Campus Botucatu INTERAÇÕES MULTITRÓFICAS: O EFEITO DE FORMIGAS, BESOUROS E MARIPOSAS SOBRE A REPRODUÇÃO E A MORFOLOGIA FLORAL DE TOCOYENA FORMOSA (RUBIACEAE) PRISCILA ANDRE SANZ VEIGA Dissertação apresentada ao Instituto de Biociências, Câmpus de Botucatu, UNESP, para obtenção do título de Mestre no Programa de Pós-Graduação em Ciências Biológicas (Botânica), Área de concentração Morfologia e Diversidade Vegetal. BOTUCATU – SP 2016                                                   Campus Botucatu UNIVERSIDADE ESTADUAL PAULISTA “Júlio de Mesquita Filho” INSTITUTO DE BIOCIÊNCIAS DE BOTUCATU INTERAÇÕES MULTITRÓFICAS: O EFEITO DE FORMIGAS, BESOUROS E MARIPOSAS SOBRE A REPRODUÇÃO E A MORFOLOGIA FLORAL DE TOCOYENA FORMOSA (RUBIACEAE) PRISCILA ANDRE SANZ VEIGA PROF. Dr. FELIPE WANDERLEY AMORIM ORIENTADOR Dr. SANTIAGO BENITEZ-VIEYRA COORIENTADOR Dissertação apresentada ao Instituto de Biociências, Câmpus de Botucatu, UNESP, para obtenção do título de Mestre no Programa de Pós-Graduação em Ciências Biológicas (Botânica), Área de concentração Morfologia e Diversidade Vegetal. BOTUCATU – SP 2016 FICHA CATALOGRÁFICA ELABORADA PELA SEÇÃO TÉC. AQUIS. TRATAMENTO DA INFORM. DIVISÃO TÉCNICA DE BIBLIOTECA E DOCUMENTAÇÃO - CÂMPUS DE BOTUCATU - UNESP BIBLIOTECÁRIA RESPONSÁVEL: ROSANGELA APARECIDA LOBO-CRB 8/7500 Veiga, Priscila Andre Sanz. Interações multitróficas : o efeito de formigas, besouros e mariposas sobre a reprodução e a morfologia floral de Tocoyena formosa (Rubiaceae) / Priscila Andre Sanz Veiga. - Botucatu, 2016 Dissertação (mestrado) - Universidade Estadual Paulista "Júlio de Mesquita Filho", Instituto de Biociências de Botucatu Orientador: Felipe Wanderley Amorim Coorientador: Santiago Benitez-Vieyra Capes: 20300000 1. Rubiaceae. 2. Cerrados. 3. Mutualismo. 4. Polinização. 5. Mariposa. 6. Flores - Morfologia. 7. Evolução (Biologia). Palavras-chave: Cerrado; Esfingofilia; Mutualismo defensivo; Polinização; Seleção fenotípica.   i    Escrever a meu ver é sentir brincar É dar ao pensamento asas para voar Escrever é indiretamente conviver com o alheio È tê-lo sempre junto como esteio Escrever é criar versos em prosa de mística emoção Que envolva o espírito e atinja a imaginação Escrever é o imprevisível que se manifesta em realidade Vem de nosso ego e levar para outros a felicidade Escrever é uma situação que se inventa e se arrebenta Como um furacão no centro da tormenta Escrever é proporcionar ao leitor novos entretenimentos È comunicar a todos agradáveis passa tempos Enfim, o que é escrever? È difícil de dizer, mas é fácil... È só escrever o que você deseja que outros venham a conhecer Mario Andre     Aos meus queridos avós Mario e Maria, exemplo do mais puro amor e felicidade. Estiveram presente em todos os momentos da minha vida. Infelizmente não puderam ficar para mais um capítulo. A eles, a minha querida Mãe e ao meu querido Pai dedico meu esforço e conquistas.   AGRADECIMENTOS À Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES) pela bolsa concedida durante o mestrado. Ao CNPq pelo apoio financeiro no âmbito do Universal CNPq, processo: 484469/2013-14. Aos proprietários da “Fazenda Palmeira da Serra” por permitir a realização do trabalho de pesquisa na reserva de Cerrado. Em especial agradeço à minha amada mãe e ao meu namorado pela enorme ajuda com o trabalho de campo, por terem me escutado, entendido e apoiado, mesmo quando nem eu mesma me aguentava. Vocês foram fundamentais, sem vocês nada teria sido possível. Mãe sua ajuda foi essencial, seu amor, atenção e apoio me deram forças para continuar e enfrentar todas as dificuldades. Obrigada por todo carinho e principalmente pela paciência! Ao meu guerreiro e inspirador pai, cuja dedicação e foco me inspiram e motivam cada dia. Obrigada pelo apoio, pelas valiosas discussões e contribuições filosóficas! Ao professor Dr. Felipe W. Amorim pela orientação e oportunidade de mestrado. Muito obrigada pelos ensinamentos, dedicação, discussões, incentivo e, principalmente pela paciência!! Obrigada por compartilhar seu conhecimento e essa paixão pelas interações planta-animal. Ao Dr. Santiago Benitez-Vieyra pela excelente coorientação, pela atenção, ensinamentos e indispensável contribuição. Ao Dr. Leonardo Ré pela colaboração, atenção e essencial contribuição. À Profa. Dra. Marlies Sazima, muito obrigada pelo carinho, incentivo e por ter aberto a primeira porta ao fascinante mundo da biologia da polinização. A todos do laboratório de Biossistemática da Unicamp (Fer, Coquinho, Vini, André, Pietro, Pedro, Marina) obrigado pelos cursos oferecidos, pelos debates e pelo apoio. A dedicação desses alunos apaixonados pelo que fazem foi o estimulo fundamental para o meu mestrado. Agradeço ao Dr. Rodrigo Feitosa da Universidade Federal do Paraná pela atenção e contribuição na identificação das formigas. Ao Dr. Sérgio Vanin (Universidade De São Paulo) pela atenção e ajuda na identificação da espécie de curculionídeo. Ao Dr. Sebastian e ao Javier (Unicamp), aos professores Dr. Rodrigo Medel, Dr. Luis Navarro, Dr. Wesley Dáttilo e Dr. Anselmo Nogueira pela atenção, considerações e sugestões que, de alguma forma, contribuíram para esse trabalho. À Fer pela hospitalidade, carinho, atenção, pelo imprescindível apoio durante todo o meu mestrado! À July pela ótima convivência, paciência e ajuda. À Maella pelos momentos de descontração e risadas! Aos queridos papas de July que me acolheram em sua casa na Argentina. À July, Heloísa, Alan, Salvador e também aos alunos do curso de ciências biológicas da UNESP-Botucatu (Caio, João, Carolina, Andrei, Gustavo, Larissa ) pela ajuda em campo e na triagem e coleta dos insetos. Obrigada a todos do Laboratório de Ecologia Evolutiva e Biologia Floral da Universidad de Córdoba por terem me recebido durante minha estadia em Córdoba. A todos os amigos e familiares que, de alguma forma, me ajudaram durante o meu mestrado. Muito Obrigada! ÍNDICE RESUMO 1 ABSTRACT 3 INTRODUÇÃO GERAL 5 REFERÊNCIAS BIBLIOGRÁFICAS 11 CAPÍTULO I The role of ant-tended pericarpial nectaries on fruit protection against pre-dispersal seed predation in a widespread cerrado shrub ABSTRACT 17 INTRODUCTION 18 MATERIAL AND METHODS 20 RESULTS 27 DISCUSSION 39 REFERENCES 44 CAPÍTULO II Efeito das interações multitróficas sobre a seleção mediada por polinizadores em Tocoyena formosa (Rubiaceae) RESUMO 52 INTRODUÇÃO 53 MATERIAL E MÉTODOS 55 RESULTADOS 63 DISCUSSÃO 74 REFERÊNCIAS BIBLIOGRÁFICAS 81 MATERIAL SUPLEMENTAR 87 CONSIDERAÇÕES FINAIS 89 REFERÊNCIAS BIBLIOGRÁFICAS 92 1 VEIGA, P. A. S. INTERAÇÕES MULTITRÓFICAS: O EFEITO DE FORMIGAS, BESOUROS E MARIPOSAS SOBRE A REPRODUÇÃO E MORFOLOGIA FLORAL DE TOCOYENA FORMOSA (RUBIACEAE). 2016. 93p. DISSERTAÇÃO (MESTRADO) – INSTITUTO DE BIOCIÊNCIAS, UNESP – UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO”, BOTUCATU. RESUMO - Polinizadores são tidos como os principais agentes seletivos da morfologia floral. Tocoyena formosa (Rubiaceae) é uma espécie esfingófila, autoincompatível, completamente dependente de mariposas com probóscide longa para sua reprodução sexuada. A presença de variação interindividual na morfologia floral sugere que o comprimento do tubo da corola pode estar sujeito à seleção natural imposta por polinizadores. Entretanto, sua reprodução também é afetada pela atividade de predadores de sementes pré-dispersão. Por outro lado, os frutos em desenvolvimento possuem nectários pós-florais pericárpicos (NPP) que atraem formigas, cuja interação mutualística deve mitigar a atividade dos predadores de sementes. Logo, o resultado da pressão seletiva imposta pelos polinizadores através do componente materno do fitness pode depender do efeito mútuo de antagonistas e formigas. Nesse contexto, para testar se formigas associadas aos NPPs de T. formosa conferem proteção aos frutos e sementes em desenvolvimento, e se predadores de sementes e formigas interferem no resultado da seleção imposta pelos polinizadores, nós realizamos experimentos de exclusão para avaliar o efeito de cada interagente sobre o êxito reprodutivo feminino (ERF) de T. formosa. Através de análise de caminhos e modelagem de equações estruturais determinamos a importância simultânea dos caracteres florais, mutualistas e antagonistas para o ERF da planta, e estimamos a ocorrência de seleção natural sobre o comprimento do tubo da corola. As formigas associadas aos NPPs não conferiram proteção aos frutos e sementes em desenvolvimento contra os principais predadores de sementes. Nem a abundância dos NPPs, tampouco a composição da fauna de formigas influenciaram no resultado da interação. Apesar dos predadores de sementes pré-dispersão terem tido um efeito negativo sobre o ERF através da diminuição do número de sementes por fruto, eles não influenciaram nem na direção, tampouco na magnitude da seleção fenotípica imposta pelos polinizadores sobre a morfologia floral. Provavelmente, a elevada frequência de visitas dos esfingídeos e a efetividade do serviço de polinização compensaram os efeitos negativos dos predadores de sementes sobre a reprodução de T. formosa. A natureza do sistema de polinização, no qual o pólen é transportado em massas que formam uma unidade discreta que se adere à probóscide dos polinizadores favorece a transferência de pólen entre flores cujo comprimento do tubo da corola é igual ou maior do que o da flor doadora. Dessa forma, plantas com tubos florais longos atuam principalmente como receptoras de pólen, podendo formar proporcionalmente mais frutos do que plantas com tubos curtos. Por sua vez, flores com tubo curto atuam 2 principalmente como doadoras de pólen, sendo favorecidas, provavelmente, via êxito reprodutivo masculino. Logo, o efeito líquido da seleção fenotípica mediada por polinizadores sobre o comprimento do tubo floral, também pode depender da direção e magnitude das pressões seletivas atuando mutuamente sobre os dois componentes do êxito reprodutivo. Além de trazer evidências de que os polinizadores atuam como os principais agentes seletivos da morfologia floral via ERF, o nosso estudo também destaca a importância da elevada efetividade do serviço de polinização para a reprodução de Tocoyena formosa. Palavras-chave: Esfingofilia, Cerrado, Curculionidae, mutualismo defensivo, polinização, seleção fenotípica. 3 VEIGA, P. A. S. MULTITROPHIC INTERCTIONS: THE EFFECT OF ANTS, BEETLES AND MOTHS ON THE REPRODUCTION AND FLORAL MORPHLLOGY OF TOCOYENA FORMOSA (RUBIACEAE). 2016. 93p. DISSERTAÇÃO (MESTRADO) – INSTITUTO DE BIOCIÊNCIAS, UNESP – UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO”, BOTUCATU. ABSTRACT - Pollinators are considered the main selective agents of floral morphology. Tocoyena formosa (Rubiaceae) is a self-incompatible sphingophilous species, completely dependent on long-tongued hawkmoths for sexual reproduction. Interplant variations in floral morphology suggest that the corolla tube length may be subjected to natural selection imposed by pollinators. However, its reproduction is also affected by the activity of pre- dispersal seed predators. On the other hand, the developing fruits possess post-floral pericarpial nectaries (PPN) which are constantly visited by mutualistic ants that should mitigate the activity of such seed predators. Therefore, the result of pollinator-mediated selection through the female component of fitness may depend on the mutual effect of antagonists and ants. In this context, to test whether PPN-associated ants provide protection to the developing fruit and seeds, as well as, whether pre-dispersal seed predators and ants affect the result of selection imposed by pollinators, we performed exclusion experiments to assess the effect of each interactor on the female reproductive success of T. formosa. For this end, we used path analysis combined with structural equation modeling to determine the simultaneous importance of floral traits, mutualists and antagonists to the female reproductive success, and estimated the natural selection acting on flower tube length. The PPN-attracted ants did not provide protection to the developing fruits and seeds against the main seed predators. Neither the abundance of NPPs, nor the composition of the ant fauna affected the outcome of the interaction. Despite the negative effect of pre-dispersal seed predators on plant reproductive success by reducing the number of seeds per fruit, they did not influence on the direction or the magnitude of the phenotypic selection imposed by pollinators on flower morphology. Probably, the high frequency of hawkmoths visits and the effectiveness of the pollination service may have compensated the negative effects of seed predators on T. formosa reproduction. The nature of pollen transfer mechanism, by which pollen grains are clumped in discreet units adhered to the pollinator proboscis, may favor the pollen flow from short-tubed to long-tubed flowers in the population. Thus, long-tubed flowers are more effective in pollen receipt, and thus may produce proportionately more fruits than short-tubed ones. Short-tubed flowers, in turn, may act mainly as pollen donors being, probably, favored through the male component of the reproductive success. Therefore, the net effect of pollinator-mediated selection on flower tube length may also depend on the direction and 4 magnitude of the selective pressures acting simultaneously through both female and male components of fitness. Despite evidencing that pollinators act as the main selective agents via female reproductive success, this study also highlights the importance of the high pollination effectiveness to the reproduction of Tocoyena formosa. Keywords: Cerrado, Curculionidae, defensive mutualism, phenotypic selection, pollination, Sphingophily. 5 INTRODUÇÃO GERAL Estudos sobre as interações mutualísticas entre plantas e insetos são de fundamental importância para a compreensão dos processos relacionados à evolução e diversificação das plantas (Strauss & Irwin 2004; Bronstein et al. 2006). Associações entre plantas e polinizadores estão entre os tipos de interações mutualísticas mais bem documentadas. Neste sentido, diversos estudos têm apresentado fortes evidências de que caracteres florais evoluem como adaptações aos polinizadores (Galen 1996; Fenster et al. 2004; Harder & Johnson 2009). O que reforça a influência dos polinizadores na diversificação e radiação das angiospermas (Stebbins 1970; Dodd et al. 1999; Kay et al. 2006; Whittall & Hodges 2007). Sistemas de polinização são, em sua maioria, generalistas e envolvem a associação com diversos grupos de polinizadores que podem exercer pressões de seleção distintas sobre os caracteres florais, que inclusive podem ser anuladas entre si (Waser et al. 1996; Johnson & Steiner 2000; Fenster et al. 2004; Sahli & Conner 2011; Sletvold et al. 2012). Nestes sistemas de polinização a importância de cada visitante floral como agente seletivo da morfologia floral não é tão evidente, uma vez que a contribuição de cada grupo de polinizadores para o êxito reprodutivo pode ser variável (Gómez et al. 2009). Por outro lado, em sistemas de polinização especializados as características florais geralmente refletem respostas adaptativas diretas à interação com um grupo específico de polinizadores (Galen 1996; Johnson & Steiner 2000). A seleção natural, por sua vez, opera quando a interação planta-polinizador confere maior êxito reprodutivo (fitness) a plantas com determinado fenótipo floral em detrimento dos demais fenótipos presentes na população. Logo, a seleção natural produz efeitos fenotípicos imediatos em cada geração, que podem ser medidos de forma independe dos princípios da hereditariedade e da evolução (Land & Arnold 1983). Porém, para que a seleção provoque mudanças na distribuição do fenótipo na população de uma geração a outra, é necessário que uma proporção significativa das diferenças fenotípicas seja herdável. Desta forma, mudanças evolutivas no fenótipo floral podem refletir respostas adaptativas à seleção imposta pelos polinizadores (Land & Arnold 1983; Strauss et al. 2005; Gómez et al. 2009). Desde a introdução de métodos que possibilitam inferências quantitativas da ação da seleção natural (Land & Arnold 1983; Arnold & Wade 1984; Schluter 1988; Morrissey & Sakrejda 2013), alguns estudos sobre evolução floral têm abordado o efeito das pressões seletivas impostas por polinizadores sobre caracteres florais relacionados, principalmente, à atração e ao ajuste morfológico entre a flor e o agente de polinização (veja Maad 2000; Benitez-Vieyra et al. 2006; Sletvold et al. 2010; Moré et al. 2012; Sletvold et al. 2012). Caracteres florais de atração podem influenciar a reprodução da planta através de seu efeito sobre o 6 comportamento dos polinizadores, por outro lado, caracteres de ajuste atuam sobre a efetividade da remoção e deposição do pólen. Estes estudos têm evidenciado que caracteres florais, tais como, número de flores, tamanho, forma, cor, recurso energético e fenologia estão sujeitos à seleção imposta pelos polinizadores (revisão em Harder & Johnson 2009). Longos tubos da corola e esporões atuam no ajuste morfológico entre a planta e o polinizador, logo, sua evolução parece estar diretamente relacionada às pressões seletivas impostas pelos polinizadores (Nilsson 1988; Johnson & Steiner 1997; Maad 2000; Alexandersson & Johnson 2002; Pauw et al. 2009; Moré et al. 2012). Polinizadores possuem uma importância inquestionável para a reprodução da maioria das angiospermas (Ollerton et al. 2011). No entanto, as plantas são constantemente visitadas por uma enorme diversidade de organismos cujos efeitos podem influenciar diretamente ou indiretamente seu fitness (veja Gómez 2008). Além do que, cada interagente pode modificar, reforçar e, inclusive anular de forma recíproca, o impacto individual do outro organismo sobre a planta (e.g. Mothershead & Marquis 2000; Gómez 2005; Strauss et al. 2005). Por exemplo, através do efeito sobre a taxa de frutificação, polinizadores podem influenciar a taxa de predação dos frutos, uma vez que antagonistas podem danificar preferencialmente frutos de plantas com maior abundância e/ou maior tamanho de frutos (e.g. Herrera 2000; Cariveau et al. 2004). Os predadores, por sua vez, podem anular os benefícios relacionados à maior taxa de polinização (Herrera 2000; Herrera et al. 2002). Assim, apesar da importância dos polinizadores como agentes seletivos da morfologia floral, outros fatores ecológicos podem afetar a intensidade da seleção natural agindo sobre os caracteres florais e influenciar a evolução floral (revisão em Strauss & Whittall 2006). Antagonistas também podem exercer pressões seletivas diretamente sobre os caracteres florais (e.g. Galen & Cuba 2001; Cariveau et al. 2004). Neste sentido, os caracteres florais podem sofrer pressões seletivas opostas caso polinizadores e antagonistas atuem sobre os mesmos caracteres (Gómez 2003; Toräng et al. 2008; Pérez-Barrales et al. 2013; Sletvold et al. 2015). Em determinados casos, a importância de antagonistas para processos de seleção fenotípica, pode ser inclusive maior que a dos polinizadores (Cariveau et al. 2004; Parachnowitsch & Caruso 2008). Por outro lado, o efeito dos antagonistas sobre a seleção natural em caracteres florais será indireto se sua atividade influenciar o efeito dos polinizadores (e.g. Mothershead & Marquis 2000). Predadores de sementes ainda em desenvolvimento nos frutos (daqui em diante ‘predadores de sementes pré- dispersão’) possuem um efeito direto sobre o componente feminino do fitness, e podem reduzir a oportunidade da seleção por polinizadores, caso sua atividade leve a diminuição de variações no fitness geradas através da interação com polinizadores (Herrera 2000; revisão em Strauss & Irwin 2004). Dessa forma, a importância de um organismo como agente seletivo 7 dependerá da presença e do efeito de outros organismos (Strauss et al. 2005). Assim, mudanças evolutivas no fenótipo floral serão determinadas pelo efeito conjunto dos múltiplos organismos sobre o fitness da planta (Galen & Cuba 2001; Herrera et al. 2002; Ehrlén et al. 2002; Gómez 2008). Tocoyena formosa (Cham & Schltdl.) K. Schum. (Rubiaceae) está entre as espécies lenhosas com distribuição mais ampla no bioma cerrado (Ratter et al. 2003). A espécie possui uma morfologia floral muito especializada que compreende uma flor com corola tubular, cujo tubo pode alcançar até 15 cm de comprimento (veja Silberbauer-Gottsberger 1972; Silberbauer-Gottsberger & Gottsberger 1975; Oliveira et al. 2004). Além da morfologia floral especializada a espécie é autoincompatível, o que torna sua reprodução completamente dependente de algumas espécies de mariposas da família Sphingidae, em especial aquelas com probóscide muito longas (> 6,0 cm). A ocorrência de variação interindividual no comprimento do tubo da corola em populações de T. formosa (numa amplitude entre 6,0 e 15,0 cm) possibilita, entretanto, que a espécie esteja sujeita a pressões seletivas exercidas pelos polinizadores. Contudo, o êxito reprodutivo feminino de T. formosa, i.e. formação de frutos e sementes, também está sujeito ao efeito de antagonistas, uma vez que durante o desenvolvimento dos frutos, as sementes são intensamente predadas por larvas de diversas ordens de insetos. Assim, caso determinados caracteres florais favoreçam a produção de mais frutos e sementes, indiretamente, também podem favorecer a atração de predadores de sementes pré-dispersão, cujo efeito pode influenciar no resultado da interação com polinizadores sobre o êxito reprodutivo feminino (Herrera 2000; Pérez-Barrales et al. 2013). Porém, após a senescência da flor que é caracterizada pela queda da corola, o ovário permanece aderido à planta e o nectário floral segue em funcionamento. Em flores polinizadas esse nectário permanece no fruto em desenvolvimento e passa a constituir o nectário pós- floral pericárpico (NPP), que é constantemente visitado por formigas (Santos & Del-Claro 2001, mas veja também Del-Claro et al. 2013; Falcão et al. 2014). Nesse sentido, a associação entre formigas e os NPPs pode representar um mutualismo defensivo e estar relacionada à diminuição da predação de sementes, o que poderia influenciar o resultado líquido do efeito dos predadores e polinizadores sobre o fitness feminino. Mutualismos com formigas através de nectários extraflorais representam um mecanismo de defesa, no qual, em troca de proteção, plantas fornecem recurso energético às formigas (Bentley 1977; Koptur 2005). No cerrado, o mutualismo defensivo entre formigas e nectários extraflorais é um mecanismo amplamente distribuído entre as espécies vegetais (Oliveira & Leitão-Filho 1987) e possui um importante papel na defesa das plantas contra herbívoros foliares (Del-Claro et al. 1996; Oliveira 1997; Oliveira & Freitas 2004; 8 Nascimento & Del-Claro 2010; Alves-Silva & Del-Claro 2013). Em muitas espécies da família Rubiaceae após a abscisão da corola, o nectário floral permanece ativo sobre o ovário durante o desenvolvimento dos frutos (e.g. Amorim & Oliveira 2006; Del-Claro et al. 2013; Falcão et al. 2014). Porém, pouco se sabe acerca dos efeitos da presença dos NPPs sobre a predação de sementes (Santos & Del-Claro 2001; Del-Claro et al. 2013). Neste contexto, o resultado líquido das pressões seletivas impostas por polinizadores sobre determinados caracteres florais, também pode depender do resultado da interação com formigas e predadores de sementes pré-dispersão. Mesmo que vários estudos tenham abordado o impacto de diversos interagentes sobre o fitness da planta (Brody & Mitchell 1997; Herrera 2000; Mothershead & Marquis 2000; Ehrlén et al. 2002; Herrera et al. 2002), poucos têm avaliado de forma conjunta o resultado líquido dessas interações sobre a seleção natural em caracteres florais. A influência da interação planta-polinizador sobre o fitness geralmente é avaliada separadamente da influência de outros interagentes, o que pode levar a resultados espúrios, já que o efeito líquido sobre o fitness e consequentemente sobre a seleção natural depende das interações multitróficas (Gómez 2003; Strauss & Irwin 2004; Strauss et al. 2005; Gómez 2008; Toräng et al. 2008). No Brasil pouco tem sido estudado sobre seleção mediada por polinizadores, e segundo um levantamento recente (Benitez-Vieyra et al. 2014), dentre os estudos sobre seleção fenotípica mediada por polinizadores realizados nos últimos 20 anos, apenas um trabalho foi realizado no Brasil (Moré et al. 2012). Por outro lado, estudos envolvendo o efeito de antagonistas e outros mutualistas sobre a seleção imposta por polinizadores ainda são inexistentes. Logo, Tocoyena formosa, cuja frutificação é afetada pela atividade concomitante de polinizadores, predadores de sementes e formigas, representa um bom modelo para o estudo do efeito das interações multitróficas sobre a morfologia floral. Embora a seleção natural possa ser interpretada como a relação entre os valores de fenótipo e seu respectivo êxito reprodutivo, a seleção também pode atuar de forma indireta através de caracteres correlacionados (e.g. Gómez 2000). Neste sentido, a existência de associação entre caracteres fenotípicos pode invalidar os resultados da análise de seleção sobre determinado caractere fenotípico. A fim de solucionar tais ressalvas, diversas técnicas baseadas em análises multivariadas foram propostas para quantificar a ação da seleção sobre um caractere de forma independente do seu efeito indireto operando através de outro caractere correlacionado (Land & Arnold 1983; Mitchell-Olds & Shaw 1987). Adicionalmente, tais metodologias podem ser utilizadas em associação com técnicas alternativas que representam soluções às limitações estatísticas inerentes à regressão múltipla, como por exemplo, técnicas 9 não paramétricas (veja Schluter 1988; Brodie et al. 1995; Morrisay & Sakrejda 2013; Benitez- Vieyra et al. 2014). Análises de rotas (path analysis) e modelagens de equações estruturais (SEM), por sua vez, permitem testar relações causais multivariadas complexas entre os interagentes, os caracteres florais e o fitness da planta, revelando as relações diretas e indiretas entre as variáveis (Kingsolver & Schemsk 1991). Além do que, também possibilitam testar hipóteses sobre a importância dos distintos interagentes para seleção fenotípica atuando em determinado caractere. Desta forma, tais metodologias constituem uma importante ferramenta para estudos de seleção em caracteres quantitativos (e.g. Gómez 2000; Cariveau et al. 2004). Finalmente, em conjunto com estudos de genética, essas técnicas permitem testar hipóteses adaptativas e inferir mudanças evolutivas nas características florais (Gómez 2000). A ação de outros interagentes que também pode influenciar na direção e força da seleção fenotípica, o que pode dificultar a interpretação dos resultados das análises de seleção, uma vez que os gradientes de seleção não trazem informação sobre a identidade dos agentes da seleção fenotípica observada. Assim para quantificar a importância de cada interagente para seleção é necessário abrir mão de metodologias que envolvam manipulação experimental (Parachnowitsch & Caruso 2008; Sandring & Ågren 2009; Sletvold et al. 2010; Sletvold et al. 2015). Porém, poucos trabalhos têm quantificado experimentalmente a importância simultânea de polinizadores e antagonistas para a seleção sobre a morfologia floral (Gómez 2003; Ågren et al. 2013; Sletvold et al. 2015). A seleção imposta por polinizadores pode ser identificada através da comparação da seleção estimada em plantas expostas à polinização natural e plantas após a suplementação manual de pólen (Galen 1996; Fishman & Willis 2008; Sandring & Ågren 2009; Sletvold et al. 2010). De forma similar, o estudo de mutualistas e antagonistas como agentes de seleção requer abordagens experimentais que permitam quantificar o efeito isolado, assim como a interação dos múltiplos interagentes (e.g. Sletvold et al. 2015). Neste contexto, o presente estudo teve como principais objetivos testar o efeito simultâneo da interação com polinizadores, predadores de sementes pré-dispersão e formigas mutualistas sobre a reprodução de Tocoyena formosa, assim como, quantificar a importância dos mutualistas e antagonistas para a seleção fenotípica sobre os caracteres florais. Este trabalho está divido em dois capítulos, nos quais nós utilizamos diferentes abordagens metodológicas e experimentais para investigar o efeito de mutualistas e antagonistas sobre a reprodução de T. formosa. No Capítulo I testamos experimentalmente o efeito da interação com formigas e predadores de sementes sobre o êxito reprodutivo feminino (i.e. produção de frutos e sementes). Especificamente, nós identificamos a fauna de formigas e dos predadores de sementes pré-dispersão associados aos frutos em desenvolvimento. 10 Determinamos experimentalmente se a interação com formigas mediada pelos nectários pós- florais pericárpicos (NPPs) confere proteção aos frutos e sementes contra predadores pré- dispersão. Adicionalmente, nós também testamos se a espécie de formiga e a disponibilidade de recursos, i.e. néctar, influenciam o efeito das formigas sobre a frutificação. 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Nature 447: 706-710. 16 CAPÍTULO I The role of ant-tended pericarpial nectaries on fruit protection against pre-dispersal seed predation in a widespread cerrado shrub 1 Priscila Andre Sanz Veiga1*, Leonardo Ré Jorge2, Santiago Benitez-Vieyra3, Felipe Wanderley Amorim1 1 Departamento de Botânica, Instituto de Biociências, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu, São Paulo, Brazil 2 Departamento de Biologia Animal, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil 3 Instituto Multidisciplinario de Biología Vegetal (CONICET), Universidad Nacional de Córdoba, Ciudad de Córdoba, Córdoba, Argentina * Corresponding author E-mail: psanzveiga@gmail.com (PASV) 1 Capítulo apresentado conforme as normas da Plos ONE 17 Abstract Extrafloral nectaries (EFNs) related to ant-plant defensive mutualisms can occur in both vegetative and reproductive plant structures. The presence of post-floral pericarpial nectaries (PPNs) might be strategical for fruit protection against seed predators, as plants are expected to invest higher on more valuable and vulnerable parts. In many Rubiaceae species in the Brazilian Cerrado, after corolla abscission, the floral nectary continues to secret nectar throughout fruit development forming PPNs which commonly attract many ant species. Here, we experimentally investigated whether the interaction with ants mediated by the PPNs of Tocoyena formosa influenced plant reproductive success by reducing the number of seed predators on fruits. We also assessed whether ant protection was dependent on ant species composition and resource availability. Although most of the plants were visited by large and aggressive ant species, such as Ectatomma tuberculatum and Camponotus spp., ants did not protect fruits against seed predators. Furthermore, the result of the interaction was neither related to ant species composition nor to the availability of resources. We suggest that these results may be, at least in part, related to the nature and behavior of the most important seed predators, like the Hemicolpus abdominalis weevil. On the other hand, at the community level, not explored factors, such as reward quality local ant abundance, ant colony characteristics and/or the presence of alternative energetic sources could also account for variations in ant frequency, composition, and ant protective effects, revealing the great conditionality of facultative ant-plant mutualisms. Finally, because variations in fruit production due to pollinator´s temporal fluctuation are common in highly specialized pollination systems, we suggest that ant defense could be more necessary or important during periods of low fruit set or higher seed predation. Keywords: Ant-plant interactions; Ectatomma tuberculatum; Hemicolpus abdominalis; mutualism; Tocoyena formosa; weevil 18 Introduction Ant-plant defensive mutualisms represent an indirect defense strategy widely distributed among angiosperms in which plants provide energetic resources and/or housing for ants that in turn provide protection against herbivores [1-4]. Ant-plant associations range from facultative to obligate (reviewed in [5]) and plants involved in obligate and more specific associations generally invest more energy on food rewards and nesting structures to the mutualists, which may guarantee higher fidelity and effectiveness of ants [3, 6, 7]. On the other hand, facultative mutualisms involve looser associations, in which ants are attracted to food resource produced by the plant but do not nest on the plant, in such associations plants can interact with many different and unspecific ant species [8, 9]. Most ant-plant associations are facultative [3, 5] and associations with defensive ants mediated by extrafloral nectaries (EFNs) represent a classic example of such non-obligatory interaction [1, 10, 11]. Extrafloral nectar is produced in secretory glands, generally not involved in pollination [1, 12]. EFN-visiting ants can protect plants by deterring or preying upon leaf [10, 13, 14], bud and flower herbivores [11, 15-17], as well as pre-dispersal seed predators [18, 19]. However, some studies have suggested that EFN-visiting ants may be very ineffective in plant defense [20-25] or even have negative effects by repelling pollinators or the natural enemies of herbivores [26, 27]. Hence, the outcome of ant-plant interactions may vary along a continuum from positive to negative effects [28], and the net result of the interaction depends on ant density and herbivore abundance [29], ant behavior [16, 22, 25, 30, 31], herbivore vulnerability to ant predation [21, 24, 29, 32-34], plant traits and resource abundance [34-38], presence of other arthropods competing with ants for extrafloral nectar [12, 39], and abiotic conditions [40-42]. Although EFNs are present in both vegetative and reproductive structures, according to the predictions of optimal defense theory, plants are expected to invest more in protection of the most vulnerable and valuable parts, such as young leaves, buds and flowers [43, 44]. 19 The occurrence of EFNs on reproductive structures may represent one example of such investment, as these parts are directly related to plant reproductive success [45]. Some studies have assessed the role of EFNs located on reproductive parts, mainly on pedicels, sepals and bracts [18, 20]. Some EFNs located on the pedicel and bracts can extend their activity throughout the whole plant reproductive stage and attract ants to protect buds, flowers and developing seeds [43, 45, 46]. Moreover, plants can also present EFNs on fruits derived from newly developed structures [43, 47, 48] or from persistent floral nectaries [49-51]. In the last case, the so-called post-floral pericarpial nectaries (PPNs) may be related to fruit protection against pre-dispersal seed predators [19]. However, the few studies that have so far investigated the effectiveness of the association between ants and PPNs on fruit protection have provided some contrasting results [19, 50, 52]. In the Brazilian Cerrado, EFN-bearing plants are very common and widespread in many families [53, 54], and their association with ants is effective on the protection of vegetative and reproductive plant structures parts [11, 14, 16, 17, 55]. In many species among the Rubiaceae family, the floral nectaries continue to secrete nectar following the flower senescence and corolla abscission originating PPNs [e.g., 49, 50, 51]. However, only few studies have investigated the importance of PPNs on plant reproductive success [50, 52]. Tocoyena formosa (Cham. & Schlechtd.) K. Schum. (Rubiaceae) is a common and widely distributed shrub in the Cerrado (see [56]), that produces fruit-bearing PPNs which are constantly visited by ants. In contrast to the small foliar NEFs commonly found in other Cerrado plants, PPNs of T. formosa produce copious amounts of nectar throughout fruit development. Moreover, even ovaries from non-pollinated flowers continue to produce nectar for about two months after corolla abscission (P.A.S Veiga, personal observation, Fig 1). Hence, contrary to the notion that facultative ant-plant associations involve lower plant investment in energetic resource to the mutualists [6, 7, 57], these observations suggest that non-obligatory associations may also have high costs. In this context, we hypothesized that 20 such a high investment in energetic reward in T. formosa may provide fruit protection against pre-dispersal seed predators. However, since reward availability may influence in the outcome of ant-plant interaction [58, 59] and the number of nectaries are highly variable among T. formosa plants, both ant visitation frequency and species composition may also be affected by nectar availability. In this sense, we also hypothesize that plants with more nectaries will receive more visits from competitively superior ant species with higher defensive abilities [30, 60], which may result in higher reproductive success in terms of fruit and seed set. In this context, the main goals of this study was to test whether PPN-visiting ants have a positive effect on T. formosa reproductive success by reducing seed predator infestation, and whether protection is affected by ant species composition and resource availability. To this purpose, we used experimental and observational approach and addressed the following questions: (1) What are the most important pre-dispersal seed predators of T. formosa? (2) Are there differences in ant fauna composition among plants? (3) Do ants have a positive effect on plant reproduction by decreasing pre-dispersal seed predation? (4) Ant protection depends on the dominant ant species? (5) Does resource availability (number of nectaries) influence ant visitation and ant effect on plant reproduction? (6) Does nectary abundance influence at the same time ant fauna composition and the plant reproductive success? Material and methods Study site and species characterization The study was carried out during the years 2014-2015 in a Cerrado area in a private reserve at “Palmeira da Serra Farm” (22º48’50” S, 48º44’40” W). The reserve is located in the municipality of Pratânia, São Paulo state, southeastern Brazil, and is constituted by two main plant physiognomies: cerrado stricto sensu, a vegetation dominated by herbs, shrubs and small trees and, “cerradão”, a dense woodland made up of trees about 10 m (for further details see [61]). The total area covers approximately 224 ha. with an elevation of 720 m a.s.l., and 21 the climate is warm temperate (Cwa according to Köppen 1984) and characterized by two well-stablished seasons, i.e., a dry winter from March to September and a hot summer from October to April. The mean annual temperature is 21ºC and annual precipitation is around 1450 mm [62]. At the study area T. formosa is a caducifolious shrub (< 3 m) which leaf loss occurs during the driest months (June to August), and new leaves are produced throughout spring (September-October). Flowers are produced in inflorescences at the apex of branches and flowering occurs in November-January, and fruiting in March-April. Flowers are tubular and the corolla tube length ranges between 70 and 140 mm. Pollination and sexual reproduction of T. formosa relies exclusively on long-tongued hawkmoths [63, 64]. In this species, after corolla abscission the floral nectary remains active and becomes an extrafloral nectary with post-floral secretion (Fig 1) [52]. 22 Fig 1. Post-floral pericarpial nectaries of T. formosa at different developmental stages. (A) Active post-floral nectaries just after corolla abscission (arrow heads). Note the corolla tube of flower before abscission (arrow). (B) Post-floral pericarpial nectary (PPN) at the initial stage of development after one month of flower pollination (arrow). (C) PPN after two months of flower pollination (arrow), and post-floral nectaries derived of non-pollinated flowers (arrow heads). (D) Fruits at advanced stages of development with active PPN (arrow) and a still active post-floral nectary after three months of corolla abscission of a non- pollinated flower (arrow head). 23 Ant exclusion experiment To test the role of ants in fruit defense, we conducted exclusion experiments that allowed us to compare plant reproductive success (i.e., fruit and seed formation) at different conditions (presence and absence of ants) in the same plant. In December 2014, we tagged 42 randomly marked plants. In each plant we chose two branches with flower buds and randomly assigned them as control (free access of ants) or treatment (ants excluded). Ants were prevented to access the PPNs of fruits by applying a nontoxic resin (TanglefootTM) on the branch basis. Branches or leaves that could be used by ants as bridge to access the experimental branches were removed. We assessed ant effectivity by comparing the number of seed predators per fruit, number of fruits started, i.e., number of fruits in the initial stage of development, number of developed fruits, i.e., fully developed fruits in pre-mature stage, and the mean number of seeds per fruit between the treatment (ants excluded) and control (free access of ants) branches. At the end of the fruiting season (from March to April of 2015) pre- mature fruits were collected and kept on plastic containers, for approximately one month, until the emergence pre-dispersal seed predators. In addition, we checked all the fruits for the presence of dead larvae and insects that did not emerge. To test the influence of the number of nectaries on ant effectiveness, we quantified the total number of nectaries on each individual plant. We considered the total number of flowers as a proxy for nectary abundance since our observations indicated that even those nectaries from ovaries that do not develop into fruits remain active during the period fruit development (see Fig 1). Plant reproductive success The reproductive success was evaluated by the proportion of fruits started (number of fruits in the initial stage of development / total number of flowers produced per plant), proportion of developed fruits (number of pre-mature fruit / number of fruits started) and seed 24 set (mean number of seeds produced per fruit). The mean number of seeds per fruit was determined on collected fruits after insect emergence in 2 to 5 fruits per treatment. Only fully developed and intact seeds were counted. Seed predator fauna The collected insects (adults and larvae) found on fruits were preserved in 70% ethanol and subsequently classified at the lowest taxonomic level possible. Voucher specimens were deposited in the insect collection of the Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu – SP. Hemicolpus abdominalis (Hustache, 1938) (Coleoptera: Curculionidae) voucher specimens were deposited in the zoology collection of the Universidade de São Paulo, São Paulo – SP, Brazil. Seed predator relative frequency was determined for each insect group on control branches. Insect infestation was estimated as the number of seed predators per fruit (i.e., total number of seed predators emerged from fruits divided by the total number of fruits collected) of a given branch of the experiment. Infestation was measured both for total number of predators and for each taxonomic category. Ant fauna and natural history observations To assess the PPN-visiting ant fauna, we performed weekly censuses of ant activity on control branches of all tagged plants from February to April of 2015. The observations were carried out between 08h00 and 18h00 and lasted 2 minutes per plant. On average, 10 censuses per plant were performed, totaling 14 hours of observations. Ants were collected, preserved in 70% ethanol, and identified with the aid of specialists. Voucher specimens were deposited in the insect collection of the Laboratory of pollination ecology and interaction at Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu-SP, Brazil. We determined the frequency of visits of each ant species in the tagged plants dividing the number of times a certain species was recorded by the total number of censuses made. 25 Then, we considered as dominant, those ant species that occurred in most of the censuses in a particular plant, and thus had the highest visitation frequency value in a given individual plant. While recording ant visitation, we also recorded the behavior of ants and seed predators. We characterized the behavior of the PPN-visiting ants by describing the activity of each species around the nectaries, as well as their response to the herbivores. The behavior of the pre-dispersal seed predators was investigated by describing, when it was possible, their foraging and feeding habits as well as their response to encounters with ants. Analysis To determine whether the number of seeds per fruit is related to fruit infestation, we tested the relationship between the total number of seed predators per fruit and the mean number of seeds per fruit with a simple linear regression, and the relationship between all predators groups and the mean number of seeds per fruit with a multiple regression approach. Finally, we used a simple linear regression to define the relationship between the main seed predator (Curculionidae beetle) and the number of seeds. We used the mean number of total seed predators per fruit, the mean number of seed predators from each taxonomic category and the mean number of weevils per fruit as predictor variables and the mean number of seeds per fruit as response variable. In order to test for the occurrence of different ant composition among plants (N= 42 plants), we performed a Non-metric Multidimensional Scaling (NMDS) based on the frequency of PPN-visiting ants in each plant using Bray-Curtis distances. We also used generalized linear mixed models (GLMM) to test the effect of ants on seed predators and plant reproductive success (i.e., proportion of fruits started, proportion of developed fruits and the mean number of seeds per fruit).We used a Gaussian model for the quantitative response variables, i.e., number of seeds per fruit, number of total seed predators per fruit and number of weevils per fruit. While for proportions of fruits started and final fruits, we used binomials models. In all cases, the treatment (ant presence / absence) was 26 considered as fixed effect and plant individual was assigned as random effect. We used the Akaike Information Criterion (AIC) to compare a model including treatment with a null model with the random effect only [65]. To test whether the ant effect on the number of seed predators per fruit depend on dominant ant species, we used GLMM in which plant individuals were assigned as random factors, while treatments (ant presence / absence) and dominant ant species as fixed effects. The mean number of seed predators per fruit was the response variable. We also used model selection to test whether including treatment and/or dominant ant species and the interaction between treatment and ant species improved the prediction over a null model with only random variation among plants. Ant species that appeared as dominant in only one plant were removed from the analysis. To test the hypothesis that plants with more nectaries were also the most visited by ants, we used a simple linear regression model with the total number of nectaries (i.e., total number of flowers produced per plant) as predictor variable and total frequency of PPN- visiting ants as a dependent variable. To test whether resource availability influenced the effect of ants on plant reproductive success, we used linear mixed models and model selection. We fitted Gaussian models to predict the mean number of seeds per fruit. Plant individuals were considered as random effect and the number of nectaries and ant exclusion treatment as fixed effects. Models with all the possible combinations of the fixed effects were considered. These models were also compared with a null model including only the random effect and a fixed intercept. Then, we compared the models above by means of the AIC. To assess whether resource availability influences the composition of the ant fauna and if composition affects plant reproductive success, we performed a Canonical correspondence analysis (CCA) using the number of nectaries and the mean number of seed per fruit as environmental matrix. 27 Results Seed predator fauna The main seed predators found on developing fruits (N=245 fruits) of control branches belong to Lepidoptera (0.97%), Diptera (7.5%), Coleoptera (43.3%) and Hymenoptera (48.2%). Hymenoptera were represented by at least two species of wasps, possibly from Ichneumonidae family, while Hemicolpus abdominalis (Hustache, 1938) (Coleoptera: Curculionidae) comprised 97% (N=129 specimens) of the Coleoptera. Beetles were observed in 69% of the plants, wasps in 48%, Diptera in 29% and Lepidoptera only in 7% (Table 1). When considering all seed predators, regardless of the taxa category, we found no relationship between the number of seeds and the number of total seed predators per fruit (R2=0.04, F(1,40) = 2.79; p=0.103). Also, there was no relationship when we considered the effect of each seed predator group (R2=0.10, F(4,37) = 2.11; p=0.099). However, we found a negative relationship between the number of seeds and the number of beetles per fruit (R2=0.12, F(1,40) = 6.82; p=0.013; Fig 2). 28 Table 1. Abundance, relative frequency and mean number per fruit (±SD) of seed predators found in Tocoyena formosa fruits. Abundance Relative frequency (%) Mean number / fruit/ plant Lepidoptera 3 0.97 0.01±0.04 Diptera 23 7.49 0.11±0.24 Coleoptera 133 43.32 0.59±0.62 Hymenoptera 148 48.21 0.60±1.48 Insects were collected on fruits from control branches (N= 245 fruits in 42 plants) 0.0 0.5 1.0 1.5 2.0 2.5 0 20 40 60 80 Number of beetles per fruit M ea n nu m be r of s ee ds pe r fr ui t Y= -8.8326X + 45.001 R2= 0.12; p = 0.013 Fig 2. Relationship between the mean number of seeds per fruit and number of beetles per fruit. 29 Ant fauna We recorded 10 ant species distributed in seven genera (Ectatomma, Brachymyrmex cf., Camponotus, Cephalotes, Crematogaster, Neoponera, Pseudomyrmex), and five subfamilies (Ectatomminae, Formicinae, Myrmicinae, Ponerinae, Pseudomyrmecinae) (Table 2). Among large (7-15 mm) and medium size ants (4-7 mm), Ectatomma tuberculatum (Olivier, 1792) and Camponotus spp. (N=225 and 223 total observed specimens) were the most abundant and frequent ants (Table 2). Ectatomma tuberculatum was recorded in 60% of the plants and was the dominant ant species in 55%, while Camponotus crassus (Mayr, 1862) occurred in 19% of the plants being the dominant species in 14%. Among the small ant species (1-3mm), Crematogaster goeldii (Forel, 1903) together with Brachymyrmex sp. aff. were the most abundant in the plants where they occurred, but they presented a low frequency in the whole population (Table 2). The first occurred in 7 plants, being the dominant species in six of them, while the second also occurred in 7 plants, but was dominant in only one plant. Neoponera villosa (Fabricius, 1804) was observed as a dominant species in two plants, although in only one, it occurred with relatively high frequency, thought presenting a very low abundance in both plants. Camponotus rufipes (Fabricius, 1775) was dominant in two plants. Camponotus renggeri (Emery, 1894) and Cephalotes sp. were dominant in only one plant each. Although Camponotus ager (Smith, 1858) was observed in one plant during day censuses, it was not the dominant species. NMDS ordination showed that most differences in ant composition among plants were due to the presence of E. tuberculatum, C. crassus and Crematogaster goeldii (stress: 0.05) (Fig 3). Plants in which these ants occurred were most similar in composition. Generally, when one of these three ant species was present in a given plant, few or no other ant species co-occurred. Furthermore, plants visited by the other recorded species did not show clear differences on ant composition. 30 Table 2. Ant species observed visiting PPNs on control branches of Tocoyena formosa. Mean abundance (±SD) per plant in each census and mean (±SD) visitation frequency per plant. Ants Mean abundance per plant Mean visitation frequency Ectatomma tuberculatum (Olivier, 1792) 0.46±0.63 0.27±0.29 Camponotus crassus (Mayr, 1862) 0.34±0.86 0.10±0.25 Crematogaster goeldii (Forel, 1903) 0.76±2.03 0.05±0.13 Camponotus rufipes (Fabricius, 1775) 0.08±0.36 0.03±0.16 Brachymyrmex sp. aff. 0.74±2.01 0.03±0.08 Neoponera villosa (Fabricius, 1804) 0.02±0.09 0.02±0.09 Cephalotes sp. 0.06±0.22 0.01±0.06 Camponotus renggeri (Emery, 1894) 0.04±0.27 0.01±0.08 Pseudomyrmex gracilis (Fabricius, 1804) 0.01±0.04 0.01±0.03 Camponotus ager (Smith, 1858) 0.01±0.04 0.006±0.04 Fig 3. Non-metric multidimensional scaling (NMDS) showing individual plants dissimilarities in ant composition fauna. Plants are displayed as numbers. 31 E. tuberculatum and N. villosa were the largest T. formosa PPN-visiting ants. N. villosa was never observed foraging in groups, neither attacking other insects, while E. tuberculatum seldom foraged in groups, although in some occasions many ants were recorded in the same branch. E. tuberculatum presented the most aggressive behavior and in many occasions were seen standing on the fruits with their antennae and mandibles held wide open (Fig 4A). In some occasions this position preceded attacks to other insects which approached the fruits. We recorded antagonistic behavior toward flies and an effective predation of H. abdominalis (Fig 4B). However, in most cases this behavior did not result in pray capture. C. crassus were classified as a medium sized ant and foraged mainly in groups of two or three individuals. They were more active and present a more exploratory behavior on branches and fruits when compared to E. tuberculatum. Although they were not as aggressive as E. tuberculatum in some cases these ants were observed attacking flies on the fruits. Among the small sized ants, few agonistic interactions were recorded, but we recorded them attacking another ant species (Cephalotes sp.) and a Lepidoptera larva once. During day censuses, H. abdominalis weevils were seen ovipositing inside holes which they made with their mouth parts on fruit pericarp, that caused visible damage on fruits (Fig 4C). Despite this, the incidence of weevils apparently did not affect the presence of other predator groups, since we recorded the simultaneous presence of different groups in the same fruit. Weevil larvae feed on seeds and each larva consumed more than one seed per fruit. Although a single fruit could bear up to four larvae, most fruits harbored only one or two. The larvae pupated inside the fruit and after reaching the adult phase perforated the pericarp and left the fruit (Fig 4D). However fruits infested by weevil larvae usually reached full development. Moth larvae were the largest in size seed predators found in T. formosa developing fruits. These insects presented the greatest potential of fruit and seed damage, as some infested fruits had 100% of the seeds consumed which always led to fruit abortion. Adults of 32 flies were also observed feeding on the PPNs and, when they occurred in high abundances, their larvae could consume all fruit pulp and seeds, also leading to fruit abortion. Wasp oviposition on fruits also occurred during daylight (Fig 4E) and their larvae fed exclusively on a single seed throughout their whole development (Fig 4F). We found up to 20 wasp larvae in a single fruit, and after development into adults, wasps left the fruit by perforating its pericarp without causing fruit abortion. 33 Fig 4. Tocoyena formosa interactions with the most aggressive ant species and pre- dispersal seed predators. (A) E. tuberculatum with its jaws open on a T. formosa fruit. (B) E. tuberculatum preying on and carrying the most common seed predator of T. formosa, the weevil H. abdominalis (arrow). (C) H. abdominalis perforating the fruit pericarp with its 34 rostrum. (D) An adult of H. abdominalis emerging from the fruit. (E) A wasp ovipositing in a T. formosa fruit, arrow showing its ovipositor. (F) An adult wasp emerging from a seed (arrow). Scale: A, B, C, D, E–5 mm; F–1 mm. Ant effect on seed predators and plant reproductive success The number of total seed predators per fruit was not affected by the presence of ants (branches without ants: 1.25 ± 1.32; branches with ants: 1.30 ± 1.62) (Fig 5A). Also, there was no effect of ants on the occurrence of weevils (without ants: 0.64 ± 0.58; with ants: 0.59 ± 0.62; Fig 5B).Thus, regardless of the occurrence of ants, fruits presented similar levels of seed predator infestation. Furthermore, neither the initial proportion of fruits produced (without ants: 0.46 ± 0.20; with ants: 0.47 ± 0.16) nor the final (without ants: 0.89 ± 0.17; with ants: 0.91 ± 0.13) were affected by the presence of ants (Fig 6A). Finally, we found no differences on the mean seed number per fruit between branches with (39.83 ± 14.16) or without ants (39.44 ± 17.69; Fig 6B). In all cases the null model presented the lowest AIC value (Table 3). The effect of ants on the number of seed predators per fruit did not depend on the dominant ant species, as the null model performed better than the ones that included both the effect of treatment and ant species (Table 4). All these results agree that ants had no effect on plant reproductive success. Also, we found no influence of the total number of nectaries per plant on ant visitation frequency (Fig 7). Nectary abundance did not influence ant effectiveness in plant defense, since models not including the effect of ant occurrence performed better than those that included (Table 5). CCA analysis also showed no relationship between variations in nectary abundance and ant fauna composition, which in turn, did not affect plant reproductive success (Fig 8). 35 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 1 2 3 4 5 6 7 Ants Absence Presence To ta l s ee d pr ed at or s pe r fr ui t A Ants Absence Presence W ee vi ls pe r fr ui t B Fig 5. Results of the ant exclusion experiment on fruit infestation. (A) Ant effect on the total number of seed predators per fruit and (B) on the number of weevils per fruit, in fruits of branches without and with ants (N = 42 plants). 0.2 0.4 0.6 0.8 1.0 1.2 Ants Absence Presence Pr op or ti on of f in al f ru it s 0 20 40 60 80 100 Ants Absence Presence M ea n se ed nu m er pe r fr ui t Fig 6. Results of the ant exclusion experiment on fruit production. (A) Ant effect on the proportion of developed fruits and (B) on the mean number of seeds per fruit, in branches without and with ants (N = 42 plants). 36 Table 3. Models testing ant effect on seed predators and plant reproductive success. Comparison of models including the effects of treatment (with and without ants) and a null model with the random effect only. Models AIC ΔAIC d.f. Total seed predators per fruit Model with treatment 303.5 2.2 4 Null Model 301.6 0.0 3 Weevils per fruit Model with treatment 157.3 2.0 4 Null Modell 155.5 0.0 3 Proportion of started fruits Model with treatment 397.4 1.8 3 Null Model 395.7 0.0 2 Proportion of developed fruits Model with treatment 204.1 1.3 3 Null Model 203.0 0.0 2 Mean seed number per fruit Model with treatment 3389.5 1.7 4 Null Model 3387.8 0.0 3 Table 4. Models testing the effect of dominant ant species on the number of seed predators per fruit. Comparison of models including the effects of treatment (with and without ants), dominant ant species and the interaction between treatment and dominant ant species with a null model with the random effect only. Models AIC ΔAIC d.f. Treatment + Dominant ant species + Interaction 286.3 5.6 12 Treatment + Dominant ant species 291.0 7.6 8 Treatment 287.1 2.2 4 Null Model 285.1 0.0 3 37 0 50 100 150 200 250 300 350 400 450 0.0 0.2 0.4 0.6 0.8 1.0 Y= -0.0006 X + 0.5912 R2= 0.03; p = 0.123 Total number of nectaries F re qu en cy of a nt vi si ta ti on Fig 7. Relationship between the total number of nectaries and the frequency of ant visitation. Table 5. Ant exclusion experiment and nectary effect on mean seed number per fruit. Comparison of models including the effects of treatment (with and without ants), nectary abundance and interaction between treatment and nectary with a null model with the random effect only. Models AIC ΔAIC d.f. Treatment + Nectaries + Interaction 705.7 2.7 6 Treatment + Nectaries 705.6 2.2 5 Treatment 706.7 3.1 4 Nectaries 703.6 0.0 4 Null Model 704.7 0.9 3 38 Fig 8. Canonical correspondence analysis (CCA). Diagram showing the ordination of individual plants according to ant species composition, nectary abundance and mean number of seeds per fruit. Individual plants are displayed as numbers. 39 Discussion Although we have hypothesized that the presence of pericarpial nectaries would represent an indirect defense mechanism against pre-dispersal seed predators, T. formosa PPN-attracted ants did not reduce seed predation nor increased plant reproductive success. Neither the large and aggressive ants nor the abundance of nectaries affected ant performance on protecting seeds. Developing seeds suffered predation mainly due to the activity of weevils and wasps larvae. Although, infestation by seed predators did not result in fruit abortion most of time, increasing beetles number per fruit led to a decreasing number of seeds, indicating that H. abdominalis is the most important pre-dispersal seed predator of T. formosa. To the best of our knowledge, this is the first study to report the behavior and feeding habits of this weevil, which might present host specific oviposition preferences. Similarly to our results, Del-Claro et al. [50] found that the interaction with ants mediated by the PPNs of Palicourea rigida, another Rubiaceae species, did not provide protection from seed-parasitic wasps. Instead they concluded that ants have an indirect effect on plant reproductive success by protecting leaves against foliar herbivores. In another population of T. formosa in Minas Gerais state, southeastern Brazil, Santos and Del-Claro [52] also did not find any differences in plants with and without ants. However, in Mentzelia nuda (Loasaceae) there was a positive effect of ants on pods and seed formation, which suggest that PPN-ant associations may represent an effective strategy of seed defense against pre-dispersal predators [19]. The absence or the reduced effectiveness of ants in protecting plants against herbivores may be related to the facultative nature of the ant-plant interaction mediated by EFNs [4, 7, 28, 29]. In some cases, however, effectiveness in plant defense against herbivores depends on the composition of the ant fauna [25], being protection related to ant size and/or recruitment capacity [9, 16]; but see also [66]. Here, though the dominant ant species differed in size and foraging behavior among individual plants, they did not effectively protected the 40 developing seeds in any case. Seed protection was ineffective even in those plants dominated by E. tuberculatum, which is reported as a highly aggressive ant species see [67, 68] and very effective in flower and fruit protection in other species [16]. However, in other Cerrado areas, ant benefits on plant reproductive success is reported to be temporal variable, with different ant species being associated with higher seed production in successive years [42]. Camponotus spp. are among the most common and effective EFN-visiting ants in the Brazilian Cerrado [14, 17, 69]. Nevertheless, in some systems they fail to protect plants against foliar and flower herbivores [33, 34, 70], and also against seed predators [50]. Indeed, the few plants dominated by C. crassus did not show lower fruit infestation by seed predators. Maybe the fact that only few plants were dominated by C. crassus might have hampered the detection of positive effects. However, in another T. formosa population which presented a higher frequency of C. crassus, this species did not provide plant protection from seed consumers or foliar herbivores [52]. The identity and behavior of the most important seed predator may also have accounted for the absence of ant protection, since specialized antagonists can present adaptations to escape plant defenses [71]. The sclerotized body of weevils may provide protection against ant attack [26, 33]. For instance, Alves-Silva et al. [33] recorded that C. blandus attacks to florivorous beetles never resulted in weevil injury or predation. However, the sclerotized body alone may not account for all beetle immunity, since ants are very effective against beetles in other systems [11, 16, 19, 32]. In the case of Hemicolpus abdominalis weevil, we observed once, the production of a drop-like secretion by the weevil pygidium after an encounter with an Ectatomma sp., which resulted in the ant leaving the branch without disturbing the beetle. As observed for other groups like sawflies and trips, we suspect that this secretion may have a repellent effect on ants (see [21, 34]). Additionally, the endophytic habit of these beetles could also have negatively influenced ant effectiveness because the larvae which develop inside fruits are out of the reach of ants, and thus ant 41 protection may only take place when ants directly interfere with oviposition [24, 26]. Another mechanism by which antagonists usually escape ant attack is related to the behavioural ability of seed predators to avoid ants [21, 34]. When visiting the PPNs of large fruits, ants usually are positioned on the top of the fruit where the PPNs are located, and thus may not notice insects located at the base of the fruit. However, despite of the defense strategies of weevils, as we observed in the T. formosa, sometimes aggressive ants as E. tuberculatum can successfully prey upon H. abdominalis weevils. Despite the fact that intrinsic differences in the amount of reward among plants can influence ant visitation frequency and the ant-plant interaction outcome [35, 70, 72], our results indicate that neither ant visitation frequency nor their effect on plant reproduction were influenced by the abundancy of nectaries. On the other hand, since plants can concentrate nectar production in more vulnerable periods of plant development, like the initial stage of fruit development [46, 51], we suggest that further studies should explore the existence of differences in nectar production in different ontogenetic stages of fruit development in T. formosa. Variations in resource abundance between plants also had no effect on ant fauna composition inhabiting T. formosa plants. Similarly, Dáttilo et al. [69] did not find any relationship between EFN abundance and ant richness in three Cerrado plant species. Instead, as we observed in T. formosa, they found that few dominant ant species were the main visitors in most plants. Although competitively superior species are thought to compete for higher quality plants [9, 44, 60], neither the presence nor the frequency of E. tuberculatum were related to nectar availability. Finally, ant composition had no effect on seed set, indicating that even when considering the amount of reward and the ant fauna composition together no differences in seed set due to ant presence were detected. Although plants are expected to invest more in defense of structures directly related to reproduction [e.g., 43, 73,] the presence of nectar secreting structures that attract aggressive 42 ants to developing fruits of T. formosa did not provide protection against pre-dispersal seed predators during the reproductive period studied. Though we cannot rule out these results, a possible explanation is that this absence of protection reflects the great conditionality of facultative mutualisms [28]. In this sense the low interaction intimacy of this facultative ant- plant association, i.e., ants do not rely exclusively on T. formosa for energetic provision, predicts that the interaction outcome can be highly variable [4, 41]. Even though T. formosa apparently invests in high amounts of resources for the mutualists, this ant-plant association does not seems to represent an exception from the notion that facultative ant-plant interactions are highly variable [28]. However, at species level, the prevalence of large ants between most of T. formosa individuals support the existence of links between plant traits and ant composition, since plant species with higher amounts of nectar may better fit the higher energetic requirements of large and competitively superior ants [9]. Our observations could also indicate that the absence of protection may be related to seed predator characteristics which may confer to weevils, protection against ants. On the other hand, at the community level, yet to be explored factors, such as reward quality [38], local ant abundance [22, 74], ant colony characteristics [8, 58, 67], abiotic factors [41] or the presence of alternative energetic sources [35, 58] could also account for variations in ant frequency, composition and ant protective effects [44, 60]. Furthermore, an interesting approach would be to investigate whether this lack of protection entails cost for plants, i.e., balance of cost and benefits of ant-plant association [e.g., 70] and whether these results are consistent in space and time. During the year of study the high fruit set observed in the population of T. formosa could have decreased the importance of seed predators [24]. Indeed, ants might be more effective in conditions of low fruit formation, since fruits can be better defended. In this occasion, the prevalence of active nectaries from ovaries that do not develop into fruits may guarantee the presence of ants even in conditions of low fruit production. Though, here we do 43 not have information on whether the permanence of post-floral nectaries depends on the presence of developing fruits in the same inflorescence. In addition, because pollinator fauna is prone to spatio-temporal fluctuations, in especial long-tongued hawkmoths [75, 76], fruit set can be highly variable, especially in highly specialized long-tubed flowers which depend on specific pollinators [77, 78]. Additionally, pre-dispersal seed predators may also be highly variable in time [79]. Thus, we speculate that ant defense could be more necessary and important in periods of low fruit formation and higher seed predation. 44 References 1. Bentley BL. Extrafloral nectaries and protection by pugnacious bodyguards. Annu. Rev Ecol Syst. 1977; 8: 407–428. 2. Beattie AJ. 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