and fruit (mg) for cypselae and proportion of seed/fruit (%). Followed by the results of the Welch T-test between the mean of the two cypsela types within the species. Standard error reported in brackets. Species Types Seed Fruit Seed/Fruit B. alba Cen. 0.560(0.015) 0.540(0.0114) 1.038(0.026) Per. 0.414(0.013) 0.522(0.009) 0.793(0.022) t=7.337, t=1.203, df=7.902, df=7.799, p<0.001 p=0.264 B. pilosa Cen. 0.716(0.014) 0.654(0.008) 1.095(0.022) Per. 0.592(0.026) 0.686(0.021) 0.869(0.059 t=4.133, t=-1.4142, df=6.182, df=5.158, p=0.006 p=0.2147 B. subalternans Cen. 0.974(0.027) 0.922(0.019) 1.058(0.037) 0.596(0.029) Per. 0.634(0.038) 1.062(0.024

t=7.3292, t=-4.596, df=7.274, df=7.710, p<0.001 p=0.002 B. bipinnata Cen. 3.259(0.0216) 1.949(0.059) 1.678(0.050) Per. 2.304(0.065) 1.960(0.111) 1.191(0.075) t=13.997, t=-0.087, df=4.878, df=6.105, p<0.001 p=0.933 B. frondosa Cen. 2.048(0.073) 1.054(0.0250) 1.944(0.056) Per. 1.822(0.053) 1.048(0.033) 1.743(0.061) t=2.492, t=0.145, df=7.274, df=7.449, p=0.040 p=0.888 B. gardneri Long 2.653(0.027) 2.060(0.013) 1.288(0.013) Short 2.196(0.106) 2.242(0.055) 0.978(0.027) t=4.172, t=-3.236, df=4.520, df=4.453, p=0.011 p=0.027 B. aristosa Cen. 2.277(0.102) 1.015(0.022) 2.242(0.082) Per. 2.368(0.076) 1.048(0.030) 2.266(0.087) t=-0.713, t=-0.877, df=7.388, df=7.219, p=0.497 p=0.409 Bidens sp. 2.109(0.015) 1.331(0.027) 1.588(0.039) 36

Table S2: Germinability (%) and germination rate (days-1) Bidens cypselae types. Follows the results of the Welch T-test between the mean of the cypsela types within the species, and the one-way ANOVA to B. gardneri. Standard error reported in brackets. Species Types Germinability Germ. Rate B. alba Cen. 96.667(2.357) 0.191(0.014) Per. 91.667(0.962) 0.134(0.014) t=1.964, t=2.928, df=3.973, df=5.993, p=0.122 p=0.026 B. pilosa Cen. 89.167(2.846) 0.008(0.001) Per. 59.167(3.155) 0.009(0.001) t=7.060, t=-0.578, df=5.938, df=5.616, p<0.001 p=0.586 B. subalternans Cen. 80.833(5.672) 0.002(0.0002) Per. 42.5(4.589) 0.002(0.0003) t=5.254, t=0.708, df=5.75, df=5.829, p=0.002 p=0.506 B. bipinnata Cen. 100(0) 0.017(0.0004) Per. 41.234(6.185) 0.016(0.0002) t=9.502, t=3.499, df=3, df=4.678, p=0.002 p=0.019 B. gardneri Long 91.25(2.741) 0.108(0.006) Inter. 87.083(2.630) 0.067(0.003) Short 68.75(3.329) 0.040(0.001) F2,21=16.84, F2,21=77.51,

p<0.001 p<0.001

A E

B F

200µm

C G ex mm om ex om im ts en mm im

ts en es

D H ex om mm v 40µm ex im en im mm om en co v ts ts co

Figure S1. Immature cypsela transversal slides of Bidens alba. Left: central cypsela: A and C proximal region slices, near to the radicle, B and D medial region slices. Right: peripheral cypsela: E and G proximal region slices, F and H medial region slices. Black arrow points to phytomelanin deposition (co = cotiledons; ex = exocarp; om = outer mesocarp; mm = median mesocarp; im = inner mesocarp; en = endocarp; ts = seed testa; v = vascular axis).

A E

B F

200µm

C G

ex en om mm ex en ts ts

mm om

D H ex 40µm ex om ts mm om en ts en co mm

Figure S2. Immature cypsela transversal slides of Bidens aristosa. Left: central cypsela: A and C proximal region slices, near to the radicle, B and D medial region slices. Right: peripheral cypsela: E and G proximal region slices, F and H medial region slices. Black arrow points to phytomelanin deposition (co = cotiledons; ex = exocarp; om = outer mesocarp; mm = median mesocarp; im = inner mesocarp; en = endocarp; ts = seed testa).

A E

B F

200µm

C G ex om

mm mm im

om exe en en ts

D H en om ex mm ts im 40µm ex en om mm

ts im co

Figure S3. Immature cypsela transversal slides of Bidens bipinnata. Left: central cypsela: A and C proximal region slices, near to the radicle, B and D medial region slices. Right: peripheral cypsela: E and G proximal region slices, F and H medial region slices. Black arrow points to phytomelanin deposition (co = cotiledons; ex = exocarp; om = outer mesocarp; mm = median mesocarp; im = inner mesocarp; en = endocarp; ts = seed testa).

A E

B F

200µm

C G v ex ex om om mm en ts en ts mm

D H exe om 40µm ex om mm ts mm en ts co im

Figure S4. Immature cypsela transversal slides of Bidens frondosa. Left: central cypsela: A and C proximal region slices, near to the radicle, B and D medial region slices. Right: peripheral cypsela: E and G proximal region slices, F and H medial region slices. Black arrow points to phytomelanin deposition (co = cotiledons; ex = exocarp; om = outer mesocarp; mm = median mesocarp; im = inner mesocarp; en = endocarp; ts = seed testa; v = vascular axis).

E A

B F

200µm

G C ex om 40µm mm im im en om ts ex ts en mm em

D H ex om ex om im

mm mm im ts ts en en

Figure S5. Immature cypsela transversal slides of Bidens pilosa. Left: central cypsela: A and C proximal region slices, near to the radicle, B and D medial region slices. Right: peripheral cypsela: E and G proximal region slices, F and H medial region slices. Black arrow points to phytomelanin deposition (co = cotiledons; ex = exocarp; om = outer mesocarp; mm = median mesocarp; im = inner mesocarp; en = endocarp; ts = seed testa).

E A

B F

200µm

C G om ex ex mm

mm im im om en en ts

ts 40µm

ex H D om ex mm om im im mm en en ts

ts co

Figure S6. Immature cypsela transversal slides of Bidens sp. Left: central cypsela: A and C proximal region slices, near to the radicle, B and D medial region slices. Right: peripheral cypsela: E and G proximal region slices, F and H medial region slices. Black arrow points to phytomelanin deposition (co = cotiledons; ex = exocarp; om = outer mesocarp; mm = median mesocarp; im = inner mesocarp; en = endocarp; ts = seed testa).

CAPÍTULO 2

GERMINATION CONSTRAINTS OF DICARPIC CYPSELAE OF Bidens pilosa

Paulo Roberto de Moura Souza Filho, Massanori Takaki

Departamento de Botânica, Universidade Estadual Paulista, Rio Claro, São Paulo, 13506- 125, Brazil.

ABSTRACT

Bidens pilosa is a heterocarpic weed species with two cypsela types which have clear morpho-physiological differences, which the peripheral type is smaller and slower germination than central one. We aimed to verify by which mechanisms their germination behavior varies. We focused on two mechanisms: (1) pericarp constraints (physical and chemical) and (2) hormonal stimulation (Abcisic acid [ABA] and Gibberellin [GA]). Both cypselae types are mechanical constrained by the pericarp, when the seed were excised, both types increase their germination, but the behavioral differences remained. The pericarp of peripheral type has chemical inhibitors that effectively inhibited the intact central cypsela. That inhibitor is still unidentified, but we discussed by which way it possibly works. To test the hormonal effects, we focused on the ABA/GA control. Both cypselae responded to exogenous ABA concentration gradient, however there is no variation among types on the sensitivity to it. Also both cypsela types were indifferent to Fluridone addition (ABA inhibitor), which indicates that the dormancy is not maintained by de novo ABA synthesis. The cypsela types had different sensitivity to the exogenous GA3 gradient, which the central type was more sensitive to the treatment than the peripheral one. But when the endogenous GA synthesis were blocked (by Paclobutrazol) both types responded equally to same GA concentrations. It indicates that endogenous GA synthesis may be related to difference on germination of cypsela types. To sum, the seed types differ on it growth potential to overcome the pericarp resistance, while the inhibitor in the peripheral fruit reduces the potential, the GA increases it.

Key-words: hormonal controlling, pericarp inhibition.

INTRODUCTION

Many higher plants produce diaspores with different morpho-physiological traits and behaviors to favor seed establishment and plant dispersion, a strategy known as seed heteromorphism. This reproductive strategy is found in many plant families and it is particularly common in Asteraceae (VENABLE 1985, IMBERT 2002). In Bidens, the accepted dicarpy model is that the central cypsela are bigger and germinate faster than the 47 peripheral ones (FORSYTH and BROWN 1982, DE MARINIS 1983, BROWN and MITCHELL 1984; CORDIKI et al. 1991, ROCHA 1996, AMARAL and TAKAKI 1998, BRÄNDEL 2004a). The central type thus has more chances to widen its spatial dispersal area (ROCHA 1996). On the other hand, a germination delay allows the peripheral one to increase its temporal spread (FORSYTH and BROWN 1982). However, the mechanism inducing different physiological behaviors in each cypsela type is still unknown. Some authors suggest that the peripheral cypsela pericarps are thicker (ROCHA 1996) or have an inhibitor (FORSYTH and BROWN 1982), while others propose that both types have a different environmental sensitivity (AMARAL-BAROLI and TAKAKI 2001). To avoid early establishment and environmental threats, fresh seeds easily germinate after some requirements are met. Non-deep physiological dormancy is known in most higher plant species and it is related to the mechanical constraints of the tissues surrounding the embryo. This barrier is alleviated by environmental signals that trigger inner embryo mechanisms provoking root protrusion, which is germination stricto sensu (FINCH-SAVAGE and LEUBNER-METZGER 2006). In many species reproducing through cypselae (or achenes), the pericarp restricts embryo growth by two main ways: mechanical and/or chemical. It limits water and air exchanges, causing a delay in seed germination (VENEBLE and LEVIN 1985, AGUADO et al. 2011). It also impedes embryo growth mechanically through the presence of fiber layers or hard tissues (MCEVOY 1984). On the other hand, the pericarp may contain chemical inhibitors that block the progress of embryo development (MATILLA et al. 2005). In Salsola komarovii, dormant seed perianths present high traces of abscisic acid (TAKENO and YAMAGUCH 1991). Other authors found that soluble leachates retard cypsela germination (BENEKE et al. 1993, FORSYTH and BROWN 1982). Hougue (1972) proposed that the pericarp and seed coat of Bidens cernua contain an unidentified inhibitor and that this chemical constraint is alleviated by exogenous hormones and tegument removal. Forsyth and Brown (1982) observed that the peripheral cypsela leachates of Bidens pilosa contain a germination inhibitor. When this extract is applied on central cypselae, their germination rate is slowed, however is not clear the nature of it. In another approach, Felippe (1978) stimulated dark germination of the central types, via pericarp removal. In addition, Amaral-Baroli and Takaki (2001) showed that cypsela types present different responses to light. Dormancy varies through endogenous changes within the embryo whose growth potential against pericarp mechanical constraints will determine germination stricto sensu (NONOGAKI 2006). Growth potential is determined by the balance of two hormones: (1) gibberellins, which trigger germination, and (2) abscisic acid, which maintains embryo dormancy. Furthermore, in the seeds of B. pilosa, non-deep physiological dormancy 48 responds to environmental cues: red light stilmuli (VALIO et al. 1972, FELLIPE 1978; FORSYTH and BROWN 1982, AMARAL-BAROLI and TAKAKI, 2001), after-ripening (WHITAKER et al. 2010), and, possibly, stratification (in B. frondosa [BRÄNDEL 2004a]). These signals trigger the embryo germination process and/or alleviate dormancy. So our objective verify how both cypsela types of B. pilosa differ on it germination mechanisms. We try to elucidate the mechanisms of variation in germination behavior through hormonal and fruit removal manipulations. Thus, we firstly verified how their behavior naturally vary among populations. Then we focus on the ABA/GA hormonal balance, which is a known mechanism that control the germination. We verified the sensitivity to inhibitor, i.e. exogenous ABA and Fluridone, ABA synthesis inhibitor, among cypsela types and populations. Then we tested the effect of the mechanical and chemical constraints of the fruit on the germination, via manipulative treatments, aiming to verify if there is any difference on the cypsela types. In addition, the sensitivity of the germination promoter hormone, GA3, on their germination were verified. Either we worked with

Paclobitrazol, GA inhibitor, exogenous GA3 and the pericarp removed seeds to verify their effects on cypsela type’s growth potential. We summarized the proposed mechanism on the Figure 1 (based on Cadman (2006) and Finch-Savage and Leubner Metzger (2006). It represents how the cypsela types behaves. In the model the fruit inhibitor induces the ABA syntheses on peripheral cypselae which moves the hormonal balance to a dormant state. On the other hand, the absence of that inhibitor makes the central type germination faster than peripheral ones.

Figure 1. Proposed model of the germination mechanism of dimorphic cypselae in B. pilosa. There is a balance between the endogenous hormones: abscisic acid (ABA) induces cypsela dormancy, and gibberellins (GA) stimulate germination, favoring embryo growth potential. The physiological mechanisms (e.g. after- ripening, stratification, and light responses) alleviate dormancy through GA synthesis or sensitivity. On the other hand, the presence of inhibitors in the fruit induces the de novo ABA synthesis or increases sensitivity to it. The model was based on Cadman (2006) and Finch-Savage and Leubner Metzger (2006).

MATERIALS AND METHODS

Cypsela collection

Cypselae of Bidens pilosa were collected from populations growing in rural or urban sites at Rio Claro, SP, Brazil (Table 1). The cypselae of each plant, or small group (when they were tangled), were stored in paper bags under room conditions for less than one month. When needed for a new experiment, fresh cypselae were collected at the same site to avoid after-ripening effects on stored seeds.

Table 1: Location and features of populations.

Pop. Place Coord. Features Cana Road to Ipeúna 22°26'7.42"S; 47°37'54.05"W Roadside near a sugar cane crop Site surrounded by sugar cane Horto Av. Saúde 22°25'28.99"S; 47°30'5.92"W crop. Municipal street Site surrounded by sugar cane Ajapi 22°20'49.36"S; 47°32'42.87"W to Corumbataí crop. 11-B 11-B Street 22°24'20.57"S; 47°32'52.31"W Urban wasteland site.

Germination variation among populations

To analyze the physiological variations among populations, we carried out germination assays with freshly collected cypselae (one day storage). Prior the assay the cypselae were selected by each types, and then twenty-five of it were put into 90mm Petri dishes lined with two layers of filter paper (Prolab, Brazil) and moistened with deionized water. We used five dishes for each type and population, as replicates, which were kept under constant fluorescent white light (approx. 30 µmol.m2.s-1) or in the dark (in a black polystyrene box), kept in a 25±1°C germination room. Censuses were carried out daily for light treatments and weekly for dark ones, the latter occurring in a dark room under dim green safe light. At the end of the assay (after 3 weeks), the viability of ungerminated cypselae was tested with tetrazolium (0.5% in phosphate buffer kept at 41°C for 24h).

Variation of sensitivity to abscisic acid

To evaluate the effect of abscisic acid (ABA) on the germination of the cypsela types of B. pilosa, we applied exogenous ABA or Fluoridone (FLU), an inhibitor of endogenous ABA synthesis. Twelve cypselae of each type were placed in 5mm Petri dishes lined with two layers of filter paper (Prolab, Brazil) moistened with 3mL of different solutions, four replicates were made for that assay. The assays were performed for the four populations. Solutions were: 0.05, 0.5, 5µM ABA (Sigma-Aldrich Co., US); 0.1, 1, 10µM FLU (Fluka; Sigma-Aldrich Co., US), and both stock solutions were diluted in ethanol (max. 0.1%), the concentration were chosen based on previous assays and literature. The dishes were maintained under the same conditions (light and darkness) described above. Every week, cypselae were placed into dishes with a newly prepared solution. Censuses and dish exchange were performed at room temperature for light treatments, and in the darkroom for dark treatments. The assays lasted three weeks and the viability of ungerminated cypselae was tested, as mentioned before.

Pericarp influence in seed germination

To analyze the role of pericarp in seed germination, we used the “Horto” population due to high cypsela availability and to their physiological responses. Freshly collected cypselae were kept for 4 to 12h in darkness in a humid chamber (no direct contact with liquid water) prior to pericarp removal. Then, in the dark room, under dim green safe light, the pericarps of 12 cypselae per dish were carefully separated from the seeds (testa, endosperm and embryo) using forceps and a stereoscope. Treatments were: (1) intact cypselae; (2) intact cypselae with the crushed pericarp of the other type (crossed fruits 51 influence); (3) seeds only; and seeds of both types with crushed central (4) and peripheral (5) pericarps. Pericarps were crushed with a mortar and silica sand was added. The cypselae or seeds were put in 5mm Petri dishes lined with one layer of filter paper (Prolab, Brazil), under which one layer of silica sand (treatments 1 and 2) and powdered pericarps (treatments 3 and 4) were added. All dishes were moistened with deionized water and maintained under the same conditions (light and darkness) detailed above. Germination time was recorded for further analysis twice a day for the light treatments, and once a week for the dark ones.

Gibberellic acid effects on germination

For the first assays, we used the “Horto” population. Four replicates of 30 cypselae of each type were put inside 90mm Petri dishes lined with two layers of filter paper (Unifil,

Germany) moistened with 7mL of GA3 solution. The GA3 (VETEC, Brazil) work solutions were diluted in ethanol and deionized water was added to reach final concentrations of 50, 100, 500, and 1000µM. The assays were maintained under the same conditions mentioned above (light and darkness); dishes were renewed twice a week and scored daily for both treatments. At the end of the assay (after 30 days), the remaining ungerminated cypselae were tested with tetrazolium.

Sensitivity to exogenous gibberellic acid

“Horto” and “Ajapi” populations were used in this second assay. Four replicates with 12 cypselae or seeds of each type were placed in 50mm Petri dishes lined with one layer of filter paper. The manipulation treatments were: (1) intact cypsela, (2) isolated seeds, and (3) seeds with crushed pericarp. Fruit removal followed the protocol described above. All dishes were then moistened with 3mL of a solution. Solutions were Paclobutrazol (PESTANAL®; Sigma-Aldrich Co., US) 200 µM diluted in acetone (PAC), and 10, 100µM of

GA3 diluted in ethanol. The final work solution concentrations were 0.03% acetone and 0.1% ethanol (to 100µM GA3). We used an endogenous GA inhibitor (PAC) to avoid the bias caused by GA synthesis variations among embryo types (detailed below). The dishes were renewed weekly to ensure constant solution concentrations. At the end of the assay, the remaining cypselae and seeds were tested with tetrazolium for check it viability.

Statistical analysis

We tested the germination behavior: (1) final germination proportion and (2) germination rate, by two modeling approaches: (1) Generalized Linear Mixed Models (GLMM) and (2) Time-to-event Analysis, respectively, both focusing the cypsela or seed as sampling unit. These methods was preferred to deal with individual seed behavior rather than with the population inside dishes (SILESHI 2012; HAY et al. 2014). All analyses were performed with R (R Core Team, 2013). The final germination proportion of the treatments was analyzed by multiple regression generated by GLMM or GLM (General Linear Models), the latter when the random effect was not significant. To make the models we used Binomial distribution of the errors with log link (‘lme4’ package [BATES et al. 2014]), when it was overdispersed we used the Negative Binomial distribution (‘MASS’ package [VENABLES and RIPLEY 2002]). Model building followed Zurr et al. (2009) recommendations, as follows. First, all the explanatory variables (according the assay) were included, also the addition of all interactions and the random effects (i.e. dishes and/or populations). For most models, the cypselae type and light treatment variables were included, and other fixed explanatory variables were added depending on the assay. When an interaction variable was not significant to the final model, it was removed for selection procedure (for details see ZUUR et al. 2009). For example the final model for the population variation assay was:

Gi∼NB(μi , k) 2 E(Gi) = μi and Var(Gi) = (μi+μi )/k

log (μi) = α + β1 x CYPSELAE TYPE + β2 x LIGHT TREATMENT + β3 x

POPULATION + β4 x TYPE:LIGHT + β5 x LIGHT:POPULATION + β6 x

TYPE:POPULATION + β7 x TYPE:LIGHT:POPULATION

The germination probability (Gi) was Negative binomial distributed (NB). It mean (μi) 2 and variance ((μi+μi )/k) changes in function of the experimental factors, in this case: cypselae type, light treatment and their interaction. When chosen, via model comparison excluding the non-significant variables (not the case for the above model), the final model coefficients (β) were analyzed by multiple pairwise Z-test comparison with the Bonferroni adjustment of the p-value (‘multicomp’ package [HOTHORN et al. 2008]). Value and statistical significance are also presented. For germination behavior responses, we used Time-to-Event analysis, also known as survival analysis (‘survival’ package [THERNEAU and GRAMBSCH 2000]), which allows to 53 analyze when events, such as germination, that occur over the time, and to quantify the effects of contributing factors (ONOFRI et al. 2010, HAY et al. 2014). The germination times of the individual seeds were used to calculate the probability that one seed germinates after a specific time t once the assay began. The time the seeds take to germinate is preferred to final germination proportions to detect variations in seed behaviors. The censuses were considered to yield “exact data” due to daily verification. Non-parametric Kaplan-Meyer estimator before modeling to estimate germination probability. Parametric Accelerated Failure Time (AFT) modeling was then run underlying the best distribution (Weibull, log logistic, logaritimic, exponential). Based on Akaike´s Information Criterion we chosen the best fitted the non-linear regression models to cumulative germination data. The AFT is known as fully parametric because the survival functions follows the parameters of the chosen distributions. It also assumes there is a linear relationship between the logarithm of time to germinate and the analyzed factors. The latter accelerate or decelerate germination curves according to a coefficient (b). To the models, we used fixed explanatory variables: cypsela types, light treatments, the assay treatment (depending on the assay), and all the interactions. Replicates and/or population were tested as clustering random effects and included in the model using the frailty function of the survival package with Gamma distribution for it significance to the model, if not it was removed from the model (ONOFRI et al. 2010; DÉLYE et al. 2013).

S(t) = S0(φt) and t = t0φ

φ = exp (b1 x CYPSELAE TYPE + b2 x POPULATION + b3 x TYPE:POPULATION)

log(t) = b0 + b1 x CYPSELAE TYPE + b2 x POPULATION + b3 x TYPE:POPULATION + ε

Where t is time, S(t) is the germination function (in one of the parametric forms), S0 is the baseline germination function for the reference seed. Also t are related to the to the corresponding germination times for the reference seed lot (t0) by way of a relative constant amount, called “time ratio” (φ). And the time ratio is made based on the experimental factors in this case cypselae type, populations and it interaction. Also considering the time logatimic distributed we have the log(t) (see ONOFRI et al. 2010). The F-statistics were used to verify the significance of the variable inputted in the model. When ready, the final model were Z- tested the factor effects on the germination curve coefficients via multiple comparison with Bonferroni p-value adjustment. For both model-building procedures, the non-viable cypselae, which were tested with tetrazolium at the end of each assay, were excluded from the analysis. We considered that 54 their embryo were low developed or damaged which it is not detectible due to the pericarp structure.

RESULTS AND DISCUSSION

Variation of germination among populations

The final germination proportion model included cypselae type, light treatment, population and all the interactions as fixed effects and Negative Binomial distributed, as presented above. The B. pilosa cypsela types germination proportion varied (β=- 1.386±0.559, z=-2.480, p=0.013), and the peripheral cypsela presented lower proportions than the central (Figure 2A). Also for the light treatments there were a significant difference between white light and darkness (β=2.031±0.266, z=7.647, p<0.001). Just the in “11-B” population the germination proportion did not differ between the cypselas types on the white light (β=-0.104±0.131, z=-0.795, p=1.000), while for “Ajapi” (β=1.091±0.179, z=-6.076, p<0.001), “Cana” (β=-0.633±0.146, z=-4.340, p<0.001) and “Horto” (β=-0.992±0.173, z=- 5.724, p<0.001) the types differed. Under the darkness, despite the low germination, there is no variation on cypsela germinability, thus both types requires white light for germination. However there is low germination on “Rua 11-B” and “Ajapi” populations, and I may happened due to: (1) they were more sensitive to green light, through very low fluence response (VLFR) of phytochrome (AMARAL-BAROLI and TAKAKI 1996), (2) or light requirement was alleviated prior to cypselae collection, and phytochromes were previously activated. The AFT model for the germination rate were built accounting for populations, cypsela types and their interaction, and it was log logistic distributed, as presented above. Under white light, the cypsela types differed for all the populations, represented for T50, i.e. time to 50% germination (Figure 2B). The central cypsela type germinated faster than peripheral ones for all the populations (b=0.4015±0.068, z= 5.95, p<0.001). However, the rate varied among the population which the “11-B” have the faster germination for both cypsela types. Other authors (FORSYTH and BROWN 1982, ROCHA 1996, AMARAL and TAKAKI 1998) already reported the difference of the cypsela type behavior. However, no previous works reported the difference on the dicarpy among populations. Our results showed that the deppending on the population the behavior among types may vary. Also the variability on the germination is related to the peripheral cypselae than the central ones. In Crepis sancta populations, the germination rate of both cypsela types varied (DUBOIS and CHEPTOU 2012). Variation in peripheral cypsela delay, or dormancy deepness, may be 55 related to a strategy to maintain populations (ROCHA 1994). Germination delay is a response to environmental effects on the mother plant. This plasticity optimizes sibling establishment and guarantees the maternal population maintenance (DONOHUE 2005). In Suaeda aralocaspica (Amaranthaceae), the black seed, with deeper dormancy, it germination was affected by the fertility of the soil where mothers grew. When soil nutrient were highly available, black seed germination decreased, while that of brown ones remained unaffected (WANG et al. 2012).

A B

Figure 2. Final germination proportion (A) and Time to 50% germination (T50) generated by the AFT analysis of cypselae collected in the four populations (see Table 1). Symbols = final germination proportion within the replicates (A) and estimated T50 (B), the error bars = standard error, Uppercase letters = difference among cypsela types (p<0.05), Lowercase letters difference among populations (p<0.05) and N.S. = no significant difference.

Variation of sensitivity to abscisic acid (ABA)

The ABA sensitivity assay model were made with three main effects, i.e. cypsela type, light and hormone treatments, and the interaction of the cypsela type:light treatment and hormone:light treatment, also the populations were added to the model as random effect. Tt was Binomial distributed linked with log function. The ABA concentration rose affected the germination of the cypsela by reducing it (β=-0.491±0.129, z=-3.783, p<0.001) (Figure 3A). The cypsela type (β=-1.092±0.325, z=-3.360, p<0.001) and light treatment (β=4.098±0.389, z=10.537, p<0.001) were also significant different. However, the triple interaction were not significant to the model, thus is not possible to test for the pairwise comparison. On the other hand, the AFT germination rate model all the treatments and it interactions were significant: cypsela type, hormone and interaction, by log logistic distribution. The population was added as random effect. Both main effects cypsela type (b=0.779±0.094, 56 z=8.310, p<0.001) and hormonal concentration (b=0.118±0.024, z=4.908, p<0.001) were significant different (Figure 3B). In addition, the interaction showed a low significant difference between the cypselas types within the ABA gradient (b=0.077±0.0396, z=1.943, p=0.0521), maybe related to intrinsic cypsela germination delay. To verify the among populations variation, we made a new AFT model with just the higher ABA concentration, i.e. 5µM. The population, cypsela types and their interaction were the fixed effects variables, and it was log logistic distributed. By pairwise comparison, the peripheral types of all population was slower germination than central ones (Figure 3C). But just for the peripheral there is a significant difference (p<0.05) on germination delay among populations. This variation may be related to its maintenance strategy were the maintainer type should “fit better” to the surrounding environment. The inhibitory effects of ABA on seed germination delay radicle expansion and weaken endosperm, in addition to enhancing the expression of transcription factors (MIRANSARI and SMITH 2014). This signaling can be affected by outer ways that can increase or release seed dormancy deepness. Some Arabidopsis ecotypes presented different ABA sensitivity, via DOG1 (Delay of Germination) expression, whose highest expression increases seed dormancy. On the other hand, there is little variation in exogenous ABA response among Arabidopsis genetic backgrounds and their responsiveness seems to be related to gibberellins pathway (BARUA et al. 2011). Both cypsela types were insensitive to Fluridone (FLU), indicating that no de novo ABA synthesis was observed in the cypselae of B. pilosa (Figure 4A and B). For the germination proportion model (Binomial distributed), the main effect of the FLU concentration were not significant, in other words, there is no variation on the germination response with it concentration. Also, the AFT model showed no response to FLU, i.e. there are no variations in germination probabilities as FLU concentration raises (b=-0.0022±0.011, z=-0.0174, p=0.986, log normal distributed), either. This may indicate that there is no ABA de novo synthesis. We expected the FLU effect mainly to the peripheral ones, as stated by the model (Figure 1). Endogenous ABA content, maintained by its anabolism during seed imbibition, is responsible for dormancy maintenance in several species (GUBLER 2005; FINCH-SAVAGE and LEUBNER-METZGER 2006). However, it was not the case with mature fresh seeds of B. pilosa. We observed a positive response of the FLU concentration through its effects on seedling photosynthetic pigments. Under higher concentrations, leaf colors changed. Thus, ABA anabolism is not related to the maintenance of primary dormancy in B. pilosa. However, we do not discard that a secondary dormancy or dormancy cycling, caused by unsuitable environmental conditions, promotes ABA de novo synthesis, generating a dormancy cycling 57 via environmental signaling (FOOTITT et al. 2013). Carmona and Bôas (2001) observed that B. pilosa central cypselae increased their dormancy in the dry season and alleviated it in raining season. Our graphical model (Figure 1) considers this dormancy cycling, which may be regulated by ABA content.

A B C

Figure 3. Final germination proportion of the cypsela types on an exogenous ABA gradient (A), Time to 50% germination (T50) generated by the AFT analysis of cypselae (B), T50 of the populations submitted to 5µM of ABA (B). Symbols = final germination proportion within the replicates (A) and estimated T50 (B and C, by different models), the error bars = standard error, Uppercase letters = difference among cypsela types (p<0.05), Lowercase letters difference among populations (p<0.05).

A B

Figure 4. Final germination proportion (A) and time to 50% germination (T50) generated by the AFT analysis of cypselae submitted to a Fluridone gradient Symbols = final germination proportion within the replicates (A) and estimated T50 (B), the error bars = standard error.

Pericarp influence on seed germination

The influence of the pericarp final model accounted for the cypsela type, light treatment, pericarp restriction treatments, and cypsela type:light treatment interaction. It was Binomial distributed and linked by logarithmic function. In the model, the mechanical removal of the pericarp increase it germination proportion (β=-4.679±0.892, z= 5.243, p<0.001, Intact vs. No fruit) (Figure 5A). The chemical restriction was not observed for the intact cypsela (β=-0.931±0.868, z=-1.073, p=0.812, Intact vs. Crossed), neither for the isolated seed on central (β=1.049±0.683, z=1.537, p=0.525) or peripheral (β=-0.769±0.6846, z=-1.123, p=0.785) extract treatments. Under darkness treatments, the isolated seeds showed higher and more variable response. This behavior may be related to the alleviation of light requirements for germination when there is no physical barrier. When it is not impeded, the embryo becomes more sensitive to germination stimuli (see further results) and lower light stimuli can trigger germination, even the green safe light through Very Low Germination Response (VLGR) (AMARAL-BAROLI and TAKAKI, 2001). The AFT model for the pericarp influence accounted for the two main variables treatment and cypsela type, and their interaction. The pericarp removal did not differ the germination rate when compared to the control (b=-0.029±0.100, z=-0.287, p=0.774). But focusing in the peripheral type there is a significant difference on the mechanical constraints (b=-0.639±0.115, z=-5.572, p<0.001, Intact vs. No fruit). The crossed chemical treatment on the intact cypsela was effective on slow down it germination rate (b=0.296±0.101, z=2.922, p=0.003, Intact vs. Crossed), and that is related mainly to the central cypselae affected by peripheral extract (b=0.296±0.101, z=2.922, p=0.098). On the other hand, the isolated seeds were not affected by the central (b=0.050±0.102, z=0.494, p=0.621, No fruit vs. Central Fruit) nor peripheral (b=-0.119±0.096, z=-1.245, p=0.213, No fruit vs. Peripheral Fruit) fruit extracts.

A B

Figure 5. Mean final germination proportion (A) and time (hours) to 50% of seed germination of AFT model (B) of fresh cypselae and seed types according to fruit manipulations. Treatments were: intact cypselae; crushed fruit of the other type on the cypsela (Crossed); separated seed (No fruit); seed with crushed central fruits (Cen. fruit), and seed with crushed peripheral fruits (Per. fruit). Symbols = final germination proportion within the replicates (A) and estimated T50 (B), the error bars = standard error, Uppercase letters = difference among cypsela types (p<0.05), Lowercase letters difference among treatments (p<0.10).

Root protrusion occurs when the increase in embryo growth potential overcomes pericarp blockage (SUN et al. 2009). Most fruits are not hard-coated or impermeable and, when the seeds are free, dormancy is alleviated (BEWLEY 1997, FINCH-SAVAGE and LEUBNER-METZGER 2006). Some dicarpic species also present cypsela types with thicker pericarps that maintain dormancy (TANOWITZ et al. 1987, AGUADO et al. 2011).Thus, peripheral cypselae have a low growth potential as compared to central ones. The thickness of both pericarp types neither their dry mass differ for B. pilosa (central: 0.654±0.008mg; peripheral: 0.686±0.021mg; F1,8=2, p=0.195) (SOUZA-FILHO et al. Chapter 1). Yet seed dry mass varies (central: 0.716±0,014mg; peripheral: 0.592±0.026mg; F1,8= 17.08, p= 0.003). In Hemizonia increscens, the pericarp of peripheral cypsela physically impedes embryo growth and, when excised or nicked, germination is quicker, whereas removing the pericarp of central cypselae has no particular effect (TANOWITZ et al. 1987). In Anthemis chrysantha, the pericarps of peripheral cypsela are thicker than those of the central ones. They present more cell layers with secondary cell walls and their germination is slower (AGUADO et al. 2011). In B. pilosa, the dry mass ratio of seed/cypselae of the peripheral type is lower than that of the central one for “Horto” population. This was already observed in Senecio jacobaea cypselae, whose more dormant, peripheral cypselae presented lower ratios (MCEVOY 1984). The relative embryo size of Aster pilosus var. pilosus cypselae influences 60 germination rate. The bigger, tetraploid seeds germinate faster than the smaller, diploid ones (PRINZIE and CHMIELEWSKI 1994). The authors attributed lower resource allocation to the pericarps of tretraploid seeds, which makes them thinner than those of the diploid ones. However in B. pilosa, both pericarps possibly impose the same resistance to the embryo (not measured). In B. pilosa, the extracts of peripheral cypselae, rather than central ones, contain unknown compounds (or group) that reduce the germination of the central type (FORSYTH and BROWN 1986). We obtained similar results with intact central cypselae (Figure 5B), but the excised seeds were less responsive to chemical effect. When the physical barrier is released, central cypsela seeds lose their sensitivity to chemical inhibition. Previous experiments indicated that peripheral fruits contain flavonoids, e.g. quercetin (which may inhibit embryo growth potential (SOUZA-FILHO et al. [Chapter 1]). Further identification and biological test assays will be performed to identify these effective compounds and test it effects.Some secondary compounds influence seed dynamics, e.g. proanthocyanidins, which are, present in the testa of Arabdopsis thaliana. They induce ABA anabolism in the embryo, thus maintaining dormancy. Nevertheless, this pathway was not observed in the germination mechanism of seeds of B. pilosa, because FLU was not effective to alleviate dormancy (Figure 4). However, we cannot exclude this hypothesis because we did not test the effects of FLU and the peripheral leachate on central seeds, but it had a clear chemical inhibition. New assays should be carried out to determine whether or not ABA is related to chemical pericarp inhibition. Flavonoids are known to be antioxidant compounds that can act scavenging directly the reactive oxygen species (ROS) (RICE-EVANS et al. 1996). ROS drive some pathways that are related to seed germination stimulation: they oxidize specific proteins and mRNAs and are involved in cell wall loosening (HALLIWELL 2006, POURCEL et al. 2007, BAILLY et al. 2008, GOMES and GARCIA 2013, LARIGUET et al. 2013, EL-MAAROUF-BOUTEAU et al. 2014 and references within). For instance, the dormancy of Helianthus annus at 15°C is alleviated by ROS action through the oxidation of specific embryo proteins (ORACZ 2007). On the other hand, at 40ºC, their accumulation causes cell damages (CORBINEAU et al. 2002). In Arabidopsis the ROS accumulation is required for the seed germination, it acts signalizing the germination in a light-dependent manner via phytochrome interaction (LARIGUET et al. 2013). Whitaker et al. (2010) showed that the germination of peripheral cypselae of B. pilosa is stimulated by some ROS donors, which alleviate their dormancy. Superoxide (methyl viologen dichloride hydrate [MV]) affects both after-ripening dormancy and light requirement processes. On the contrary, hydroxyl radical generators, i.e. hydrogen peroxide and Fenton reagent, induce little germination. Thus, the antioxidant action may 61 scavenge the endogenous superoxide from the embryo and inhibit its germination. The presence of antioxidant flavonoids, e.g. quercetin (RICE-EVANS et al. 1996), may buffer ROS accumulation thus delaying the peripheral type germination. The addition of peripheral fruit leachates on intact central cypselae may also affect germination via superoxide scavenging (Figure 5). In the pericarps of B. pilosa, antioxidants act as in those of Hordeum vulgare glumellae (MENDIONDO et al. 2010), whose phenolic compounds react to oxygen, leading the seed to a hypoxic state, thus affecting ABA synthesis and catabolism genes. Antioxidant in the pericarp may also increase seed longevity (LONG et al. 2014) and resistance to oxidative stresses (JIA et al. 2011), but this still needs to be further analyzed.

Response to germination hormonal promoter

The model for the first GA3 sensitivity assay accounted for the three main variables: cypsela type, light and hormonal treatments, and their interactions. Tt was Negative

Binomial errors distributed linked by log link. The rose on the GA3 concentration increased the germination proportion of both cypsela (β=0.002±0.0002, z=9.897, p<0.001) (Figure 6A). The cypsela type (β=-0.740±0.207, z= -3.581, p<0.001) and the light treatment (β=1.395±0.130, z=10.717, p<0.001) significatively differed. Under light treatments, the final germination for both types did not differed, despite the low germination of the peripheral on the control treatment (β=-0.323±0.164, z=-1.965, p=0.8957). However, under darkness, the hormone addition overcame the light requirement for the germination. Both central (β=2.103±0.352, z=5.978, p<0.001, 0 vs. 500µM) and peripheral (β =1.860±0.471, z=3.949, p<0.05, 0 vs. 500µM) cypsela presented significant germination at 500µM. The AFT model was made with all the same variables as previous model, i.e. cypsela type, light and hormonal treatments, and their interaction, plus the replicates as random variables. This model was log logistic distributed. The germination probability did not differed between the types (b=0.373±0.394, z=0.947, p=0.344), however both treatments: light (b=-

2.492±0.379, z=-6.569, p<0.001) and GA3 (b=-0.002±0.0005, z=-2.909, p=0.004) differed. Under light, the germination rate differed for both types, however their sensibility to the hormone varied: the peripheral type accelerated it germination with the increase of GA3 concentration, while the central do not varied. The addition of 50µM was enough to increase the peripheral´s germination rate (b=-0.509±0.098, z=-5.203, p<0.001). On the other hand the addition of 1000µM did not increase the central germination rate (z=0, p=1). Under darkness, the germination rate of both types significatively increased with the hormonal treatment. The central type were more sensitive because it suffered increase on it 62 germination at 100µM (b=-0.583±0.137, z=-4.256, p<0.05), while the peripheral type increase was at 500µM (b=-0.779±0.162, z=-4.810, p<0.01). The GA triggers various mechanisms that allow germination stricto sensu, or root protrusion: both embryo growth and endosperm weakening overcome the mechanical resistance of the envelope and need GA anabolism (YAMAGUCHI and KAMIYA 2002). GA also downregulates ABA-upregulated genes: (1) GA reduces ABA levels by affecting ABA biosynthesis; (2) GA negatively regulates the ABA response pathway; and (3) GA and ABA signals are targeted independently of the distinct cis-regulatory sequences of a single gene (OGAWA et al. 2003). In ungerminated seeds, GA concentrations are maintained by endogenous homeostasis which limits them. In A. thaliana, these mechanisms are controlled by GA 20-oxidases and GA 3-oxidases that are negatively regulated by GA activity through feedback inhibition, whereas GA upregulates the genes encoding GA-deactivating GA 2- oxidases through a positive feed forward loop (OLSZEWSKI et al. 2002). The addition of exogenous GA3 to the medium increases GA concentration and forces GA-mediated genes to express and trigger root protrusion. In the cypselae of B. pilosa, exogenous GA increased: peripheral type germination rate under light, and the germination proportion and rate in darkness. However, we observed that the central type is more sensitive to GA3 than the peripheral one (Figure 6B). Yet it is not clear whether this central cypsela sensitivity is caused by hormone signaling or by its low physical pericarp constraint. To answer this question, we made another assay controlling the endogenous synthesis of GA and pericarp effects.

A B

Figure 6.Final germination proportion (A) and time to 50% germination (T50) generated by the AFT analysis of cypselae submitted to GA3 gradient. Symbols= means (A) and medians (B), error bars = standard error. 63

The requirement for GA3 assay model accounted for the seed types, fruit removal and hormonal treatments (PAC; PAC+10µM GA3; PAC+100µM GA3), the possible interactions were: homone:fruit removal treatments and fruit treatments:seeds type. The seed type: hormonal treatment was meaningless to the final model, thus it was not significant difference by removing it (p=0.565). The model was Negative Binomial distributed linked by log function. This assay was carried out to verify the GA to the germination progress of intact cypselae and excised seeds. It differs from the GA3 sensitivity assay because it is not a dormancy breaking treatment. The Paclobutrazol (PAC) treatment which inhibits the monooxygenases involved in the oxidation of ent-kaurene into ent-kaurenoic acid - a GA pathway precursor (JACOBSEN and OLSZEWSKI 1993) - alone was effective to inhibit germination and maintained seed viability.

The increase of the GA3 induced the germination for all the treatments (β=0.018±0.006, z=2.927, p=0.003). The removal of the pericarp did not significant increased the GA3 sensitivity (β=-0.006±0.006, z=-0.875, p=0.382). However, there is a significant difference between the sensitivity of the isolated seed and the seed with the fruit extract (β=-

0.007±0.002, z=-2.790, p= 0.005) indicating there was a chemical effect on the GA3 sensing mechanism.

A B C

Figure 7. Final germination proportion of the seeds and cypselae submitted to hormonal treatments. Manipulation treatments were: (A) Intact cypsela; (B) seed alone; (C) Seed plus crushed fruit. Solutions were: deionized water (control); 200μM of Paclobutrazol (PAC); PAC plus 10 and 100μM of GA3. Symbols= Means (A), error bars= standard error.

In both cypselae of B. pilosa, pericarps act mechanically to inhibit seed germination and the peripheral type is relatively more impeded (Figure 5). Felippe (1978) also showed that mechanical scarification (fruit apex removal) of cypselae of B. pilosa overcame photodormancy. The difference in cypsela germination is not directly related to pericarp mechanical constraints. One possible explanation is that embryo growth is also constrained by the endosperm. In Lactuca sativa, few-cell-layered endosperm mechanically impedes embryo growth. Endosperm thickness is not homogeneous: close to the micropyle, it has more cell layers rich in mannan. When stimulated (GA or red light), endo-β-mannanase is produced, which weakens the tissues surrounding the embryo root. Since isolated endosperm can also inhibit seed germination, it has a chemical crosstalk with the embryo, probably through ABA synthesis (BEWLEY 1997). Another hypothesis is that the embryos of peripheral fruits are previously inhibited by a chemical inhibitor, maybe during seed development, that affects the further germination of peripheral seeds. On the other hand, the central embryos not affected by the inhibitor have a high growth potential. However, when peripheral leachate is applied, this potential decreases. In Dimorphotheca sinuate (monocarpic), D. polyptera, (dicarpic) and Artotis fastuosa (dicarpic), intact cypselae are dormant and dormancy is released when the pericarp is damaged or removed, the latter method being more effective (BENEKE et al. 1993). The addition of pericarp leachate inhibited their embryo germination. The addition of more pericarp leachate (three times more than usual) further inhibited germination. For them dormancy maintenance is attributed to both mechanical and chemical germination resistance. Pericarp inhibitors are condensed tanniferous substances (proanthocyanidins). However, these authors did not cross fruit leachates to test their effects (BENEKE et al. 1993). In the deeper dormant achene types of Salsola komarovii (Chenopodiaceae), higher amounts of ABA were found in the perianths. Nevertheless, the chemical difference had no effect on achene type germination, and dormancy was attributed to other factors (TAKENO AND YAMAGUCHI 1991). The low response of excised B. pilosa seeds was not observed because releasing the mechanical resistance overstimulated the embryo. We expected that the germination process would be more affected as more pericarp is added, as observed by Beneke and co-authors (1992).

CONCLUSIONS

Bidens pilosa dimorphic cypsela share physiological traits: light requirement; they do not catabolize ABA for dormancy maintenance; the GA3 stimulates the darkness germination. However, there is some behaviors that differed between the cypsela types: their germination rate; peripheral type delay varied among the populations; central type were 65 sensitivity to chemical inhibitor. The difference between them may be related to the chemical inhibitor on the pericarp that affects the peripheral´s embryo growth potential and the central´s when manipulated. Thus, the pericarp plays an important role in chemical inhibition via embryo weakening.

REFERENCES For the reference, please check it at page 95.

CAPÍTULO 3

AMONG-POPULATION DICARPY VARIATION IN Bidens bipinnata L.

Paulo Roberto de Moura Souza Filho, Chunhui Zhang, Massanori Takaki, Kathleen Donohue.

Departamento de Botânica, Universidade Estadual Paulista, Rio Claro, São Paulo, 13506-125, Brazil. State Key Laboratory of Grassland Agro-ecosystems, School of Life Sciences, Lanzhou University, Lanzhou, Gansu 730000, People`s Republic of China. Department of Biology, Duke University, Durham, North Carolina 27708, United State of America.

ABSTRACT

The Bidens bipinnata dicarpy consists of two mopho-physiological distinct cypsela types. The central cypselae has bigger size and faster germination than the peripheral ones. We aimed to verify dicarpic traits: (1) morphology and (2) seed behavior variation among the populations, also identify which abiotic factors are correlated to them. Fourteen populations were measured for correlation analysis: their abiotic factors, soil chemical composition and light availability; also their vegetative and reproductive plant features. We tested for the cypselae behavior difference in lab and field assays, the latter we also manipulated the top soil to verify its influence on the cypsela types (maternal or alien). We analyzed the effects of the hormonal stimulants (Fluridone and Gibberellins plus Paclobutrazol) on them. The plant traits direct correlate to both abiotic factors, however the reproductive output correlation was not clear. The peripheral cypselae numbers increased with the light availability. And peripheral cypselae germination varied more among populations (it were reduced by after-ripening). Both germination behavior correlates to the population resources availability. The field assay results were similar to lab ones, however, in the soil treatments, the peripheral cypselae was more responsive to soil which they came from. Also the dormancy difference among types is not related to abscisic acid de novo synthesis, neither to exogenous gibberellins sensitivity. To sum, the dicarpy varied among the populations, and some features correlated to soil nutrients and/or light availability, but both types behaved in accordance to the maintainer/colonizer model.

Key-words: cypsela behavior, hormonal balance, seed production

INTRODUCTION

Somatic heteromorphism is a reproductive strategy in which a single plant produces a variety of diaspores which have different fates and behaviors so that each type tends to establish in specific sites or environment conditions (VENABLE 1985, IMBERT 2002, BASKIN et al. 2013). Diaspore heteromorphism frequently occurs in weeds or herbs subjected to spatially and temporally heterogeneous environments, such as semi-arid to desert climate and saline soils (VAN DER PIJL 1982), as well as agricultural and disturbed sites (HARPER 1977, RUIZ DE CLAVIJO 2005, CHEPTOU et al. 2008). Asteraceae comprises more heteromophic species than any other plant family, and, within this family, diaspore variation occurs mostly among one-seeded fruits (cypsela or achenes) inside the head, or capitulum. The cypsela types vary in morphology: external structures (i.e. pappus, bracts or appendages), color, and size (IMBERT 2002). Furthermore, the seed types of heterocarpic species differ in germination behaviors, i.e. degree of primary dormancy and seed sensitivity to light quality or thermal and saline gradients (VENABLE et al. 1987, RUIZ DE CLAVIJO 2001, CORKIDI et al. 1991, AMARAL-BAROLI and TAKAKI 2001, WANG et al. 2008). Heterocarpic cypselae types usually vary according to their position within the capitulum (MATILLA et al. 2005, TORICES and MÉNDEZ 2010). Such morpho-physiological variations among the cypselae types ensure a wider dispersion both in space and time (VENABLE 1985, SNYDER 2011). Heterocarpic species are known to modulate the proportion of different diaspores they produce according to environmental stimuli (WANG et al. 2012). The bet-hedging strategy reduces the risk of extinction of a lineage in uncertain environments by dispersing progeny through space (via seed dispersal) and time (via dormancy) so that at least some progeny avoid fatal environmental disturbances. Although it may decrease the arithmetic mean progeny fitness within a given year, it increases geometric mean fitness of a lineage over multiple generations (VENABLE 1985, MARTORELL and MARTÍNEZ-LÓPEZ 2014). The optimum proportion of progeny distributed at random depends on the degree of environmental variation and on the quality of the home site (VENABLE and BROWN 1988). When the home-site quality is favorable, the proportion of non-dispersing propagules is hypothesized to increase, as articulated in the "informed dispersal theory" (MARTORELL and MATINEZ-LOPEZ 2014). Studies on desert annuals have demonstrated that some species alter the proportion of dispersed progeny to approach predicted optima (VENABLE and BROWN 1988). On the other hand, adaptive maternal effects occur when the maternal environment accurately predicts the environment that progeny will experience, and phenotypes of progeny are altered so that progeny fitness is increased in this environment 69

(CREAN and Marshall 2009). For phenological traits, such as germination timing, adaptive maternal effects can increase the probability of adaptive habitat selection, so that progeny is more likely to germinate under conditions in which they can survive (DONOHUE 2013). In the case of bet-hedging, the variance in progeny germination time is expected to increase (as well as the dispersal distance and the proportion of dispersed progeny). In the case of adaptive maternal cuing, the mean germination time is expected to change in response to the environment to improve the seed response to such signals (MARSHALL and ULLER 2007, DONOHUE 2014). Variation in seed properties in response to the environment has been studied in controlled experiments (normally in greenhouses) or natural populations. Venable and collaborators (1987) tested local adaptation of Heterosperma pinnatum in field sites and Martorell and Martínez-López (2014) recently used this species to test successfully the informed dispersal theory. They showed that under various environmental backgrounds H. pinnatum produced different low/high dispersive type ratios with low genetic relation (VENABLE et al. 1998). On the other hand, in Crepis sancta, the cypselae ratio is hereditable (IMBERT 2002). Cheptou and co-authors (2008) tested its rapid evolution under local selection in urban sites. However for both species, and others, there are no germination behavior differences in cypselae types regarding maternal effect. Most Asteraceae heterocarpic species exhibit differences in germination behavior among seed types. Two contrasting seed behaviors exist in heterocarpic Asteraceae: non- dormancy (high risk), and non-deep physiological dormancy (low risk). These germination behaviors are frequently associated with dispersal mode: high dispersive (high risk), and low dispersive (low risk), respectively (BASKIN et al. 2013). These seed behaviors represent temporal dispersion and can be a local survivorship constraint (DONOHUE 2005). The degree of dormancy acts as an allowance for the seeds not ready to germinate after dispersal, which could imply the avoidance of hazards and intraspecific competition during the germination season. Furthermore, this dormancy mechanism allows them to enter and maintain in the seed bank, mainly the low dispersion type. In addition, both types can display distinct seed bank patterns (VENABLE and LEVIN 1985b, ESPINOSA-GARCIA et al. 2003). Non-deep physiological dormancy is relieved by the processes of after-ripening (passive dormancy loss as seeds age) and dormancy breakage by warm or cold imbibition. Both processes occur via the regulation of gene expression and enzyme activity and are signaled by hormonal pathways. The ability to germinate is strongly regulated by the hormonal balance between Abscisic acid (ABA) and Gibberellins (GA) (BARUA et al. 2012; SEBASTIAN et al. 2014). ABA induces and maintains dormancy, and GA induces germination by alleviating the mechanical restriction of the endosperm and initiating embryo 70 growth. The relation between the concentration (synthesis or degradation) and the sensing of each hormone determines the germination process (KUCERA et al. 2005, FINKELSTEIN et al. 2008). Natural populations have been shown to differ in germination sensitivity to these hormones (BARUA et al. 2012) The aim of this work is to verify if there is any variation in dicarpic strategy, and by which micro-environmental cues upon mother plant controls the dimorphic cypselae quantity and quality. The questions addressed here are: (1) which environmental factors experienced during seed maturation (specifically soil components and light availability) influence the production of different heterocarpic seed types ,i.e. quantity and quality?.(2) Do different heterocarpic seed types differ in germination behavior under field conditions? (3) Does maternal soil influence the types germination? (4) Which hormonal mechanisms are associated with the differences in germination behavior among dicarpic types, and how it varies among the populations? To answer these questions we used different populations of the heterocarpic Asteraceae plant, Bidens bipinnata L. This species exhibits dicarpy in morphology, since central cypselae are longer and lighter colored than the peripheral ones, and germination behavior, as peripheral cypselae have deeper dormancy than central ones. In addition, germination presents geographic variation between Indian and South African populations (DAKSHINI and AGGAWAL 1974, BROWN and MITCHELL 1984, IMBERT 2002).

MATERIALS AND METHODS

Study species

Also known as Spanish needles, Bidens bipinnata L. is an erect annual herb/forb distributed worldwide. Native to Central America, it is an invasive species in much of its range, as are other Bidens species (SÎRBU and OPREA 2008, JING et al. 2013). Plants grow from 0.4 to 2.65 m tall and frequently occur in clumps. They occur in a great variety of habitats, but are most frequently found in anthropogenically disturbed sites. Flowers are arranged in a head, or capitulum, which presents two types of florets: yellowish-whitish sterile ligulate rays and yellowish monoecious tubular discs. Cypsela resulting from the center of capitulum are linear and differ from the peripheral ones that have a tetragonal shape. These two cypselae types are easily distinguished morphologically, without intermediates, as is the case with other Bidens species (BROWN and MITCHELL 1984, SASSAKI et al. 1999). The central type is a pale-gray, glabrous fruit that is longer than the dark-brownish, peripheral cypselae (SHERFF 1937). The awned pappi on the central cypsela are longer (3.46±0.01mm) and more numerous (3-4) than those on the peripheral 71 ones (2.63±0.01mm and 2, rare 3). Both the central position and the morphological features of central cypselae favor their attachment to animal dispersal vectors (ROCHA 1996). Also, the central cypselae germinate faster and under wider conditions than the peripheral ones (DAKSHINI and AGGAWAL 1974, BROWN and MITCHELL 1984). The species is a summer annual, and in the U.S.A., its seeds germinate in early spring (April-May) and plants flower in fall (August-October).

Plant material and micro-environmental data collection

Fourteen B. bipinnata populations (maximum 106km between sites) were found near roads, parks, farms, and other disturbed places in central North Carolina, U.S.A (Table S1 Supplementary data). In each site, we collected twelve plants with ripe seeds that were at least 1m apart. The collected plants were taken to lab and the following traits were measured: height (using a ruler); stem basal diameter (using a caliper); number of leaves; number of heads. Plant dry biomass was obtained by separating its parts and putting them into paper bags, i.e. leaves, stems, and heads, to be oven dried at 60°C until mass stabilization. Then the plant materials were weighed using analytical balance (0.01g). Three unripe full-developed heads were also taken (kept fixed in 70% ethanol) to measure their diameter and determine their number of cypselae types. Fresh cypselae, collected from all population plants, were stored in paper bags under ambient laboratory conditions for later germination studies. The length and fresh mass of the collected 100 of each cypselae type were measured for each population. Cypselae length was measured on pictures taken with a digital camera connected to a stereoscope. Fresh mass was weighed per single cypselae on an analytical balance (0.001mg). Soil samples were collected from each site. The top soil compound samples (5cm deep) collected from each site were analyzed for NH4, NO3, K, Mg, Na (salinity indicator) content in soil extracts (TRAN and SIMARD 1993, ROBERTSON et al. 1999). We also estimated the light available at each site with a fish-eye lens canopy pictures. The digital camera was placed above the plants and one picture per site was used for graphical measurement. Canopy pictures were analyzed with the GLA software (FRAZER et al. 1999).

Laboratory germination assays

To compare germination rate between cypselae types and among populations, we first conducted germination assays under controlled laboratory conditions. Cypselae stored for 4 and 20 weeks were placed into 90mm Petri dishes lined with two layers of moistened filter 72 paper (deionized water). For each population, four replicate of 25 central or peripheral cypselae from each population were kept in incubator at 22ºC with 12h photoperiod (Model GR41LX, Percival Scientific Inc., Perry, IA). Dishes were scored every two days, when the germinating seeds were removed and the dishes controlled for moisture. The plates were verified for 20 weeks after which the experiment finished. Some populations presented ungerminated cypselae, which were gently squeezed with a forceps to determine their firmness (for lab essays) and further tetrazolium tests were performed to confirm their viability.

Field germination assays

To compare germination phenology between cypselae types and among populations, we monitored germination timing under field conditions. The field experiment took place at the Duke Experimental Field in Durham, North Carolina, USA (36° 0'32.39"N 79° 1'7.75"W). The field plot was tilled to remove weeds at the beginning of the experiment in late fall 2012. Two soil treatments were used: soil from the experimental planting site (Experimental Field Soil, or EFS) and soil from the site where each population was collected (Population Site Soil, or PSS). Both types were passed through a 4mm sieve. The soil of the field planting site contained no B. bipinnata seeds because no such plants had ever grown there. The original soil of each population was previously kept for 5 weeks in a greenhouse to diminish the seed bank by stimulating seed germination before use. Twenty central or peripheral cypsela of a given population (aged between 46 and 77 days) were put on the soil inside a single 8cm diameter fiber pot (approx. 30g of soil). For each one of the ten 1.5m2 blocks one pot were planted accounting for populations (14 levels) x soil types (2 levels) x cypselae types (2 levels), totaling 56 pots per block (see Figure S3). All seeds were sown on December 10th, 2012, and each pot was scored until May 24th, 2013. Scores were conducted weekly during the first spring, and biweekly as germination rates declined over the summer. During each score, the new germinating cypselae of each pot were counted and then removed. Also the soil temperature, air temperature at 80cm above surface level, and soil moisture were recorded every hour during the experiment.

Hormonal assays

To investigate physiological mechanisms that account for differences in germination between cypselae types, we studied germination responses to the two major hormonal regulators of dormancy and germination: ABA and GA. We used cypselae from ten populations (see Table S1 Supplementary data) that still had peripheral cypselae after the 73 other experiments. These cypsela were stored for 150 to 190 days, due to different collection dates. Each population had three replicate Petri dishes with 20 seeds for each of the four hormonal treatments. Petri dishes (45 mm) were filled with 0.6% agar and a specific hormone solution was added to create the following treatments: (1) Control: 0.6% agar only; (2) 10µM Fluridone (Fluka, Sigma-Aldrich Co., US), an inhibitor of de novo ABA synthesis

(TOH et al. 2008). (3) 50µM GA4+7 (Sigma-Aldrich Co., US) plus 100µM Paclobutrazol; (4)

500µM GA3 (Sigma-Aldrich Co., US). Also there was a Negative control: 100µM Paclobutrazol (Sigma-Aldrich Co., US), an inhibitor of GA biosynthesis, but all plates had zero germination in this treatment and they were excluded from data analysis. To make the stock solutions, GAs were dissolved in ethanol, Fluridone in ethanol plus Tween 5, and Paclobutrazol in acetone. All the stock solutions were at least one thousand times more concentrated than the assay solution to reduce the solvent effect. Solutions were replaced in the plates weekly to ensure their constant concentration and the same stock solutions (kept at -20°C) were used throughout the assay. All plates with cypselae were kept at 22ºC and a 12h photoperiod in Percival growth chambers (Model GR41LX, Percival Scientific Inc., Perry, IA) and were scored every day for 30 days. In some treatments, there were non- germinated seeds and the viable ones were kept for further analysis.

Statistical analysis

To test for significant differences among the populations for plant and seed traits, we conducted one-way Analysis of Variance (ANOVA). Also, for associations between population environmental factors and plant and seed traits, we first conducted a Spearman correlation analysis for all the variables to remove those that did not show co-linearity (data not shown). We also performed a Path Analysis by Partial Least Squares Path Modeling using the correlations between the factors to show these interactions graphically (‘plspm’ package [SANCHES et al. 2013, SANCHES 2013]). We then tested for specific associations using multiple regression modeling with the Generalized Linear Mixed Models (GLMM) (‘lme4’ package [BATES et al. 2014] and ‘MASS’ package [VENABLES and RIPLEY 2002]) with appropriate link functions. All analyses were performed with R (R Core Team 2013). To GLMM modeling, all environmental factors were used as continuous explanatory variables (mineral concentrations and light data for each population). The populations were used as a random effect variable. The plant growth traits as response variables were: (1) dry biomass, (2) number of leaves, and (3) plant dry biomass/stem volume (height x root collar diameter x π) - "stem density". The reproductive output response variables were: (4) number of floral heads per plant; (5) head diameter, (6) number of central and peripheral 74 cypsela, and (7) R-ratio, or the proportion of low-dispersive (peripheral) diaspore over the total number of diaspores (CHEPTOU et al. 2008) Cypsela traits included: (8) cypselae fresh mass and (9) cypselae length. All of them were explained by the vegetative growth (dry biomass) or the environmental factors (mentioned above). Then, homogeneity and residual normality were tested graphically for confidence proof. T-tests were used to check significant effects of environmental variables on the response variables. Model building followed the Zurr et al. (2009) recommendations. To analyze germination rate, we used the Time-to-Event analysis, also known as Survival analysis (‘survival’ package [THERNEAU and GRAMBSCH 2000]). This method allows to analyze the time course of events as germination and to quantify the effects of factors on that time course (proposed by ONOFRI et al. 2010). Germination data from the lab assay were considered “exact data”, while the data from the field experiment were “interval census data”, since the experiment was scored weekly. Field data were right- censored data, although we did not search for non-germinated cypsela. The probability of germination was estimated by the non-parametric Kaplan-Meyer estimator prior to modeling. Next, parametric Accelerated Failure Time (AFT) modeling was run, utilizing the most appropriate distribution based on Akaike´s Information Criterion. Also we checked the graphical similarity between the chosen model and the Kaplan–Meier estimator curves. The AFT assumes a linear relationship between the logarithm of time to germination and the analyzed factors. These factors accelerate or decelerate the germination curves according to a coefficient (b). (Figure S1 and S2 Supplementary data). In the AFT models, we used the following as fixed explanatory variables: (1) seed type (two levels), (2) treatments (4 or 20 weeks stored for lab essays; EFS or PSS for field experiment), (3) populations, and (4) all interactions. The replicates were used as a clustering random effect and included in the model using the frailty function of the survival package with a Gamma distribution (ONOFRI et al. 2010, DÉLYE et al. 2013). Then the F-statistics were used to verify the significance of each factor and its interactions (Table 2). Additionally, the effects of the environmental factors at the seed-collection sites were tested with Z-tests, using the soil mineral content and light availability as fixed effects and the populations as random effects. GLMM was used to analyze germination responses to hormonal treatments. Germination (germinated vs. not germinated) was the dependent variable analyzed with a binomial distribution linked by logit. The hormonal treatments (PAC+GA3, PAC+GA4+7, Fluridone, and Control) and seed type were fixed factors while population and replicate were random factors. We also checked whether seed types and populations responded to hormones differently by testing for seed type x hormone x population (fixed) x interactions.

RESULTS AND DISCUSSION

The synopses the correlations among variables thought as an interaction net (see Figure 1). The morphological dimorphism, that is, the difference in size and number of cypselae types per head, is upregulated by the soil and light environment as well as by the vegetative growth of the plants. Behavioral dimorphism, i.e. the difference of the physiological responses between cypselae types, is positively related to light /availability and negatively related to plant size, reproductive output, and soil nutrient contents. To understand further these relations, we used a multiple regression modeling.

Figure 1: Path Analysis diagram of the interaction of environmental factors on dimorphic traits. The numeric values= Correlation coefficients between latent variables. Asterisks represent significant correlation: * p< 0.10, ** p< 0.05, *** p<0.001.

The vegetative growth traits varied among populations (dry biomass: F13,154=10.89, p<0.001; leaf number: F13,154=4.65, p<0.001; stem density: F13,154=5.62, p<0.001). Plant dry biomass is positively related to NO3 and Na, but negatively related to Mg soil ion concentrations (Table 1). Its relation to Na is not considered since salt content in soil is not a limiting factor for these populations. However, the relation to Mg ions is still unknown. The number of leaves was also higher in locations with high soil fertility. Light availability was correlated to plant growth traits, however it correlation is very low. Closed canopy sites probably had previous selective effect to the prior life stages by limiting seedling 76 establishment (via light requirement), thus it filters the plant for suitable sites. In most Bidens species, B. bipinnata included, cypsela, mainly the peripheral type, are highly sensitive to light environment (quality and quantity) (BROWN and MITCHELL 1984; AMARAL-BAROLI and TAKAKI 2001). Plant resource accumulation, measured as aerial part dry biomass, was related to reproductive output (Figure 1), e.g. increase in the number of floral heads per plant (β=0.89±0.02, z= 53.13, p<0.001, corrected Poisson distribution). Larger plant size was also associated with a greater number of cypsela per head (β=0.0011±0.0003, z= 3.14, p=0.002, corrected Poisson distribution), and this effect was caused primarily by its influence on the number of central cypsela (β=0.0011±0.0004, z=3.03583, p= 0.003, corrected Poisson distribution) rather than on peripheral ones (β=0.0003±0.0010, z=0.23, p=0.77, corrected Poisson distribution). Head diameter had a weak but significant positive association with plant size (β=0.026±0.008, t=1.87, p=0.06) and it was strongly correlated to the light environment of the population site (p<0.001, Table 1). Thus, in open sites head diameter was larger than in closed canopies, but this is not related to the increase of cypsela number.

Both amounts of cypselae types varied among populations (central: F13,154=12.67, p<0.001, 3 to 25 cypsela; peripheral: F13,154=8.00, p<0.001, 0 to 6 cypsela, Figure 2A). The number of central type increased according to plant size, but the number of both cypselae types is not driven by any environmental factor measured (Table 1). Despite its significant among-population variation (F13,154= 6.80, p<0.001), the R-ratio, the proportion of peripheral cypsela, did not vary according to plant size (β=0.001±0.003, t=0.51, p=0.61) or due to the measured environmental factors (data not shown). There is among-population variation in the cypselae type ratio of B. bipinnata, but which factors modulate it is still unknown. It is probably related to water availability during plant development or to another stressful unmeasured environment factor of the site (IMBERT et al. 1997, MARTORELL and MARTÍNEZ-LÓPEZ 2013). The number of peripheral cypsela per head varied more than that of central ones (CV=0.16 and 0.38, respectively). In Crepis sancta, the number of peripheral and intermediate cypsela varied according to nutrient availability treatments while that of central ones was constant (IMBERT and RONCE 2001). This contrasts with other works reporting that the number of central cypsela varied more than that of peripheral ones (BAKER and O´DOWD 1982, KINGEL 1992, MÖLKEN et al. 2005). In B. bipinnata, this may be explained by floral features: ray florets (ligulates) are sterile and disc florets, which are monoecious, produce heterocarpic fruits. In Callendulla micrantha and C. arvensis, only the disc florets (ligulates) are monoecious, while the (tubular) disc florets are androecious and both species have polymorphic cypsela (GARDOCKI et al. 2000, RUIZ DE CLAVIJO 2005, respectively). Nevertheless, to better understand how types differentiate, further studies on 77 flower and fruit ontogeny are needed. The control of cypselae production allows a "facultative" heteromorphism, in which one type, i.e. the peripheral one, occurs or not. One reported example is B. pilosa, which is known to be dicarpic, although some populations do not present the peripheral type (ROCHA 1996 and personal observation). Actually, some B. bipinnata heads did not have peripheral types. We suggest that, under stronger stressful conditions, fruit production aims local escape, thus it does not produce low dispersive types.

Cypselae size: fresh mass (F13,2772=62.19, p<0.001) and length (F13,2772= 19.39, p<0.001) varied significantly among populations (Figure 2B). The peripheral cypsela were shorter (β=-0.44±0.005, t=-86.32, p<0.001) and lighter (β=-0.78±0.03, t=-27.83, p<0.001) compared to central ones. In B. pilosa, the longer central type is more exposed inside the head than the peripheral one and it has more chances to attach to dispersal agents (ROCHA 1996), and a similar strategy is expected for B. bipinnata. Environmental factors strongly affected cypselae features and both soil fertility and light availability were associated with greater fruit mass and length (Table 1). Thus, the size of both cypselae types increases as nutrient and light availability of the site increase. As central cypsela get more numerous and longer, the plant (and the population) increases its spread distance. It is expected that the ratio of cypselae types varies among the population of most heterocarpic species in a non-hereditable manner (MATILLA et al. 2005). Thus, this variation depends on: (1) species plasticity, which is genetically limited; and (2) the surrounding environmental cues on fruit production. For example, the cypselae ratio of Hedypnois rhagadioloides varies among an aridity population gradient and the number of high-dispersive cypselae decreases in drier sites (KINGEL 1992). The variation in number of cypsela of other species follows a similar pattern: Crepis sancta according to nutrient availability, competition (IMBERT et al. 1997), and stress conditions (IMBERT and RONCE 2001). Heterosperma pinnatum among different population sites (VENABLE et al, 1987) and light and water availability treatments (MARTORELL and MARTÍNEZ-LÓPEZ, 2013). Hypochoeris glabra according to plant competition (BAKER and O´DOWD, 1982); Tragopogon pratensis varied among populations (MÖLKEN et al. 2005). Thus, under stressful or low resource conditions, plants may modulate to increase the relative amount of their high-dispersal type seeking local escape. Also, for the above cases, the spatial dispersive type is low cost to the plant. On the other hand, the cost of a single low spatial dispersed cypselae is usually high, because of some extra structure or composition, i.e. fruit protection (MAXWELL et al. 1994), germination constraints (VENABLE and LEVIN 1985a, AGUADO et al. 2011) or stocked reserves (RAI and TRIPATHI 1987). However, the opposite pattern occurs for Packera tomentosa whose heavier type is central due to its bigger embryo (LEVERETT and JOLLS 2013). In Bidens, the central cyspelae are heavier than the 78 peripheral ones, probably due to fruit length investment. Its fruit morphology, i.e. cypselae mass and length, also varies significantly among populations and it correlates to the measured environmental traits (Table 1 and Figure 2C). Thus, when resources are abundantly available, fruit size increases, which may favor longer dispersal distances. Some heteromorphic species have a gradient or continuous variation inside the capitulum following ontogenetic pathways, which translates into diverse seed types (IMBERT, 2002). Brown and Mitchell (1984) analyzed a cypselae size gradient within B. bipinnata head, but they concluded it was a physiologic dimorphism. Thus, in the present work, the intermediate morph (rare occurrence) was not accounted for. However, other species present an among-population variation in fruit morphology, i.e. the presence of intermediates or undeveloped types. For example, H. pinnatum presented variation in both: (1) type number, i.e. central, intermediate (undefined), and peripheral, and (2) morphology, when subjected to wide environmental background ranges (VENABLE et al 1987). In H. pinnatum, the spatial and environmental background correlates to cypselae morphological features, but it is poorly related to genetic distance (VENABLE et al. 1998). Its dispersive traits are explained by water availability and light stress over the plants and it agrees with the informed dispersal theory (MARTORELL and MARTÍNEZ-LÓPEZ 2013). We observed that some soil chemical composition and light availability are cues for maternal effects in B. bipinnata. Soil fertility affected both the vegetative growth and reproductive output features, but the latter were more significantly affected by the measured abiotic factors. In Lappula duplicarpa (Boraginaceae), the vegetative development was more affected by fertilization treatments than the reproductive biomass (LU et al. 2013). The salinity of the site soil (Na content) varied little (C.V. = 0.34), but it was significant for some B. bipinnata plant traits, although it did not affect seed germination. It also influenced Suaeda aralocaspica (Amaranthaceae) seed size, but the seed ratio was not significantly different. In addition, seed behavior was maternally affected in highly saline soil treatments, since seeds germinated more than in other treatments. Furthermore, the high saline soil seeds were not affected by salt concentration in the germination essay (WANG et al. 2008).

Figure 2: Dimorphic seed traits among the collected populations, and their germination in lab conditions. A - Boxplot of the number of each type of cypsela per floral head (N=36 heads/population). B – Boxplot of the length of each cypselae type (N=100 cypsela of each type/population). C - Median estimated time at which 50% of fresh seeds germinated in controlled lab conditions, as modeled with an AFT model with no random effect. Error bars= standard error.

Lab germination assays

As expected, the peripheral type germinated more slowly, as estimated by T50 of AFT models, than the central one under lab conditions (Table 2 and Figure S1). The degree of dormancy varied among populations (Figure 2C), as did the proportion of peripheral seeds that germinated by the end of the experiment, which ranged between 87.76±3.11% (population 14) and 3.04±1.92% (population 2). By contrast, final germination proportions of central cypsela were high in all populations.

Higher NH4 in maternal soil was associated with slower peripheral germination, while higher light availability was associated with it faster germination (Table 1). On the other hand, the germination rate of central seeds was not associated with any environmental factor. This may be related to the longer dormancy of the low-dispersive type that favors the insurance for the next generation. However, as the light available increases, primary dormancy decreases and this maternal effect is related to a facilitation for a ready establishment in open sites. Bidens species are strongly influenced by the quality and 80 quantity of light (BROWN and MITCHELL 1984; AMARAL-BAROLI and TAKAKI 2001). Thus, no B. bipinnata populations could be found under extremely closed canopy, i.e. within forests. We found populations bordering forests or in open places. However, we could observe that canopy openness or plant distance from shade affected the head diameter and the cypselae germination behavior. The light environment where the plant grows can affect the cypselae type ratio in heteromorphic species, e.g. Synedrella nodiflora shade tolerant heterocarpic species (SOUZA-FILHO and TAKAKI, 2011). It can also influence the offspring germination behavior, e.g. Plantago lanceolata (Plantaginaceae) seeds were more dormant when the mother plant grew under shade (VAN HINSBERG 1998). After-ripening strongly affected germination rates and proportions (Table 2). The physiological dormancy was alleviated after room dry storage in both seed types (F1,144= 4143.33, p<0,001 and Figure S1). For example, the germinability of population 3 increased from 12.00±1.63% to 63.00±4.43%. The rate of dormancy alleviation differed between seed types (Figure 3). The estimated T50 of peripheral seeds decayed faster than that of central ones (central: b=-44.52, peripheral: b=-181.02, linear model). The among-population after- ripening rates also varied (F11,144=829.70, p< 0.001). Despite the few treatments, dry storage under dark at room temperature (approx. 20ºC) was effective to alleviate seed dormancy and these processes varied among types and populations. The peripheral seeds presented a higher increase in germinability than the central ones. Garhadiolus papposus produced three cypselae types and it displayed different dormancy alleviation rates during storage. The central and intermediate ones, which were less deeply dormant, lost their dormancy faster than the peripheral ones, which after 3 month storage showed no germination (SUN et al. 2009). The after-ripening process is related to the GA/ABA hormonal amount and it sensitivity depends on seed maturation conduction (IGLESIAS-FERNÁNDEZ et al. 2011, BARUA et al. 2012). B. bipinnata peripheral cypsela had lower germination rates than the central ones. However, there is a difference between seed type germinability whose rates varied among populations. Some present low differences between types, while others, due to the lower germination of their peripheral type, show a higher germination difference (Figure S2). This may explain the germination variation reported by Dakshini and Aggawal (1974), and Brown and Mitchell (1984). We observed that both central and peripheral seed germination varied among populations, but peripheral germination varied more and it was related to the measured environmental traits. These signs, perceived by previous stages or generations, cause changes in the way peripheral seeds will behave and it agrees with the developmental niche construction. 81

Germination behavior is an important plant life trait that defines local survival, mainly for short-lived species (annual and biannual herbs). Their establishment mechanism thus evolved to be highly synchronized to environment cues to favors the next generations. Therefore, they need to adapt quickly to environmental changes (DONOHUE, 2005). For that, they need to: (1) have a fine tune mechanism to “understand” the surrounding cues, (2) anticipate environmental changes, and (3) prepare their successors for them. Hence, under local constraints, each population will present different seed behaviors. The variation of seed behavior is partially controlled maternally during the late stages of seed maturation (BARUA et al. 2012). In heterocarpic species, the nature of variation among seed germination and dormancy types has been little documented (DUBOIS and CHEPTOU 2012). This analysis is important to verify the population dynamics in order to understand temporal dispersion spread, i.e. the difference in germination delay among types. Our data also show how important it is to work with more than one population to avoid biases on heteromorphic seeds behavior. As expected, there is a natural variation of dimorphic B. bipinnata features among types and among populations. The among-type morpho-physiological variation was expected because it had been previously observed (DAKSHINI and AGGAWAL 1974, BROWN and MITCHELL 1984). Most heterocarpic Bidens species are also dicarpic and morpho-physiological features differ between the two diaspores, but there is, at least, one known species with a continuous variation inside the capitulum (B. gardneri [SASSAKI et al. 1999]). B. bipinnata central cypselae presents high dispersion and low dormancy. On the other hand, the peripheral one shows the opposite pattern. Thus, the species follows the H/L-L/H strategy pattern (BASKIN et al. 2013), which is quite uncommon among Asteraceae (IMBERT 2002; MATTILA et al. 2005). Other Bidens dimorphic species present the same pattern as well (CORKIDI et al. 1991, ROCHA 1996, AMARAL-BAROLI and TAKAKI 2001), with one known exception: B. frondosa, whose central type has a slower germination, H/H- L/L (BRÄNDEL 2004a). We observed that there is some among-population variation in the dicarpic strategy. 82

Figure 3. After-ripening rates for the different cypselae types, central left (black dots) and peripheral (grey dots) of the different populations (Except 1 and 2 populations).

Field germination assay

Seed germination started in the beginning of spring, after winter chilling and when temperatures were increasing (Figure 4), leading to a summer annual life-history. The two cypselae types differed through their germination timing under field conditions. The average emergence rate of the peripheral seeds (estimated T50: 152.04 ± 2.15d) was slower than that of the central ones (estimated T50: 174.26 ± 2.77d; Table 2, Figure 4). Soil treatments affected the germination behaviors of peripheral seeds (b=0.028±0.007, χ2= 14.2, DF=1, p<0.001) but not that of the central ones (b=-0.03±0.02, χ2= 2.13, DF=11.00, p=0.14) cypsela (Table 2). Peripheral seeds germinated faster in their native PSS soil than in the EFS soil. The AFT analysis considers that all seeds will germinate in this season, however they can be dormant and only germinate the next year, or die. The results indicate the peripheral type is more responsive to the maternal site than outside soil. Thus, it undergoes developmental niche construction effects more than the central one. Since the central type is considered to colonize new sites and it is a high risk diaspore, its fate is not (or little) maternally affected. The peripheral type is phenotypically modified by the mother plant to behave (germinate) according to environmental signals. The peripheral type was also responsive to the soil surrounding it. It has a higher germination proportion on the soil of the maternal site (PSS) than in outside soil treatment (EFS). These responses reaffirm the peripheral strategy: maintaining local population. On the other hand, the central cypselae behavior was little affected by the measured abiotic factors and germinated similarly independently of soil treatments. As a colonizer, it is expected to germinate whatever condition it faces. Dubois 83 and Cheptou (2012) also observed variations in among-population seed behavior regarding plant competition. However, the interaction between type and population was not significant, i.e. the between-type germination difference was constant among populations. Kingel (1992) did not observe variation in the among-population germination pattern, even though their production varies. The difference of fresh seed behaviors in lab assays decreased in field experiment with after-ripening and cold stratification processes, but a difference of about 20 days in germination timing still remained according to the T50 of AFT models. This estimation gives an idea of the real natural variation of the among-type germination phenology. The primary dormancy induction may occurs during seed maturation for both seed types, but the peripheral type undergoes to a higher induction and is dispersed into deeper dormancy than the central one. Probably, in next early spring, when dormancy alleviated via after-ripening and chilling (and other environmental stimuli, e.g. light availability), the low dispersed central type may germinate quickly and can inhibit the peripheral germination (through shade effects). When they are not able to germinate, mainly the peripheral ones, they can enter in a secondary dormancy and then in a seed bank. All these requirements can turn small germination rate differences among-types into greater delay, which can be amplified under natural conditions.

Figure 4. Field experiment AFT estimated curves for the central (solid lines) and peripheral (dashed lines) for the EFS (bold black lines) and PSS (bold grey lines) soil treatments. Dotted lines indicate standard errors of the estimated function. Mean daily temperature (°C) at each time point is in light grey. 84

Hormonal assays

To test whether the two basic hormonal pathways of ABA-regulated dormancy and GA- mediated germination vary among natural populations and contribute to the observed differences in the germination of the two types of B. bipinnata heterocarpic seeds, we manipulated the exogenous GA and ABA concentrations and measured germination. Paclobutrazol inhibited the germination of all the seeds (results not shown). Addition of GAs to Paclobutrazol increased seed germination, but the two GA treatments did not differ (GA3 vs. GA4+7: β= 0.01±0.15, z=0.07, p= 0.99). The central cypsela were more sensitive to exogenous GAs than the peripheral ones (GA3: β=-3.40±0.43, z=-7.871, p<0,001; GA4+7: β=-3.39±0.43, z=-7.849, p<0,001; Figure 5). In addition, populations varied in their germination response to both GA treatments (p<0.001). They also varied in the degree to which their seed types differed in response to GA treatments (population x seed type: p=0.04, subset model with just the GAs). Fluridone, an inhibitor of de novo ABA synthesis, stimulated the germination of the central seed type (β=0.54±0.15, z=3.58, p<0.001, Figure 5), but it had no significant effect on that of peripheral seeds (β=0.05±0.14, z=0.36, p=0.92, Figure 5). Thus, ABA contributes to maintain the dormancy of central cypsela. In contrast, dormancy of the peripheral seeds appears to be maintained independently of de novo ABA synthesis. The among-type germination behavior differs in most heteromorphic species, resulting in a wide temporal spread (IMBERT 2002, MATILLA et al. 2005). In most cases, the deepest dormant type is the low dispersed one (IMBERT 2002, BASKIN et al. 2013). The degree of dormancy is somewhat imposed by the fruit: mechanical (VENABLE and LEVIN 1985a) or chemical (BENEKE et al. 1993) germination constraints may be present. Thick or lignified pericarps, which are found in low dispersed cypsela, act as a barrier to embryo growth (AGUADO et al. 2011). Thus, the root will protrude: both embryo strengthening and barrier weakening can be hormonally induced (KUCERA et al. 2005, FINKELSTEIN et al. 2008). Despite the hormonal stimulus, the seed type germination rates kept different, although the treatments alleviated dormancy of both seed types. On the other hand, the central type was more sensitive to GA stimulus than the peripheral one, due to its response to exogenous GA and to the inhibition of endogenous synthesis by Paclobutrazol. Previous works showed exogenous GA positive effect on B. bipinnata peripheral germination (BROWN & MITCHELL, 1984). However, its action in these processes has not yet been specified. We showed that a constant concentration of GA is necessary to the germination progress, not just as a dormancy breaking stimulus, but the central type requires lower concentrations to germinate. This can be a clue to answer why the central type is more sensitive to some 85 environmental stimuli (BROWN & MITCHELL, 1984, AMARAL-BAROLI and TAKAKI, 2001). In Suaeda salsa (Chenopodiaceae), the gibberellin treatments affected differently the heteromorphic seeds, since the deep dormancy type, the black ones, were more sensitive to the GA treatment than the brown ones (LI et al. 2005).

REFERENCES For the reference, please check it at page 95.

Figure 5. Final germination proportions under different hormonal treatments. All the treatments showed a significant difference between cypselae types. The letters represent significant differences between seed types and among treatments.

Table 2. Results of the AFT modeling for the Lab assay and Field experiment. The fixed response variables are: type= central and peripheral cypselae types; treatment= Lab essay (fresh [4 weeks] and stored [20 weeks] seeds); and the populations= see Table S1. The random intercept response variable were the replicates added to the model by the frailty function Gamma distributed. The Lab assay model was log logistic distributed and the Field were Weibull distributed. Lab essay Field χ2 d.f. p χ2 d.f. p Type 752.84 1 <0.001 1364.82 1 <0.001 Treatment 2162.69 1 <0.001 2.31 1 0.12 Population 1265.25 11 <0.001 365.40 12 <0.001 Type:Treat. 16.66 1.03 <0.001 13.64 1 0.002 Type:Pop. 257.45 11.1 <0.001 219.32 12 <0.001 Treat:Pop. 195.86 8.93 <0.001 98.28 12 <0.001 Type:Treat:Pop 287.67 13.16 <0.001 29.96 12 0.0008 Random effect 7.21 2.00 0.027 0.0005 0.00009 <0.0001

Table 1. Summary for environmental effects on the vegetative, reproductive outputs and germination of B. bipinnata models. Soil traits Light availability NO3 NH4 K Na Mg Vegetative β(SE) t p β(SE) t p β(SE) t p β(SE) t p β(SE) t p β(SE) t p traits Plant dry 0.51 3.39 <0.001 0.18 1.40 0.16 -0.98 - 0.02 2.17 3.04 0.002 0.18 0.42 0.68 0.02 2.20 0.03 massa (0.15) (0.13) (0.43) 2.30 (0.71) (0.43) (0.01) 0.22 3.47 <0.001 -0.001 -0.01 0.99 -0.14 - 0.42 1.32 4.47 <0.001 -0.27 - 0.13 0.004 1.05 0.29 Leaf numbera (0.06) (0.055) (0.18) 0.81 (0.29) (0.18) 1.51 (0.004) 0.11 2.75 0.007 0.08 2.35 0.02 -0.25 - 0.03 0.76 3.93 <0.001 0.02 0.19 0.85 0.008 3.27 0.001 Stem densitya (0.04) (0.04) (0.12) 2.18 (0.19) (0.12) (0.002) Reprod. β(SE) t p β(SE) t p β(SE) t p β(SE) t p β(SE) t p β(SE) t p outputs 0.07 3.39 0.01 0.03 1.82 0.12 -0.16 - 0.04 0.31 3.13 0.02 0.05 0.78 0.46 0.003 2.35 0.06 Head numbera (0.02) (0.02) (0.06) 2.65 (0.09) (0.06) (0.001) Head -0.06 -0.59 0.58 -0.05 -0.57 0.59 -0.19 - 0.50 0.42 0.92 0.39 0.19 0.71 0.50 0.014 2.47 0.05 diametera (0.09) (0.08) (0.27) 0.71 (0.46) (0.27) (0.006) Total cypsela 0.05 1.55 0.17 -0.05 -1.68 0.14 -0.13 - 0.21 0.15 1.00 0.35 0.06 0.64 0.54 0.003 1.68 0.14 numberc (0.03) (0.03) (0.09) 1.40 (0.15) (0.09) (0.002) Central 0.06 1.64 0.15 -0.05 -1.73 0.13 -0.12 - 0.24 0.14 0.89 0.40 0.06 0.59 0.59 0.003 1.29 0.24 numberc (0.03) (0.03) (0.09) 1.31 (0.16) (0.09) (0.002) Peripheral 0.01 0.16 0.88 -0.01 -0.20 0.84 -0.16 - 0.42 0.24 0.77 0.47 0.08 0.44 0.68 0.008 2.01 0.09 numberc (0.07) (0.06) (0.19) 0.85 (0.31) (0.19) (0.004) Cypsela 0.15 2.15 0.04 0.21 3.54 0.002 0.29 1.53 0.14 0.67 2.07 0.05 -0.40 - 0.05 0.008 1.88 0.08 massad (0.07) (0.06) (0.19) (0.32) (0.19) 2.08 (0.004) Cypsela 0.001 0.21 0.84 0.015 3.29 0.001 0.03 2.20 0.03 0.03 1.32 0.19 -0.006 - 0.67 0.0005 1.75 0.08 lengthbd (0.005) (0.004) (0.01) (0.02) (0.015) 0.42 (0.0003) Germination b(SE) z p b(SE) z p b(SE) z p b(SE) z p b(SE) z p b(SE) z p 0.05 2.66 0.007 0.11 4.94 <0.001 -0.15 - <0.001 0.86 7.18 <0.001 -0.18 - <0.001 0.008 8.29 <0.001 Centrale (0.02) (0.02) (0.02) 6.01 (0.12) (0.04) 5.02 (0.001) 0.06 2.93 0.003 0.09 4.41 <0.001 -0.14 - <0.001 0.92 8.01 <0.001 -0.20 - <0.001 0.008 8.89 <0.001 Peripherale (0.02) (0.02) (0.02) 6.13 (0.12) (0.03) 5.57 (0.001) a Gaussian distribution. b Gamma distribution with log link. c Poisson distribution corrected for overexpression with log link. d Added type as intercept random variable to the model. e Accelerated failure time model analysis using log-normal distribution.

SUPPLEMENTARY MATERIAL

Table S1: B. bipinnata population places and collection dates. Elev Seed Plant Pop Place Coord. Features (m) collection Collection 35º 50’14” N 1 Manns Chapel Rd / Pittsboro 162 09/31/2012 31/09/2012 Shaded roadside. 79º 08’ 9.8” W 35º 24’ 38” N 134.1 2 Willett Rd / Sanford 09/31/2012 31/09/2012 Between crop and roadside. 79º 12’ 57” W 1 35º 24’ 5.5” N 129.2 3 Joe Matthews Rd. / Sanford b 09/31/2012 31/09/2012 Between crop and roadside. 79º 12’ 10” W 3 Washington Golf Club / 35º 59’ 20” N Golf field surrounded by Duke 4 90.83 10/02/2012 10/02/2012 Durham b 78º 57’ 24” W Forest Erwin Rd cross with W 35º 59’ 40” N 101.4 5 10/03/2012 10/03/2012 Shaded roadside. Cornwallis / Durham 78º 58’ 30” W 98 36º 00’ 11” N 104.2 6 Phytotron / Durham 10/05/2012 10/05/2012 Near building. 78º 56’ 40” W 4 Bolinwood Dr. cross Hllsboro 35º 55’ 24” N 7 92.04 09/25/2012 10/07/2012 Near urban park. St. / Chapel Hill b 79º 03’ 5.1” W S columbia St. far from 35º 53’ 54” N 128.6 8 09/25/2012 10/07/2012 Shaded roadside. Pittsboro Rd / Chapel Hill b 79º 03’ 27” W 26 Orange Grove Rd. / Chapel 35º 59’ 29” N 186.5 Between pasture and 9 10/07/2012 10/07/2012 Hill b 79º 11’ 6.6” W 3 roadside. 35º 35’ 44” N 10 Brack Penny Rd / Raleigh b 101.8 10/20/2012 20/10/2012 Shaded roadside. 78º 36’ 29” W 35º 35’ 13” N 11 Old Fair Ground Rd / Angier b 78.63 10/20/2012 20/10/2012 Shaded roadside. 78º 35’ 58” W 35º 33’ 45” N 12 Boswell Road / Kenly b 42.36 10/20/2012 20/10/2012 Shaded roadside. 78º 02’ 55” W 13 Antioch Church Rd. / Erwin ab - - 10/20/2012 - Shaded roadside. 35º 58’ 36” N 14 Forest Hill Park / Durham b 90.22 10/26/2012 26/10/2012 Inside urban park. 78º 54’ 47” W a Only the seeds were collected. b Seeds were used in the hormone assay.

Figure S1: AFT modeling graphical proof of the fitted model to the Kaplan-Meyer estimator for the field germination experiment, pooled across all populations.

Figure S2: AFT modeling graphical estimation for the lab assays.

Figure S3: graphical representation of the Block 1 in the field experiment, note that there is another 9 block within randomly distributed by soil treatments (P=PSS and S=EFS) and population (numbers).

CONSIDERAÇÕES FINAIS

Como foi possível perceber, a heterocarpia em Bidens não está associada diretamente a restrição mecânica imposta pelo fruto. Essa conclusão se deve ao fato de que os pericarpos das espécies heteromórficas não apresentaram estruturas, visíveis ao microscópio, que sugerissem tal variação. Em outras espécies heterocárpicas é bem clara a variação, o que não foi visto nas espécies de Bidens estudadas. De modo geral os pericarpos das espécies seguem padrões estruturais semelhantes, com algumas variações no número de camadas, como era de se esperar. A maior massa em relação ao tamanho do pericarpo das cipselas periféricas maduras nos levou a crer que as estruturas internas seriam diferentes, mas isso não foi evidenciado. Desse modo atribuímos essa maior massa a permanência das camadas externas nos frutos periféricos. Porém quando removidas, as sementes do fruto de B. pilosa ambos os tipos tiveram incremento na germinação, apesar de permanecer a variação entre os tipos. Assim podemos inferir que o fruto tem influência, mesmo que indireta na dicarpia. Mesmo para as cipselas centrais, com menor atraso na germinação, o pericarpo inibe sua germinação. Atribui-se essa inibição a restrição mecânica que deve ser superada pelo embrião afim de prosseguir o processo de germinação. Se as restrições foram semelhantes em ambos tipos, o que deve variar desse modo é o potencial o qual o embrião vai superar a restrição do pericarpo. A resistência do pericarpo deve ser melhor analisada e se possível testado para confirmação da hipótese. A variação na composição química entre os frutos já era esperada devido a referências bibliográficas anteriores. Contudo a ação dos mesmos nas sementes ainda não está clara. É esperado que as sementes de Bidens se comportassem como as de Arabidopsis, com a síntese de novo de ABA, contudo a hipótese foi descartada devido a ineficácia da Fluridona tanto para B. pilosa como para B. bipinnata. Outra influência que possivelmente esses compostos tem sobre as sementes é a de eliminar as espécies reativas de oxigênio (Reactive Oxygen Species). Esses compostos, que ao longo dos anos tiveram uma importância marginal nos modelos dos mecanismos de germinação, vem adquirindo certa importância como ativadores do processo de germinação, ou aliviadores da dormência para muitas espécies vegetais. Além de atuar diretamente na degradação de envoltórios do embrião, sinalizam ou estimulam genes responsáveis pelo processo. Assim, a eliminação dos mesmos reduz a germinação das sementes, via adição de compostos doadores de radicais livres, o que corrobora com nossa hipótese. Contudo isso deve ser melhor testado para comprovação, principalmente a nível molecular. Utilizando-se B. pilosa como modelo, verificamos que o potencial de crescimento do embrião é minimizado pelo inibidor o que inviabiliza a germinação do tipo periférico. Esse 92 potencial quando “igualado”, sob o tratamento de inibição do GA endógeno e aplicação do

GA3, mostraram que ambos tipos de sementes tiveram igual capacidade de germinação. Além do mais, ambos tipos são igualmente sensíveis ao ABA e insensíveis à Fluridona, desse modo podemos concluir que a aplicação ou a nova síntese de ABA não está diretamente relacionada com a variação na germinação. Contudo não foi verificado os níveis do ABA nas cipselas maduras o que poderia explicar os diferentes graus de dormência. Algo que certamente não era esperado é o grau de variação na germinação das cipselas heterocárpicas de B. bipinnata. Poucos trabalhos na literatura fizeram um apanhado das respostas germinativas de tantas populações, e menos ainda verificaram tamanho grau de variação, principalmente, para populações tão próximas geograficamente e ainda com a comprovação da variação da germinação em campo. Tentamos atribuir à variação fisiológica a um fator ambiental específico, mensurando algumas características do ambiente em que a planta mãe cresceu, contudo não foi encontrado um único fator responsável pela variação na germinação. Ao contrário, encontramos influencias específicas dos fatores a determinados atributos vegetais. Por exemplo, a disponibilidade de luz do ambiente da planta mãe influencia a germinação do tipo periférico mas não do central. Outra característica que foi pouco trabalhada anteriormente para espécies heteromórficas é a comprovação empírica da preferência germinativa diferenciada. O tipo periférico tem uma preferência a germinar em solo materno do que em ambiente desconhecido enquanto o tipo central não apresentou preferência pela origem do solo. Isso reitera a hipótese de colonizador/mantedor, no qual o tipo central germina em quaisquer locais quando comparado com o periférico, que é restrito as condições maternas. Essas conclusões devem ser melhor exploradas e testadas para ter maior consistência, contudo esses indícios já servem de alicerces para novos trabalhos.

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