ii

iii Agradecimentos

Gostaria de agradecer a todos que me ajudaram e incentivaram ao longo desse trabalho:

Em primeiro lugar, aos meus pais, Virgilia e Michael, por me apoiarem em todos os momentos, pelos conselhos e consolos. Aos meus irmãos, Leonardo e Anthony, que mesmo de longe torceram sempre por mim.

Ao Igor, minha força e minha calma, que chegou no meio dessa etapa da minha vida e sempre acreditou em mim.

Ao meu orientador, Prof. Dr. José Roberto Trigo, pela oportunidade e pelos muitos ensinamentos.

Aos membros da banca e pré-banca, Dr. Alberto Arab, Dra. Daniela Rodrigues,

Prof. Dr. Eduardo L. Borba, Prof. Dr. Flavio A. M. dos Santos e Dra. Karina L. Silva-

Brandão pelos comentários e sugestões importantes.

À FAPESP, pela bolsa concedida.

Aos companheiros de laboratório: Kamila, Alberto, Carlos, Marcela, Adriano,

Tamara e Aline, pelas discussões, pela ajuda em laboratório, pela amizade e por tornarem o laboratório um ambiente de trabalho divertido.

Aos amigos que me ajudaram em campo: Alberto, Ana, Henrique, Thiago e também ao funcionário Paulo Roberto Manzani.

Ao funcionário José Carlos da Silva, o Zé, por me ajudar a carregar vasos pesados, quebrar muitos galhos e me ensinar quase tudo o que eu sei sobre cuidados com plantas.

À Tamara pela ajuda indispensável com as extrações na etapa final.

Às amigas da graduação (Ana, Alê, Bruna, Gaby, Karen, Marilini, Stef e Yara) por estarem comigo nos momentos bons e ruins. Aos muitos amigos do Rio, por entenderem

iv minhas longas ausências e sempre me receberem de braços abertos quando eu volto. Aos amigos da pós da Ecologia pelas intensas discussões sobre ecologia ou sobre besteiras, pelos cafés e as cervejinhas.

v

Sumário

RESUMO ...... 7 ABSTRACT...... 9 INTRODUÇÃO GERAL...... 11 REFERÊNCIAS BIBLIOGRÁFICAS ...... 18 CAPÍTULO I: “SIMULATED HERBIVORY IN CURASSAVICA (: ) AFFECTS GROWTH AND REPRODUCTION BUT NOT CARDENOLIDES”...... 22 INTRODUCTION...... 23 MATERIALS AND METHODS...... 26 RESULTS...... 32 DISCUSSION ...... 34 REFERENCES...... 38 CAPÍTULO II: “METHYL JASMONATE AFFECTS GROWTH, REPRODUCTION AND DEFENSES IN (APOCYNACEAE: ASCLEPIADOIDEAE)”47 INTRODUCTION...... 48 MATERIALS AND METHODS...... 50 RESULTS...... 55 DISCUSSION ...... 56 REFERENCES...... 59 APPENDIX 1: CARDENOLIDE EXTRACTION AND LEAD ACETATE CLEANUP ...... 66 APPENDIX 2: LEAF AREA MEASUREMENTS ...... 68 CONCLUSÕES GERAIS ...... 69

vi

Resumo. A produção de defesas contra a herbivoria é essencial para o sucesso reprodutivo das plantas, no entanto pode ser custosa. Esses custos resultam da redução de investimento em outras partes do metabolismo, como o crescimento e a reprodução. Nesse estudo, usamos Asclepias curassavica (Apocynaceae: Asclepiadoideae) como modelo para avaliar como uma planta divide seus recursos entre crescimento, reprodução e defesas. A. curassavica é uma planta anual que possui cardenolidas como defesas. Para verificar como esta responde a herbivoria, simulamos a mesma através de dano artificial (DA) e medimos crescimento (biomassa de folhas e raízes) e reprodução (número de flores, frutos e sementes e biomassa de sementes) em um experimento em longo prazo. Em um experimento em curto prazo, medimos a produção de defesas (concentração de cardenolidas), para avaliar se estas podem estar interferindo no crescimento e reprodução desta espécie. Correlacionamos também, em uma população natural, a concentração de cardenolidas com a percentagem de herbivoria foliar. O hormônio jasmonato de metila

(JM) é usado para induzir compostos do metabolismo secundário em plantas, sem o custo adicional da remoção de tecido fotossinteticamente ativo causada por dano artificial ou natural. Usamos esse composto em ambos os desenhos experimentais acima ao invés de remoção da área foliar para avaliar se havia indução de cardenolidas e seu efeito sobre o crescimento e reprodução de A. curassavica .

Nossos resultados demonstraram uma redução significativa do número total de frutos, sementes e da massa final de raízes em plantas com tratamento de DA em longo prazo. O crescimento do tratamento DA não diferiu do controle, sugerindo crescimento compensatório das folhas à custa do investimento em raízes e em reprodução. Os custos reprodutivos e de crescimento de raízes detectados no experimento de longo prazo podem

7

resultar da diminuição da capacidade fotossintética em plantas danificadas e investimento simultâneo em crescimento compensatório das folhas. No experimento de curto prazo, não houve alteração da razão de indução de cardenolidas sugerindo que o dano artificial não induz defesas nessa espécie. A ausência de correlação entre cardenolidas e porcentagem de dano natural em plantas coletadas em campo pode sugerir dois cenários excludentes: 1. a indução de cardenolidas não seria importante para a defesa de A. curassavica , ou 2. a ausência de correlação, associada a baixa percentagem de herbivoria implicaria em uma defesa constitutiva eficaz contra herbívoros. O tratamento com JM a longo prazo também mostrou uma tendência à redução do crescimento de raízes e redução significativa da biomassa e porcentagem de germinação de sementes. Nas plantas tratadas com JM a curto prazo, houve um aumento significativo de cardenolidas tardio (384 h após tratamento) sugerindo que estas podem contribuir para a redução de aptidão observada no experimento de longo prazo e que existem custos da produção das mesmas.

Dano artificial leva à diminuição da aptidão, através de desvio de investimento em raízes para o crescimento compensatório das folhas. No caso do jasmonato, não houve perda de massa fotossinteticamente ativa (folhas) e a redução do crescimento de raízes pode ser resultado de um efeito direto do tratamento de JM ou indireto causado pela indução de outras partes do metabolismo (p.ex. metabolismo secundário) causada por esse fitohormônio. Experimentos futuros devem comparar os presentes resultados com dano real por um dos herbívoros especialistas para avaliar a eficácia do dano artificial em induzir cardenolidas e o papel dessa indução sobre outras partes do metabolismo da mesma.

8

Palavras-chave: Alocação, Asclepias curassavica , cardenolidas, crescimento compensatório, jasmonato de metila, herbivoria, respostas induzidas.

Abstract. Although the investment in defensive traits against herbivory is essential to the reproductive success of , it may be costly. These costs result from reduced investment in other metabolic functions such as growth and reproduction. In the present study, we used the milkweed Asclepias curassavica (Apocynaceae: Asclepiadoideae) as a model to study how a divides its resources between growth, reproduction and defense.

A. curassavica is an annual weed that uses cardenolides as defenses against herbivory. To evaluate how Asclepias curassavica responds to herbivores, we simulated herbivory by artificial damage (AD) and measured growth (leaf and root biomass) and reproduction

(number of , and seeds and seed biomass) in a long term experiment. We also measured defensive traits (cardenolide concentration) in a short term experiment to verify whether there is an investment in defense that may interfere with growth and reproduction.

We also correlated cardenolide concentration in a natural population with percent leaf damage. As the plant hormone, methyl jasmonate (MJ) is commonly used to induce secondary metabolism in plants without the additional costs of tissue removal, we used this compound in the same experimental designs instead of AD.

Our results from the long term AD treatment showed a significant decrease in final root biomass and in total fruit set and seed number. Plants from the AD treatment did not differ from controls in leaf growth, suggesting that there was a compensatory growth in the former at the expense of root growth. The reproductive and growth costs detected in this experiment may result from reduced photosynthetic capacity in damaged plants and

9

concomitant compensatory leaf growth. In the short term, we found no induction of cardenolides compared to controls, suggesting that artificial damage does not induce defenses. The lack of correlation between cardenolides and percentage leaf damage in plants collected from a natural population suggest two self-excluding scenarios: 1. the induction of cardenolides is not important for the defense of A. curassavica , or 2. the lack of correlation, coupled with low herbivore damage, suggests that this plant has an efficient constitutive defense against herbivores. The long term MJ treatment showed a trend in reduced root biomass and significantly reduced seed biomass and percentage germination.

In the short term MJ treatment we found a significant increase in cardenolide concentration

(after 384 h) suggesting that the production of these defenses is costly and this may have contributed to observed costs in the long term experiment.

Apparently, the artificial damage treatment leads to reduced fitness through reduced root growth which is a consequence of compensatory leaf growth. In the methyl jasmonate treatment, there was no removal of photosythetically active tissue (leaves) and the reduced root growth may be a direct effect of this phytohormone or an indirect effect caused by the induction of other metabolic pathways (such as secondary metabolism) caused by this hormone. Future experiments should compare the present results with natural damage by specialist herbivores to evaluate the efficiency of artificial damage in inducing cardenolides and the role of the induction of these substances on other metabolic functions.

Keywords: Allocation, cardenolides, compensatory growth, herbivory, induced responses, milkweed.

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Introdução Geral

Plantas sofrem uma variedade de pressões seletivas bióticas e abióticas que podem afetar sua aptidão (Cipollini e Levey 1997, Belsky 1993). Dentre essas pressões, os herbívoros são os que causam o maior impacto negativo na aptidão e na sobrevivência das plantas e, dentre esses, insetos fitófagos chegam a consumir mais de 10% da produção anual de plantas em sistemas naturais (Coley et al. 1985). Potencialmente, qualquer órgão da planta é alvo para um inseto fitófago e os danos causados por estes podem afetar o crescimento e a reprodução e aumentar a mortalidade das mesmas (Coley et al. 1985).

Para escapar dessas fortes pressões seletivas, as plantas desenvolveram um leque de componentes de defesa ( sensu Karban e Baldwin 1997 1) que reduzem as perdas de aptidão causadas por herbivoria comparado a plantas não defendidas (Coley et al. 1985, Karban e

Baldwin 1997). Esses componentes são convencionalmente divididos em defesas físicas

(p.ex: espinhos e tricomas não glandulares), defesas químicas (p.ex: substâncias tóxicas, deterrentes e repelentes) e defesas bióticas (p.ex: atração de inimigos naturais de insetos fitófagos). Embora essa divisão facilite a compreensão e o estudo das defesas, uma defesa pode pertencer simultaneamente a mais de uma “classe” de defesa (como p.ex., tricomas glandulares ou o uso de pistas químicas para atrair parasitóides de insetos fitófagos). Além disso, a maioria das plantas possui uma combinação de diferentes componentes de defesa, contra diversas guildas de herbívoros (Price 1986, Karban e Baldwin 1997).

1 O conceito de defesa deve ser definido do ponto de vista da planta, em situações onde o investimento em defesas provê benefícios para uma planta comparada a outro indivíduo da mesma espécie que não investiu nessas defesas. Um componente de defesa, para agir como tal, deve trazer algum benefício para a aptidão da planta quando esta for atacada, não importando se essa defesa afeta ou não a aptidão do herbívoro que está atacando a planta (Karban e Baldwin 1997).

11

A produção dessas defesas contra herbívoros pode ser custosa para a planta, por desviar recursos que seriam utilizados em outras funções metabólicos, como o crescimento e a reprodução (Herms e Mattson 1992). Dentro desse contexto, o surgimento de defesas induzidas 2 deve ter sido selecionado como um mecanismo de redução de custos da produção de defesas em ambientes onde o ataque por herbívoros é fraco ou imprevisível

(Karban e Myers 1989, Karban e Baldwin 1997).

Defesas induzidas contra a herbivoria podem incluir indução de substâncias químicas tóxicas como, por exemplo, a nicotina no tabaco (Baldwin et al. 1990), induções físicas como o aumento da densidade de tricomas não glandulares no rabanete selvagem

(Agrawal 1999), a produção de voláteis que atraem parasitas de herbívoros no tomate

(Thaler 1999) ou aumento da produção de recompensas, como o néctar, que atrai formigas predadoras em Macaranga tanarius (Heil et al. 2001).

A habilidade de responder apenas após o dano por herbivoria permite que plantas invistam pouco ou nenhum recurso em defesas antes do ataque. Como conseqüência, elas podem utilizar esses recursos para outras funções metabólicas, incluindo crescimento e reprodução. (Karban e Meyers 1989, Karban e Baldwin 1997 e referências ali contidas). A indução de defesas também foi sugerida como uma forma de reduzir metabólitos secundários disponíveis para seqüestro por especialistas (Malcolm 1995).

Tradicionalmente, as substâncias químicas do chamado metabolismo secundário são consideradas grandes responsáveis pela defesa de plantas. Essas substâncias, assim

2 Respostas induzidas podem ser definidas como mudanças no fenótipo de plantas que ocorrem após sofrerem herbivoria ou estresse (Karban e Baldwin 1997). Se essas respostas diminuem o impacto negativo da herbivoria sobre a aptidão da planta, elas são consideradas defesas induzidas, em oposição a defesas constitutivas que estão presentes nos tecidos antes do dano (Karban e Meyers 1989, Karban e Baldwin 1997).

12

denominadas por não pertencerem ao metabolismo essencial para o crescimento e reprodução das plantas (denominado metabolismo primário), influem fortemente no padrão de utilização de recursos por insetos fitófagos (Fraenckel 1959). Erlich e Raven (1964) sugeriram que substâncias do metabolismo secundário foram as maiores responsáveis pela coevolução de insetos fitófagos especialistas com suas plantas hospedeiras. Os autores se baseiam no fato destes insetos apenas consumirem grupos de plantas que produzem a mesma classe de metabólitos secundários e de serem capazes de detoxificar essas substâncias, muitas vezes seqüestrando-as e utilizando-as para defesa contra seus inimigos naturais.

Atualmente, sabemos que diversas substâncias do metabolismo secundário podem ter outras funções além de defesa, como proteção contra radiação UV, suporte estrutural e atuação contra patógenos, além da atração de polinizadores e dispersores (Herms e Mattson

1992 , Cipollini e Levey 1997, Baldwin 1998). Outros estudos indicam que o metabolismo primário também pode sofrer alterações após herbivoria e influir na resposta induzida das plantas. Essas influências podem se expressar através de alterações do conteúdo nutricional dos tecidos para o herbívoro ou através de ações sinérgicas ou antagônicas com as substâncias secundárias (Berenbaum 1995, Zangerl e Berenbaum 2004, Schwahtje e

Baldwin 2008).

Dentro desse contexto, esse estudo procurou avaliar a resposta de Asclepias curassavica Linnaeus (Apocynaceae: Asclepiadoideae) quanto a crescimento e indução de defesas quando submetida a herbivoria simulada. Asclepias curassavica (Figura 1) é uma planta anual neotropical que se caracteriza por ser um arbusto pouco ramificado, atingindo entre 1,40m e 1,80m de altura e que ocorre principalmente na beira de rios, em clareiras,

13

pastagens e áreas com distúrbio antrópico (Woodson 1954, Lorenzi 2000). Esta espécie produz inflorescências em umbela ao longo de todo o ano (Bierzychudek 1981) que possuem flores com uma coloração vermelha e amarela vistosa e néctar copioso, atraindo diversos visitantes florais (Wyatt e Broyles 1994). Assim como outras plantas do gênero

Asclepias sp., A. curassavica apresenta cardenolidas e látex, que são considerados estruturas de defesa (Martel e Malcolm 2004, Van Zandt e Agrawal 2004, Agrawal et al.

2008).

Cardenolidas são glicosídeos esteroidais (Figura 2), que podem também estar presentes nas plantas em formas não glicosiladas (geninas), os quais inibem o funcionamento da bomba Na+/K+ presente nos nervos e músculos de animais, além de ter alto potencial emético (Brower 1969, Brower et al. 1982, Malcolm 1991, 1995, Martel e

Malcolm 2004). A porção esteroidal das cardenolidas apresenta uma lactona insaturada de

5 membros no C 17 . Um ou mais açúcares podem se ligar à porção esteroidal através de um grupo hidroxila no C 3, e entre os mais de 20 tipos de açúcares que já foram isolados de cardenolidas só alguns, como glicose e frutose, são de distribuição ampla entre as plantas

(Malcolm 1991). Em A. curassavica , cardenolidas já foram detectadas no citoplasma da epiderme foliar e colênquima adjacente, nos laticíferos pressurizados e em elementos do floema de folhas e caule (Malcolm 1991). O látex dessas plantas contem cardenolidas em concentrações mais altas que nos tecidos, além de proteases, substâncias adesivas e precursores de borracha (Seiber et al. 1982, Zalucki et al. 2001, Agrawal et al. 2008). As cardenolidas encontradas em A. curassavica , assim como outras espécies de Asclepias, apresentam o açúcar ligado nos C 2 e C3 (2,3-dihidroxicardenolida, Groenveld et al. 1994,

Figura 2).

14

Através de experimentos de herbivoria simulada por dano artificial medimos o efeito do dano sobre o crescimento (ganho massa foliar e de massa de raízes) e a aptidão

(quantidade de flores, frutos, sementes e massa de sementes) de A. curassavica em um experimento de longo prazo. Além disso, em um experimento de curto prazo, verificamos se havia indução de defesas (concentração de cardenolidas) nesta espécie após dano artificial. Esse experimento foi realizado para verificar se a indução de defesas poderia estar relacionada às respostas observadas no experimento de longo prazo. Como o hormônio elicitor jasmonato de metila (JM) é geralmente usado para induzir compostos do metabolismo secundário em plantas (Baldwin 1998, Heil 2004), esse composto é útil para verificar o papel desses compostos sem o custo adicional da remoção de tecido fotossinteticamente ativo. Partindo do pressuposto de que a produção de cardenolidas pode afetar a alocação de recursos para outras funções metabólicas, utilizamos JM em ambos os desenhos experimentais acima ao invés de remoção da área foliar, para verificar as respostas de A. curassavica sem a influência de outros fatores que deve acompanhar a remoção de tecido foliar (como p.ex.: redução da capacidade fotossintética).

Correlacionamos também, em uma população espontânea de A. curassavica , a concentração de cardenolidas com a percentagem de dano por herbivoria natural.

15

Figura 1. Asclepias curassavica L. na Reserva Municipal Serra do Japi. Foto: M.A. Stanton

16

O O

17 R OH H CHO H O 2 H OH 3 OO HH

açúcar

Uscaridina R = O

S Voruscarina R =

N

S Uscarina R =

N

Figura 2. Cardenolidas mais abundantes em Asclepias curassavica (adaptado de Groenveld et al. 1994).

17

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Capítulo I: “Simulated herbivory in Asclepias curassavica (Apocynaceae:

Asclepiadoideae) affects growth and reproduction but not cardenolides”

22

Introduction

Plants suffer a wide range of selective pressures from both biotic and abiotic origin that can affect their fitness (Cipollini and Levey 1997, Belsky1993). Among which, herbivores, in particular phytophagous insects, account for the greatest negative impact on fitness (Coley et al. 1985).

The impacts on plant fitness caused by herbivores include modifications in plant growth, reproduction and maintenance (Crawley 1983). One example of these modifications is the increase in mortality and decrease in vegetative growth rates of

Aristolochia reticulata after herbivory by Battus philenor (Rausher and Feeny 1980). In other species ( Anthurium schlechtendalii ), both natural and artificial herbivory did not affect vegetative growth but significantly reduced floral display (Canto et al. 2004). Beetle herbivory delayed flowering and reduced biomass in Lythrum salicaria and increased levels of herbivory were significantly correlated with decrease in inflorescence length and plant height (Schat and Blossey 2005).

The studies above exemplify some of the consequences of herbivore pressure, against which plants have evolved several defensive traits. The production of such traits, however, incurs in costs, as a plant must expend resources to create and maintain defenses that could otherwise be used for growth and reproduction (see Herms and Mattson 1992 and references therein). Plastic responses can minimize the costs of defenses. In this way, defensive traits are expressed or increased only when herbivores are present and thus decrease negative fitness impacts of attacks on plants (induced defenses - see Karban and

Baldwin 1997). This ability to respond only after herbivore damage, allows plants to invest

23

in little or no defense before attack and consequently use available resources for other metabolic functions, such as growth and reproduction. Therefore, induced defenses are thought to have evolved as a mechanism of reducing defense costs when herbivores are absent and attack is unpredictable (Karban and Meyers 1989, Karban and Baldwin 1997 and references therein) and also as a mechanism of reducing secondary chemicals available for sequestration by specialist herbivores (Malcolm 1995).

In this context, we chose to study the impact of herbivory in a milkweed, Asclepias curassavica Linnaeus (Apocynaceae: Asclepiadoideae) to investigate two questions: does

A. curassavica respond to herbivory by reducing its fitness? If yes, in order to minimize decreases in fitness, does A. curassavica induce defensive traits when it loses tissues to herbivory?

All milkweeds produce cardiac glycosides (cardenolides), bitter-tasting steroids that have toxic effects on most by disrupting the sodium and potassium flux in cells

(Malcolm 1991). These compounds are suggested to act as an anti-herbivore defense against generalists and specialists insects (Zalucki and Brower 1992, Dussourd and Hoyle

2000, Zalucki et al. 2001, Van Zandt and Agrawal 2004). Cardenolides are present constitutively in milkweed and their production is also known to be induced following damage in A. syriaca by the foliage-chewing Danaus plexippus (Malcolm and Zalucki

1996, Mooney et al. 2008) and in A. curassavica by the phloem-sucking herbivore (Martel and Malcolm 2004). However, herbivory by A. nerii and the foliage-chewing beetle Tetraopes tetraophthalmus did not induce cardenolides in A. syriaca (Mooney et al.

2008).

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In the southeastern region of , the main herbivores found feeding upon A. curassavica are larvae of the southern monarch Cramer (:

Nymphalidae), the Danaus gillipus Cramer, the aphid Aphis nerii Boyer de

Fonscolombe (Hemiptera: Aphididae) and the Oncopeltus fasciatus

Dallas (Hemiptera: Lygaeidae) (Stanton, personal observation). All of these herbivores are aposematic specialists that are able to sequester cardenolides from their host milkweeds and use them for their own defense (Brower 1969, Rothschild and Marsh 1978, Brower et al.

1982, Berenbaum and Miliczky 1984, Cohen 1985, Malcolm 1995, Martel and Malcolm

2004).

To answer the first question we carried out a long term experiment of simulated herbivory (clipping of 50% of foliar area with scissors) where we measured rates of increase in leaf tissue as well as investment in reproductive tissues. Clipping was favored over other methods due to the ease in reproducing the amount of leaf damage and because it allowed the control of timing and location of damage on plants (Van Dam et al. 1993, Schat and Blossey 2005). Some authors have shown that artificial damage with scissors does not increase plant jasmonic acid expression or induced defenses to the same levels as natural herbivory without the addition of herbivore oral secretions (McCloud and Baldwin 1997).

This may occur due to the small amount of plant tissue injured by clipping in comparison to feeding by a chewing herbivore (Agrawal 2000), or to the timing of artificial herbivory

(Van Dam et al. 1993). Artificial damage was shown to cause no induction of defenses in another milkweed, A. syriaca (Mooney et al. 2008). However, other studies have shown a significant increase in secondary metabolism of several plants after clipping when compared to undamaged controls (Karban et al. 2004, Riipi et al. 2005, McCall 2006, Alves

25

et al. 2007), including another study with A. syriaca (Malcolm and Zalucki 1996).

Although mechanical damage does activate wound responses in plants, these responses have been reported to overlap with only part of the responses activated by chewing and for this reason it has been suggested as a poor substitute for natural herbivory (Walling 2000). However, in the cases where artificial damage does induce secondary metabolism, even if at lower levels than natural herbivory, it provides us with a useful tool for analyzing effects of induced secondary metabolism on other plant traits

(such as growth and reproduction).

Since we are unaware whether the artificial damage used in the long term experiment induces defensive traits, we investigated this question in a short term experiment, also using clipping of 50% of foliar area with scissors. In this experiment we verified whether this kind of damage induced the major milkweed defensive chemicals, cardenolides. Finally we discussed our results in the light of herbivory and cardenolide contents in natural populations of A. curassavica .

Materials and Methods

Study organism

A. curassavica is a neotropical annual shrub that occurs from the south of the United

States to , although its exact origin in the Americas is still controversial

(Woodson 1954, Wyatt and Broyles 1997). This species is a weed that thrives in sunny, humid and disturbed sites, such as pastures, and close to river beds (Wyatt and Broyles

1997, Lorenzi 2000). A. curassavica has several types of cardenolides in its latex and

26

tissues (Seiber et al. 1982, Groenveld et al. 1994) and is considered to have a medium to high range of cardenolide concentration in tissues (0.29 ± 0.07 % of dry weight; mean ± se) and low latex secretion following damage (0.74 ± 0.27 mg) compared to other milkweeds

(Agrawal and Fishbein, 2006). Latex is stored under pressure in specialized ramified cells

(laticifers) and contains a higher concentration of cardenolides than tissues, being released upon rupture of laticifers (Seiber et al. 1982, Dussourd and Hoyle 2000).

A. curassavica plants used in all experiments were raised from seeds collected from natural populations in the region of Campinas and Jundiaí, state of São Paulo, Brazil.

Plants for long term experiments were kept in 20L pots with a 3:1 mixture of soil and organic substrate for seedlings (Genebom) in a greenhouse at the Departmento de Zoologia,

Instituto de Biologia, Universidade Estadual de Campinas, under natural temperatures and were watered daily. For short term experiments, plants were kept in 1.5L pots with soil as above, were watered daily and were sprayed with Vertimec (Syngenta) and Benlate

(Dupont) to control aphids ( A. nerii ) and fungi two weeks before the beginning of the experiment.

Growth and reproductive responses in Asclepias curassavica after artificial damage

To evaluate if growth and reproduction varies after leaf loss by herbivores, we used plants that had not reached reproductive phase (approx. 5 months old) growing in 20L pots.

Plants used in this trial had 10.6 ± 3.0 leaves (means ± SD) and 477.4 ± 215.1 mg estimated biomass (means ± SD). Forty six plants were randomly assigned to one of two treatments:

1) heavy loss of 50% leaf biomass by artificial damage every two weeks and 2) undamaged control.

27

Total leaf biomass was estimated at the start of the trial (T 0 = March 16). To estimate leaf mass, we identified each leaf with a waterproof marker and measured expanded leaf lengths with a ruler along the central vein (not including petiole) in 300 A. curassavica leaves from 22 individuals. We correlated the leaf length with dry weight as follow: Leaf biomass (g) = 0.001 x (leaf length in cm) 1.92 (r 2 = 0.91). All fully expanded leaves in the artificial damage treatment were cut with scissors to simulate 50% herbivory

(clipping). Leaves were clipped halfway across their length, perpendicular to the central vein. The amount of damage was calculated for each leaf as the length along the central vein corresponding to half of the estimated leaf biomass. To estimate leaf growth, we identified and measured the biomass of new leaves produced in each sampling date (every 2 weeks). After measurement, new leaves were immediately clipped (50% leaf loss) in the artificial damage treatment. Only new unmarked leaves were measured in sampling dates after T 0.

Since there was no significant difference in total leaf biomass at the beginning of the experiment between plants before the application of treatments (means ± SD , undamaged:

493.9 ± 206.6 mg; damaged: 460.9 ± 226.7 mg; t test, t = 0.515, df = 44, p = 0.61), we used cumulative leaf biomass gain at the end of the experiment to test for differences in leaf growth between damaged and undamaged plants. We calculated cumulative leaf biomass gain as the sum of total leaf biomass at T0 and the biomass of new leaves produced in all sampling dates per individual. To estimate reproductive output, and fruit number were recorded every two weeks and seeds collected during the trial were counted and weighed in a digital analytical balance (± 0.1 mg). The data for each variable in each treatment were pooled for statistical analysis at end of experiment.

28

After a total of 13 sampling dates (Mar - Oct 2005 - data was sampled only once in the months of June and September, in one month intervals), the trial was interrupted and plants were removed from pots with care as to not damage roots. After removal from pots, we shook roots to dislodge excess soil particles without washing them and dried plants in a force-draught oven at 70ºC for 2 days. Any remaining soil was gently shaken off dry roots and these were weighed in a digital analytical balance to obtain final root dry weight.

The comparison of cumulative leaf biomass gain, root dry biomass, flower, fruit, and seed, and mean seed biomass between both treatments were analyzed with t-test; data were ln or ln (x+1) transformed when necessary to conform to assumptions of normality and homoscedasticity (Zar 1999). One individual from the undamaged treatment did not produce seeds and was excluded from seed analysis.

Plants used in the long term experiment had a smaller estimated leaf biomass than plants used in the short term experiment (see below), even though they were older. We suggest that this difference may be due to differences in seed quality used in both experiments or to increased leaf abscission in plants from the long term experiment. The latter may have occurred due to the plants used in the long term experiment being exposed to natural environmental conditions during the colder dry season in Campinas, with varying temperatures and light conditions, whereas plants used in the short term experiment were kept under controlled temperatures and constant light regime. Environmental stresses such as extreme temperatures and drought have been associated with increased leaf senescence in plants (Munné-Bosch and Alegre 2004). This difference was unimportant for the questions approached in each experiment, since data were collected and analyzed separately.

29

Do induced responses occur following artificial damage?

To assess changes in cardenolide content after artificial damage simulating herbivory, we used plants in the vegetative phase, approximately 10 weeks old and with

17.0 ± 5 .7 leaves (means ± SD) and 1179.8 ± 328.0 mg estimated biomass (means ± SD).

Plants were maintained in an acclimatized chamber (L12:D12, 25±3°C, and about 50% RH) and watered daily. Artificial damage was applied as above. Eighty plants had the distal half of all their leaves removed with a pair of scissors as above at time zero. The remaining proximal half of all leaves in the replicates (individuals) was harvested in each time: 0, 1,

24, 48, 96, 192 or 384 hours. These sampling times were chosen in order to assess whether rapid chemical induction and relaxation compared to time zero occur after damage, as has been shown to occur after hours or days in several plant species (Van Dam et al. 1993,

Malcolm and Zalucki 1996, Alves et al. 2007). Harvested tissues were immediately dried in an oven at 70ºC for 2 days and stored for chemical analysis. We clipped 12 replicates for each of the times 0, 1 and 24 h, and 11 replicates for each of the other times (48, 96, 192 and 384 h). Cardenolides were extracted from dried leaves (100 mg dry weight per sample) and quantified following Frick and Wink (1995, see Appendix 1 for details). Because the distal half of leaves were used as controls of the proximal halves in each time, it was necessary to test for possible natural differences in cardenolide concentrations between distal and proximal leaf halves; this was done at time 0 and the differences in cardenolide levels between distal and proximal half of the leaves were analyzed by paired t-test after ln

(x+1) transformation of cardenolide concentrations (Zar 1999).

Induction ratios express variation in induced responses after artificial damage by comparing levels of target chemicals before and after damage in the same individual or

30

clones (van Dam et al. 1993, Alves et al. 2007). We calculated induction ratios for cardenolides as the ratio of chemical concentrations extracted from the pooled proximal leaves with artificial damages by the concentration in pooled undamaged control (distal leaves) for each replicate at any time. We compared changes in induction ratios between times after damage by a one-way Anova with ln (x+1) transformed data (Zar 1999).

Correlation of defensive traits with herbivory in natural populations

Individual A. curassavica plants from natural populations were collected in the

Serra do Japi Reserve, near the DAE dam (23º13’21.9”S 46º58’14”W). This reserve is a fragment of semideciduous mesophyl forest in Jundiaí county, São Paulo state, Brazil. In this area, A. curassavica occurs spontaneously along the main road of the reserve and during this study was observed being attacked by three specialist herbivores: D. erippus and D. gillippus caterpillars (: ) and the aphid A. nerii (Hemiptera:

Aphididae). Plants were brought to the lab where they were pressed and oven-dried at 70ºC for 2 days. Sixteen plants were collected in one field trip in April 2007 and 18 on two field trips in March 2008. Chosen plants had visible signs of herbivory and were collected along the reserve’s entrance road that leads up to the DAE dam. As A. curassavica is an annual plant, plants collected in different years were considered separate individuals.

Herbivory was estimated as the percentage loss of leaf area (see Appendix II for details) and correlated to total leaf cardenolide concentration measured as above.

Populations were compared in respect to their herbivory percentages and cardenolide concentration using t-test for each parameter and the correlation between them was analyzed with a Pearson correlation (Zar, 1999).

31

Results

Growth and reproductive responses in Asclepias curassavica after artificial damage

The plants submitted to artificial damage treatment every two weeks showed no significant difference in cumulative leaf biomass gain at the end of the experiment when compared to undamaged plants (t-test, t = 0.297, df = 44, p = 0.77, Table 1). Root biomass at the end of the experiment was significantly smaller in the artificial damage treatment when compared to undamaged plants (ln transformed data, t test, t = -2.32, df = 44, p <

0.01, Table 1). As for reproductive traits, we found no difference in total flower set between treatments (ln transformed data, t-test, t = 1,03, df = 44, p = 0.31, Table 1), but the artificial damage plants had a significantly smaller fruit set when compared to undamaged ones (ln transformed data, t-test, t = - 3.52, df = 44, p < 0.01, Table 1). Mean seed biomass did not differ between treatments (t test, t = - 0.95, df = 42, p = 0.35, Table 1); on the other hand, the undamaged treatment produced a significantly larger number of seeds compared to artificially damaged plants (ln transformed data, t test, t = 2.38, df = 42, p = 0.02, Table

1). The reduction in seed number in artificially damaged plants apparently is a direct result of decreased fruit set, as there was no difference in mean number of seeds per fruit between both treatment groups ( means ± SD , undamaged: 93.1 ± 18.1 seeds/fruit; artificially damaged: 90.7 ± 17.3 seeds/fruit; t test, t = 0.45, df = 43, p = 0.65).

Do induced responses of cardenolides occur following artificial damage?

The concentration of cardenolides in intact leaves of A. curassavica was 199.2 ±

134.9 µg/0.1 g leaf dry weight (mean ± sd, n =23). In proximal halves (240.9 ± 186.5

32

mg/0.1 g , n = 23 ) was significantly higher than in distal halves (157.4 ± 111.7 mg/0.1 g, n =

23) (ln x+1 transformed data, t-paired test, t = 2.47, df = 22, p = 0.02). The ratio between proximal and distal concentration of cardenolides in leaves was 1.99 ± 1.96 mg/0.1 g and it was not significantly different of the hypothesized mean of 2.0 (ln x+1 transformed data, one sample t-test, t = -1.37, df = 22, p = 0.19) . Induction ratio of cardenolides, however, did not show significant differences between times after damage (ln x+1 transformed data, one-way Anova, F 6,73 = 0.57, p = 0.76, Figure 1). Pooling all times, cardenolide concentrations showed an wide range (from 21-494 µg/0.1g with a coefficient of variation of 64.6% in distal half leaves and 31-874 µg/ 0.1g, CV = 65.2% in proximal half leaves).

Correlation of defense characters with herbivory in natural populations

We found no significant correlation between cardenolide concentration and percentage herbivory, expressed as area of leaf loss, for the two samples of a natural population of A. curassavica collected in 2007 (Pearson correlation: r = 0.165, p = 0.57) and in 2008 (r = -0.252, p = 0.35). Although samples showed a significantly different percentage of herbivory when compared to each other, there was no significant difference between cardenolide concentrations in both years (Table 2). The frequency of herbivores observed in both years was low (2007: 0.4 individual/plant for Aphis nerii and 1.1 eggs/plant and no larvae for Danaus spp.; 2008: 1.8 individual/plant for Aphis nerii , 0.1 eggs/plant and 0.4 larvae/plant for Danaus spp.).

33

Discussion

Our results from the long term experiment showed no significant differences in the amount of cumulative leaf mass gain of artificially damaged A. curassavica plants compared to undamaged plants. The removal of 50% leaf tissue may be expected to alter the photosynthetic balance and consequently the growth of plants in the long term experiment. Although we did not measure photosynthetic capacity in this study, studies using natural herbivory by D. plexippus and 50% artificial herbivory (clipping) have shown a significant decrease in leaf photosynthetic capacity in injured A. curassavica and A. syriaca leaves, respectively (Delaney 2008, Delaney et al. 2008). In this way, the observed maintenance of similar levels of leaf growth in plants that had lost 50% of their photosynthetic tissues when compared with undamaged plants was considered as compensatory leaf growth and may be due an acquisition of resources from sources other than photosynthesis. Since root mass did not increase in artificially damaged plants in the same extent as in undamaged plants, root growth can represent a potential source for the observed leaf growth sink.

As for reproductive traits, we found no difference in flower set and mean seed biomass between treatments. We did, however, find a smaller fruit set and seed number in the artificially damaged plants compared to undamaged plants, suggesting reduced reproductive output caused by artificial herbivory. Reduced fruit set and biomass was also found in another milkweed, A. syriaca , after root herbivory by specialist Tetraopes tetraophtalmus larvae (Agrawal 2004), although it was not tested whether investment in defenses also influenced fitness. A. syriaca is a perennial shrub, in contrast with A.

34

curassavica which is described as an annual (Woodson 1954), and therefore reduced investment in seeds can have a higher negative impact on fitness in the latter, which is a semelparous plant that will have no future reproductive season (Hirose et al. 2005).

In the short term experiment, we found no differences between cardenolide concentrations in different sampling times after damage. A possible explanation for this lack of induction is that cardenolides are not elicited by artificial herbivory in A. curassavica . Since induced defenses are predicted to be selected when herbivores are absent or attack is unpredictable (Karban and Baldwin 1997), the application of constant herbivory during a long period in this study (in the long term experiment) may represent a scenario where induced defenses are not favorable. Previous studies with the common milkweed, A. syriaca have demonstrated varying results: a significant increase in cardenolides was reported after artificial damage by clipping (Malcolm and Zalucki 1996) and also a significant decrease in photosynthetic capacity after artificial damage (Delaney et al. 2008). Another report found that monarch larval performance on artificially damaged

A. syriaca was lower than on undamaged controls and equal to plants damaged by monarchs, suggesting that artificial damage does elicit defenses, although defensive traits were not measured in the study (Van Zandt and Agrawal 2004). However, another report showed no cardenolide induction in this same species after artificial damage (Mooney et al.

2008). These apparently contradictory results for A. syriaca suggest that the effects of artificial damage vary even within species in as with feeding by different herbivores.

The absence of a correlation between cardenolide concentration and percentage of herbivory in a natural environment observed in this study suggests that cardenolides do not vary with different levels of herbivory in Asclepias curassavica in the studied field site.

35

These results are not in agreement with the induction of cardenolides by the phloem feeding

A. nerii previously observed in this milkweed (Martel and Malcolm 2004), however we know of no studies on the effects of chewing herbivores and leaf removal on A. curassavica cardenolides. It is possible that the high constitutive cardenolide level of our field populations provided an effective defense, since observed herbivory percentage was low.

A high constitutive defense would explain the lack of induction observed in the short term experiment and in the field collected individuals and the lack of correlation between cardenolides and percentage herbivory in the latter.

Another possibility is that resistance traits not measured in this study are important for defense in A. curassavica . In a study of the effects of a community of specialized herbivores on A. syriaca , Agrawal (2005) showed that no single resistance trait explained variation in damage and investment in one type of defense was often constrained by multiple opposing selective pressures. Although cardenolides negatively affected one of the studied herbivores ( D. plexippus ), there was no correlation between cardenolides and previous damage, probably due to effects of early season damage and correlations in plant susceptibility to different herbivores (Agrawal 2005).

As A. curassavica is subject to herbivory by diverse specialist herbivores (Brower

1969, Cohen 1985, Malcolm 1991, Martel and Malcolm 2004), a further possibility is that the lack of cardenolide induction observed in laboratory and field conditions is due to investment in tolerance rather than in defense against damage. The observed compensatory leaf growth at the expense of root growth suggests that A. curassavica is able to shift resource allocation from roots to aboveground growth, which is an important prerequisite for tolerance (Strauss and Agrawal 1999, Stowe et al. 2000, Thompson et al. 2003).

36

Furthermore, the costs of investing in defenses (cardenolides) may not be an advantage when faced with specialist herbivores that are capable of sequestering and deactivating these defenses (Malcolm 1995, Dussourd and Hoyle 2000). This hypothesis, however, remains to be tested.

Previous reports have shown a similar pattern in cardenolide content in Asclepias species as found in this study. Malcolm (1991) reviewed several studies in 33 Asclepias species and reported a coefficient of variation of 43% in A. curassavica with a cardenolide concentration range of 232-1641 µg/ 0.1 g dry leaf tissue. Our results showed a high coefficient of variation in cardenolide concentration (64.6% in distal half leaves and 65.2% in proximal half leaves) and other reports have also found high intraspecific variation in cardenolide and latex content in Asclepias sp. (Agrawal 2004, Agrawal and Fishbein 2006).

In some plant species, phenotypic plasticity can create a large norm of reaction in induced responses, without altering mean population fitness (Griffith and Sultan 2006). In such cases, a genotypic approach (ex: family nested design) is more appropriate for detecting patterns without the influence of environmental factors that occurs in phenotypic traits

(Koricheva 2002, Agrawal 2005) and it would be interesting to analyze defensive traits and tolerance in A. curassavica using full-sib families.

Resource allocation to vegetative functions and defense in response to herbivory has been associated with reproductive costs that vary among different plant species (Hendrix

1988, Obeso 2002). The observed decrease in fitness of A. curassavica in our long term experiment may result from resource investment in compensatory leaf growth after artificial damage. The maintenance of leaf growth at expense of root biomass reinforces our suggestion. The role of cardenolide induction in response to simulated herbivory seems

37

to be unimportant, and this finding is supported by the lack of correlation between herbivory and cardenolide content in the field population.

Comparisons of our data with experiments involving natural herbivory by the specialist D. erippus should show if clipping is an appropriate simulation of herbivore damage. Natural herbivory would also give a measure of the relative importance of defensive traits, regrowth and the fitness consequences of these responses in local populations of Asclepias curassavica .

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Zalucki MP, Malcolm SB, Paine TD, Hanlon CC, Brower LP, Clarke AR (2001) It´s the

first bite that counts: Survival of first-instar monarchs on milkweeds. Austral

ecology 26: 547-555. van Zandt PA, Agrawal AA (2004) Specificity of induced responses to specialist herbivores

of the common milkweed Asclepias syriaca . Oikos 104: 401-409.

Zar JH (1999) Biostatistical analysis. Prentice – Hall. New Jersey, USA.

44

Figure 1. Induction ratio of cardenolides (mean ± sd of ln (x+1) transformed data) in different times after artificial damage in Asclepias curassavica leaves.

1.5

1.0

0.5

0.0

Ln (inductionLn rateofcardenolides +1) 0 1 24 48 96 192 384 Time (hours)

45

Table 1. Growth and reproduction of Asclepias curassavica plants without damage and plants with artificial damage. All values are means ± standard deviation. For statistics see

Results.

Without Damage Artificial Damage p- value

Cumulative leaf biomass gain (g) 7.3 ± 3.0 7.5 ± 2.3 0.77

Number of flowers 97.4 ± 43.4 80.7 ± 30.1 0.31

Number of fruit 14.6 ± 4.1 9.1 ± 6.1 < 0.01

Number of seeds 655.1 ± 408.0 358.9 ± 189.7 0.02

Seed biomass (mg) 2.98 ± 0.42 2.86 ± 0.39 0.35

Root biomass (g) 7.81 ± 4.79 4.82 ± 3.25 0.02

Table 2. Comparison of percentage herbivory and cardenolide concentration (means ± standard deviation of µg/100mg equivalents of digitoxin) between populations of Asclepias curassavica collected in the Serra do Japi Reserve.

2007 2008 Statistics

Mann Whitney,

Herbivory (%) 1.46 ± 2.05 10.21 ± 7.69 U = 18.0, Z = - 3.91,

p < 0.01

Cardenolide t test, t = 1.54, d.f. = 28, 139.4 ± 56.25 106.6 ± 59.71 concentration p = 0.13

46

Capítulo II: “Methyl jasmonate affects growth, reproduction and defenses in

Asclepias curassavica (Apocynaceae: Asclepiadoideae)”

47

Introduction

Plants suffer a wide range of selective pressures from both biotic and abiotic origin that can affect their fitness (Cipollini and Levey 1997, Belsky1993). Among which, herbivores, in particular phytophagous insects, account for the greatest negative impact on fitness (Coley et al. 1985). To escape from these selective pressures, plants have developed a wide array of defense components that reduce fitness losses caused by herbivory (Coley et al 1985, Karban e Baldwin 1997).

Changes in plant phenotypes expressed after damage are termed induced responses.

Induced defenses are the responses that decrease the negative fitness impacts of herbivore attacks ( sensu Karban and Baldwin 1997). Plants induced defenses are thought to have evolved as a mechanism of reducing defense costs when herbivores are absent and attack is unpredictable (Karban and Meyers 1989, Karban and Baldwin 1997). Defensive chemicals are costly because resources allocated to defense cannot be simultaneously allocated to other metabolic functions, such as growth and reproduction (Zangerl and Bazzaz 1992,

Herms and Mattson 2002).

In a previous study we chose a milkweed, Asclepias curassavica Linnaeus

(Apocynaceae: Asclepiadoideae) (see Chapter I), to evaluate whether this species responds to artificial herbivory by reducing growth or fitness, and increasing a defensive trait, cardenolides. Cardenolides are bitter-tasting steroids that have toxic effects on most animals by disrupting the sodium and potassium flux in cells (Malcolm 1991). These compounds are present constitutively in milkweeds and their production is also known to be induced following damage in A. syriaca by foliage-chewing Danaus plexippus (Malcolm and Zalucki 1996, Mooney et al. 2008) and in A. curassavica by phloem-sucking

48

herbivores such as Aphis nerii (Martel and Malcolm 2004). A. nerii and the foliage- chewing beetle Tetraopes tetraophthalmus did not induce cardenolide increase in A. syriaca

(Mooney et al. 2008). These compounds are suggested to act as an anti-herbivore defense against generalists and specialists insects (Zalucki and Brower 1992, Dussourd and Hoyle

2000, Zalucki et al. 2001, Van Zandt and Agrawal 2004).

Our previous results (see Chapter I) showed maintenance of leaf growth at the expense of root growth in artificially damaged A. curassavica plants (clipping of 50% leaf mass). This investment in compensatory leaf growth apparently caused the observed decrease in fitness (fruit and seed set) compared to undamaged plants. There was, however, no evidence of cardenolide induction in response to artificial damage.

The observed effects of artificial damage on growth and fitness (see chapter I, above) may be consequence of the loss of leaf tissue, such as a decrease in photosynthetic capacity (Delaney 2008, Delaney et al. 2008). A similar effect is expected if natural herbivory by chewing insects occurs, and it could be followed by induction of defensive compounds such cardenolides (see Malcolm and Zalucki 1996, Martel and Malcolm 2004,

Mooney et al. 2008). To separate the effects of loss of leaf tissue from other metabolic causes (such as induction of defenses) we performed two experiments following the previous design using the phytohormone methyl jasmonate (MJ) instead of clipping to simulate herbivory. Firstly, does A. curassavica respond to MJ by reducing its fitness? If so, is there a concomitant increase in A. curassavica major defensive trait, cardenolides?

MJ is a synthetic plant hormone that affects the jasmonic acid metabolic pathway in plants, inducing substances of the secondary metabolism responsible for chemical defense

(Baldwin 1998, Farmer et al. 2003, Heil 2004) and that has been reported as an elicitor of cardenolides in A. syriaca (Mooney et al. 2008). Jasmonic acid and its derivatives are also

49

reported as being involved in pathogen and wound responses and in the inhibition of growth and of root elongation (Farmer et al. 2003, Kazan and Manners 2008).

To answer the first question we carried out a long term experiment, applying 50µg

MJ every two weeks, where we measured rates of increase in leaf tissue and investment in reproductive tissues. We investigated the second question in a short term experiment, also using 50 µg MJ, where we verified if this phytohormone induces defensive traits such as cardenolides.

Materials and Methods

Study organism

A. curassavica is a neotropical annual shrub that occurs from the south of the United

States to Argentina, although its exact origin in the Americas is still controversial

(Woodson 1954, Wyatt and Broyles 1997). This species is a weed that thrives in sunny, humid and disturbed sites, such as pastures, and close to river beds (Wyatt and Broyles

1997, Lorenzi 2000). As other milkweeds, A. curassavica has several types of cardenolides in its latex and tissues (Seiber et al. 1982, Groenveld et al. 1994) and is considered to have a medium to high range of cardenolide concentration in tissues (0.29 ± 0.07 % of dry weight; mean ± se) and low latex secretion following damage (0.74 ± 0.27 mg) (Agrawal and Fishbein 2006). Latex is stored under pressure in specialized ramified cells (laticifers) and contains a higher concentration of cardenolides than tissues, being released upon rupture of laticifers (Seiber et al. 1982, Dussourd and Hoyle 2000).

A. curassavica plants used in all experiments were raised from seeds collected from natural populations in the region of Campinas and Jundiaí, state of São Paulo, Brazil.

50

Plants for long term experiments were kept in 20L pots with a mixture of soil and organic substrate for seedlings (3:1) in a greenhouse at the the Departmento de Zoologia, Instituto de Biologia, Universidade Estadual de Campinas, under natural temperatures and were watered daily. For short term experiments, plants were kept in 1.5L pots with a mixture of soil and organic substrate for seedlings (3:1), were watered daily and were sprayed with

Vertimec (Syngenta) and Benlate (Dupont) to control aphids ( Aphis nerii ) and fungi two weeks before the beginning of the experiment.

Growth and reproductive responses in Asclepias curassavica after treatment with MJ

Thirty vegetative A. curassavica plants (approx. 5 months old) growing as above were randomly assigned to one of two treatments: 1) 50.0µg of MJ in 10mL distilled water

(0.02mM, hereafter MJ treatment) every two weeks and 2) control without MJ (10mL distilled water). Plants used in this trial had 38.7 ± 8.9 leaves (means ± SD) and 2.5 ± 1.2 g estimated mass (means ± SD). There was no significant difference in the total leaf biomass at the beginning of the trial between both treatments ( means ± SD , control: 2.2 ± 0.9 g; MJ:

2.8 ± 1.4 mg; t test, t = 1.32, df = 28, p = 0.20), therefore we used cumulative leaf mass gain to compare leaf growth between treatments. The concentration of MJ used in this study was chosen following the experimental design used to induce scopolamine in

Brugmansia suaveolens by Alves et al. (2007) and after preliminary experiments that showed a significant induction of cardenolides by this concentration. MJ was suspended in water by vigorous shaking and applied in the soil at the base of plant stems, in order to be absorbed by roots as used by Baldwin (1998).

To estimate growth, we measured the cumulative leaf biomass produced every 2 weeks according to Chapter I, as well as estimates of reproductive allocation (flower, fruit

51

and seed set and seed biomass). We assumed that MJ treatment had no effect on leaf length and therefore we used the same regression used in chapter I for estimating leaf growth of artificially damaged and undamaged A. curassavica leaves. Seeds were removed from pods upon opening, counted, weighed and stored in paper bags for further germination trials.

After 6 1/2 months (Oct 2007 – Apr 2008, 14 sampling dates), the trial was interrupted and plants were removed from pots with care as not to damage roots. The root biomass was also calculated following Chapter I.

The comparison of cumulative leaf mass gain, root dry weight, flower, fruit, and seed, and mean seed biomass between both treatments were analyzed with t-test; data were ln x or ln x+1 transformed when necessary to conform to assumptions of normality and homoscedasticity (Zar 1999). Two plants from each treatment group died before the end of the experiment and thus were not included in root biomass analysis, but were maintained in relative leaf biomass gain analysis until the last time in which each was measured.

Germination trials

Trials were held in germination boxes with a layer of cotton moistened with distilled water under a piece of germination paper in a growth chamber with 12L:12D photoperiod and 22-26ºC temperature. Fifty seeds from each plant were tested during 14 days and total germination percentage and germination rate were measured (according to Evetts and

Burnside 1972). Germination rate index has been positively correlated with competitiveness in sorghum (Evetts and Burnside 1972) who suggested the rate of germination may be important for other weeds too. Germination rate index (GRI) was calculated as:

52

G G G 1 + 2 + ... + n T T T GRI = 1 2 n G% where:

G1 = the number of seeds germinated in T 1,

G2 = the number of seeds germinated in T 2 and so on

T1= the number of days until first seed count,

T2 = the number of days until second count,

Gn = the number of seeds germinated in T n,

Tn = the number of days until nth count

G% = percentage of germination

All seeds assayed from an individual plant came from the same fruit. Fruit used in the germination trial were randomly chosen among those set by an individual during the last month of the trial.

Before trials, seeds were stratified for one week in distilled water at 5ºC. Available literature on other milkweeds (Evetts and Burnside 1972, Oegema and Fletcher 1972,

Beaton and Dudley 2004) and preliminary trials suggest that this method is one of the best and easiest methods to break the dormancy that is common in milkweeds without damaging seeds. After stratification, seeds were washed with 1% sodium hypochlorite to remove possible fungi following protocol suggested by Rodrigues (1988). Germination percentage was arcsin transformed and analyzed by t test. Germination rate index for individuals with germination percentage different than 0 was ln x+1 transformed and analyzed by t test (Zar

1999).

53

Does methyl jasmonate induce cardenolide responses?

In order to assess the changes cardenolide production in response to the MJ, we measured total cardenolide concentration in A. curassavica leaves in different sampling times (1, 24, 48, 96,192 and 384 h) after treatment compared to leaves of non treated control plants at time 0 h (Control). Plants used were still in the vegetative phase, approximately 10 weeks old and with 13.3 ± 3.3 leaves (means ± SD) and 0.9 ± 0.2 g estimated mass (means ± SD). As the concentration of cardenolides did not show significant differences along 384 hours (ln x+1 transformed data, one way Anova, F 6,84 =

0.88, p = 0.52), no control was needed for each time. Plants used (n = 72) had an estimated mass of at least 800 mg estimated as Chapter I, were approximately 10 weeks old, were kept in an acclimatized chamber (L12:D12, 25±3°C, and about 50% RH) and were watered daily.

At time 0, all plants in the MJ treatment received 50.0 µg of MJ suspended by vigorous shaking in 10mL distilled water and controls received 10mL of distilled water.

The 12 replicates that received control distilled water in this time were harvested for cardenolide analysis. Plants that received MJ treatment were harvested 1, 24, 48, 96, 192 and 384 h after treatment (10 replicates for each time). The leaves were immediately dried in an oven at 70ºC for 2 days and later ground to a powder in liquid nitrogen for cardenolide analysis following Frick and Wink (1995) (see detailed description in

Appendix 1). Cardenolide concentrations were expressed in percentage of dry weight of leaves, arcsin transformed and analyzed by one-way Anova with Tukey post-hoc test (Zar

1999).

54

Results

Growth and reproductive responses in Asclepias curassavica after treatment with MJ

We found no significant difference in cumulative leaf biomass gain between plants treated with MJ and controls (t-test, t = 0.78; df = 28; p = 0.44; Table 1). MJ treated plants also showed marginally significantly smaller root biomass than those found in controls (ln x+1 transformed data, t test, t = 2.01, df = 24, p = 0.06, Table 1). Both treatments did not differ significantly in number of flowers and fruit produced (t test for flowers, t = 0.11, df =

28, p = 0.91, t test for , t = 0.94, df = 28, p = 0.36, Table 1). We also found no significant differences in seed number between MJ treatment and control (t test, t = 0.524, df = 16, p = 0.61, Table 1). Although seeds from control plants had a significantly higher mean biomass, being on average 1.6x larger than seeds from MJ treated plants (t test, t =

2.52; df = 16, p = 0.02; Table 1). In germination trials, control seeds had a significantly higher germination percentage than MJ treatment seeds (arcsin transformed data, t test, t =

3.00; df = 16; p < 0.01, Table 1), although there was no difference in germination rate index between treatments (ln x+1 transformed data, t test, t = 0.06, df = 13, p = 0.96, Table 1).

Does methyl jasmonate induce cardenolide responses?

Plants treated with MJ showed a significant increase in cardenolide concentration compared to controls (arcsin transformed data, one way ANOVA, F 6,63 = 6.407, p < 0.001).

This increase was significant at 384 h after damage when compared to time 0 h (Tukey post-hoc test, Figure 1).

55

Discussion

The results from the long term resource allocation experiment showed no significant difference in cumulative leaf mass between A. curassavica plants treated with MJ and controls. We also found a trend in reduced final root mass in the MJ treatment. Since the

MJ treatment does not remove leaf tissue, unlike clipping, reduced investment in root growth should not be a consequence of reduction in photosynthetic capacity. It is possible that the known effects of MJ in inducing secondary metabolites (Baldwin 1998, Farmer et al. 2003, Heil 2004, Mooney et al. 2008) have shifted resource investment from root growth to the secondary metabolism. Other authors have found reduced root growth after induction of defenses in Pastinaca sativa (Zangerl et al 1997) and, in induced plants in high densities, in Lepidium virginicum (Agrawal 2000), suggesting trade-offs between root allocation and defenses or competition. However, MJ has been reported as an inhibitor of root elongation (Farmer et al. 2003, Kazan and Manners 2008), and it is unclear whether the diminished root mass in the MJ treatment results from direct effects of MJ as an inhibitor or from indirect effects, by resource allocation to defense.

As for reproductive traits, we found no difference in flower production, fruit and seed set between MJ treated plants and controls, although the former group showed a significant reduction in mean seed mass suggesting a reduction in fitness compared to controls. Seeds from MJ treated plants also showed a significantly lower germination percentage compared to controls, which also gives evidence of lower fitness, but no difference was found in germination rate index, which gives a measure of the time taken for seeds to germinate. Reductions in the mass of reproductive tissues have been associated with reduced fitness in several plant species (Bazzaz and Ackerly 1992, and references

56

therein, Agrawal 2004a, Canto et al. 2004). This probably occurs because germination rate, seedling growth and competition depend on the nutritional content of seeds and reduced seed mass is suggested as an indicator of reduced nutritional content (Zangerl and

Berenbaum 2004 and references therein). The decrease in germination percentage in the lighter seeds from the MJ treated plants corroborates these findings.

The reduction in fitness of MJ treated A. curassavica probably results indirectly from the reduction in root growth. MJ treated plants sustained the same long term leaf growth as controls even though they had smaller roots, and presumably less nutrient uptake.

The maintenance of normal leaf growth in spite of smaller root growth must have taken its toll on reproductive output. It is unclear in this experiment whether the induction of secondary metabolites or other metabolic effects of MJ also contributed to reduced seed mass.

In the short term experiment, A. curassavica plants treated with MJ showed a steady increase in cardenolides, which was significant at 384 h with approximately twice as much cardenolides as controls (see Figure 1, Results). Mooney et al. (2008) reported a 33% increase in cardenolides of A. syriaca in plants treated with jasmonic acid but this increase did not affect the performance of two specialist aphids, A. nerii and A. asclepiadis .

Increase of cardenolides in milkweeds have been associated with decreases in performance of the specialist foliage eating herbivore D. plexippus, particularly in the first instars

(Malcolm 1995, Malcolm and Zalucki 1996, Agrawal 2005), and also in the phloem feeding A. nerii (Agrawal 2004b). Malcolm and Zalucki (1996) found that cardenolide concentration in A. syriaca increases significantly 24 h after damage and returns to constitutive levels afer 148 h and suggested this mechanism may decrease sequestration of cardenolides by specialists. Another study did not find an effect of time of feeding by A.

57

nerii on cardenolide concentration in A. curassavica , although cardenolides increased significantly with aphid density (Martel and Malcolm 2004). It seems that although A. curassavica has a median to high concentration of cardenolides (Martel and Malcolm 2004,

Agrawal and Fishbein 2006) and has been shown to induce cardenolides, as observed in the present study, there is no evidence that the timing of these defenses is important for this species.

The significant increase in cardenolides in MJ treated A. curassavica in the short term experiment suggests that the induction of this secondary metabolite also plays a roll in the decreased fitness of plants repeatedly treated with MJ in the long term experiment. The investment in cardenolides probably represents an additional resource sink in the MJ treated plants regardless of other metabolic effects of MJ, contributing to the observed decrease in fitness. These results suggest that the MJ induction of cardenolides is costly for A. curassavica . Nicotiana attenuata plants induced with MJ in a protected environment, showed a significant reduction in fitness compared to uninduced controls (Baldwin 1998).

These costs of induced alkaloids were offset by benefits of increased defense and higher fitness in the presence of herbivores (Baldwin 1998). Milkweed cardenolides have been shown to be effective against several specialist herbivores (Zalucki and Brower 1992,

Zalucki et al. 2001, Agrawal 2004b, Van Zandt and Agrawal 2004, Agrawal 2005) and it is possible this is also the case for A. curassavica . The question of whether the delayed increase in cardenolides of A. curassavica observed in this study is effective against these specialists remains to be tested.

In conclusion, repeated treatment of A. curassavica plants with methyl jasmonate caused a decrease in long term root growth and in the investment in seed biomass, although leaf growth was not altered when compared to untreated control plants. The induction of

58

cardenolides in MJ treated plants appears to have contributed to the observed decreases in growth and reproduction, suggesting that these defenses are costly in the absence of herbivores.

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Table 1. Growth and reproduction for Asclepias curassavica plants from control and MJ treatments. Values are means ± standard deviation.

Control Methyl Jasmonate p-value

Cumulative leaf biomass gain (g) 21.7 ± 6.4 19.8 ± 6.9 0.44

Number of flowers 223.5 ± 68.6 220.5 ± 78.0 0.91

Number of fruit 20.8 ± 6.9 24.0 ± 11.3 0.36

Number of seeds 309.1 ± 239.6 249.3 ± 241.3 0.61

Mean seed biomass (mg) 2.30 ± 0.74 1.44 ± 0.70 0.02

Germination (%) 80.1 ± 19.4 32.0 ± 36.5 <0.01

Germination Rate Index 17.5 ± 6.3 12.2 ± 10.1 0.96

Root biomass (g) 17.45 ± 7.13 11.97 ± 3.70 0.06

64

0.8 b

0.6 a a,b a aa 0.4 a a

%cardenolides 0.2

0.0 0 1 24 48 96 192 384 Time (hours)

Figure 1. Cardenolide (in percentage of dry weight) in A. curassavica leaves in different sampling times after treatment with MJ. The values are expressed in mean ± sd.

65

Appendix 1: Cardenolide extraction and lead acetate cleanup

Dried leaves of Asclepias curassavica (100 mg dry weight per sample) were ground to a fine powder in a mortar with liquid nitrogen. Individual samples were extracted in

10mL 95% ethanol at 70ºC during 45 min. After centrifugation (10 min at 3000 rpm), supernatants were evaporated and dissolved in 1mL 95% ethanol and submitted to a lead acetate clean up to remove pigments that may interfere with spectrophotometric analysis.

The first step of clean up consisted of adding 2mL of 10% aqueous lead acetate to samples, mixing for 30”, and placing in ice bath for 20’. After centrifugation (10 min at

3000 rpm), centrifuged tubes had their supernatant transferred to new tubes with 0.5mL saturated ammonium sulfate solution, which caused precipitation of lead acetate and pigments. The precipitates of centrifuged tubes were washed with 2mL of 1:2 distilled

95% ethanol and water and centrifuged again. The resulting supernatants were transferred to the tubes containing ammonium sulfate and the first supernatant, mixed (vortex 30”) and centrifuged (10 min at 3000 rpm).

The new supernatants resulting from ammonium sulfate treatment were transferred to clean tubes and precipitates were washed with 2mL 1:2 95% ethanol and saturated ammonium sulfate solution and centrifuged again (10 min at 3000 rpm). These final supernatants were transferred to the same tubes as the supernatants from ammonium sulfate treatment, corresponding to individual plant samples.

After the clean up, samples were extracted twice with 2mL dichloromethane and the dichloromethanic phase was filtered through anhydrous sodium sulfate, dried under air flow and subsequently stored in a freezer (-5ºC) until analyzed.

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To analyze cardenolide concentration, dried extracts were dissolved in 2.5mL 95% ethanol with 1mL of Kedde Reagent (1.0g 3,5-dinitrobenzoic acid in 100mL ethanol) and background absorbance was measured in a Milton Roy Genesis 5 spectrophotometer at

540nm. After measuring background absorbance, 0.5mL 1M NaOH was added to samples and absorbance at 540nm was measured after 5 min, which is the peak complexation time of cardenolides with Kedde Reagent and NaOH (Frick & Wink 1995). Absorbance values for samples were compared to a calibration curve of known concentrations of synthetic digitoxin (Sigma) and total cardenolide concentration from samples are presented as µg digitoxin equivalents/0.1 g dry weight (d.w.).

References:

Brower LP, Seiber JN, Nelson CJ, Lynch SP, Tuskes PM (1982) Plant determined variation

in the cardenolide content, thin-layer chromatography profiles, and emetic

potency of monarch butterflies, Danaus plexippus reared on the milkweed

Asclepias eriocarpa in California. Journal of Chemical Ecology 8: 579-633.

Frick C, Wink M (1995) Uptake and sequestration of Ouabain and other Cardiac-

Glycosides in Danaus plexippus (Lepidoptera: Danaidae) – evidence for a carrier-

mediated process. Journal of Chemical Ecology 21: 557 – 575.

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Appendix 2: Leaf area measurements

To measure leaf area, pressed leaves were individually scanned (HP Scanjet 2400 flatbed scanner) in b&w at 300dpi. After scanning, petioles were removed using the software Corel Photopaint (Corel Corporation) and images obtained after removal were considered as final leaf area. Initial leaf area was obtained by reconstructing damaged leaves using a similar sized undamaged leaf from the same plant to estimate original leaf size (see figure below). Area of scanned leaves (final leaf area) and estimated initial leaf area were measured with the software Area Measure (Myka Lab) that records the number of black pixels in the image and calculates the area in dots. The area in cm² was calculated using a calibration curve obtained by scanning 2 squares of black paper with a known area

(1.0 cm² and 4.0 cm²).

Individual plant herbivory was calculated as sum of Initial Leaf Areas – Sum of

Final Leaf Areas for each individual from both populations (April 2007 and March 2008).

a b

Example of scanned A. curassavica leaf showing signs of herbivory (a); and the same leaf

after reconstruction using Corel Photopaint® (b).

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Conclusões gerais

O investimento em crescimento compensatório das folhas em plantas de Asclepias curassavica após perda de 50% de biomassa foliar por dano artificial repercutiu tanto no crescimento das raízes quanto na reprodução dessas plantas no experimento de longo prazo.

A manutenção do crescimento foliar igual às plantas não danificadas em plantas danificadas deve ter desviado recursos que seriam alocados para o crescimento das raízes sendo a causa da menor biomassa final de raízes observada nesse experimento.

A diminuição do número de frutos e de sementes no grupo submetido a dano artificial simulando herbivoria sugere que o investimento em crescimento compensatório das folhas e possivelmente a redução das raízes também afetou a aptidão.

O experimento de curto prazo mostrou que cardenolidas não são induzidas por A. curassavica em resposta a dano artificial. É possível que a ausência de indução de cardenolidas após o dano artificial se deva a esse tratamento ser ineficaz em ativar a produção de defesas da mesma forma que um herbívoro. No entanto, estudos com outras espécies de Asclepias mostraram resultados variáveis em relação à indução de cardenolidas por dano artificial e por diferentes herbívoros. Além disso, a falta de correlação entre defesas e hebivoria em campo sugere que a indução de cardenolidas pode não ser importante contra herbívoros. Esses resultados no entanto não excluem a possibilidade de que a presença constitutiva dessas substâncias em altas concentrações pode ser uma defesa eficaz para esta espécie. Uma outra possibilidade a ser testada é a de que outras substâncias de defesa não analisadas nesse estudo (p.ex., proteases presentes no látex ou o próprio efeito físico do látex) sejam mais importantes para a defesa de A. curassavica .

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A aplicação do fitohormônio jasmonato de metila (JM) a longo prazo também mostrou efeitos sobre a aptidão e uma tendência a reduzir o crescimento das raízes em plantas de A. curassavica . Não foram verificados efeitos desse hormônio sobre o crescimento foliar. Não está claro se a tendência a menor crescimento das raízes de plantas tratadas com JM é um efeito direto desse hormônio ou resulta indiretamente de outros efeitos metabólicos do mesmo, como a indução de metabólitos secundários. De toda forma, a redução do crescimento das raízes provavelmente repercutiu na redução da massa média de sementes nas plantas tratadas com JM, diminuindo a aptidão das mesmas. A redução da massa média das sementes de A. curassavica após uso de JM aparentemente está relacionada à menor taxa de germinação das mesmas quando comparadas com o controle sem JM. O experimento de curto prazo mostrou um efeito tardio do hormônio JM sobre a indução de cardenolidas. Esta indução deve ter contribuído para as reduções no crescimento de raízes e na massa média de sementes no experimento de longo prazo. Esses dados sugerem que existe um custo da produção de cardenolidas, embora outras alterações metabólicas e de crescimento causadas por esse hormônio possam ter contribuído para os custos observados.

O tratamento de dano artificial e o tratamento de jasmonato de metila em plantas de

A. curassavica produziram efeitos semelhantes a longo prazo, no entanto nossos dados sugerem que esses efeitos resultam de mecanismos de ação diferentes. Os efeitos foram semelhantes na medida em que ambos os tratamentos não alteraram o crescimento foliar mas reduziram o crescimento de raízes e a aptidão das plantas tratadas comparadas a plantas controle sem dano ou sem JM. Contudo, nas plantas submetidas a dano artificial, a redução de crescimento das raízes aparentemente resulta do desvio de recursos para crescimento compensatório das folhas, que tiveram sua massa reduzida, reduzindo assim,

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tecido fotossinteticamente ativo. Já nas plantas tratadas com JM, não houve perda de massa foliar, não havendo portanto crescimento compensatório. Dessa forma, a redução do crescimento das raízes deve resultar de efeitos diretos do JM ou do desvio de recursos para outras funções metabólicas induzidas por essa substância, como p.ex., a indução tardia de cardenolidas. Além disso, a diminuição da aptidão se expressou de formas diferentes entre os dois tratamentos, havendo redução do número de frutos e sementes, sem alteração da biomassa média das sementes, nas plantas que sofreram dano artificial enquanto as plantas tratadas com JM tiveram redução da massa média de sementes, sem alterações no número de frutos e sementes produzidos. Esses dados sugerem que os mecanismos de ação do dano artificial (= remoção de área fotossinteticamente ativa) e do JM sobre o metabolismo de A. curassavica diferem entre si.

A utilização de dano por um herbívoro especialista que ocorre na região do estudo

(p.ex. Danaus erippus ) e a avaliação das mesmas respostas estudadas neste trabalho permitirá avaliação da eficácia do dano artificial e comparação com os resultados obtidos por dano artificial, JM e as observações em campo. Além disso, D. erippus é um herbívoro mastigador, que também promove a remoção de área foliar. Caso este herbívoro induza a produção de cardenolidas em A. curassavica , será interessante comparar os efeitos combinados da remoção de área foliar e da indução de cardenolidas com plantas que foram apenas induzidas por JM e com plantas que sofreram apenas a remoção de área foliar por dano artificial.

Novos estudos de campo acompanhando plantas de A. curassavica ao longo do seu ciclo de vida em seu ambiente natural e comparando as concentrações de cardenolidas em plantas protegidas e expostas a herbivoria natural seria uma forma de avaliar o papel das respostas induzidas na defesa dessas plantas.

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A variação das concentrações de cardenolidas entre plantas neste estudo sugere que plantas individuais de A. curassavica podem exibir respostas plásticas (plasticidade fenotípica) intraespecífica aos mesmos estímulos. Estudos futuros deverão avaliar o efeito de diferentes genótipos sobre as respostas medidas neste estudo (crescimento, reprodução e indução de cardenolidas) utilizando indivíduos relacionados de um genótipo A x indivíduos relacionados de outros genótipos (B, C, D etc.) . Esse tipo de estudo é facilitado pela biologia reprodutiva dessa espécie, na qual sementes provindas de um mesmo fruto quase sempre são irmãos completos ( full-sibs ), devido à transferência de pólen agregado em polínias, facilitando o estabelecimento de linhagens geneticamente relacionadas. Além disso, o uso de plantas geneticamente relacionadas permitirá a análise simultânea de diversas variáveis, e reduz efeitos fenotípicos que podem ter causado a variabilidade observada.

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