UNIVERSIDADE ESTADUAL DE CAMPINAS INSTITUTO DE BIOLOGIA
LEANDRO GIACOBELLI COSMO
DE DENTRO DA FOLHA PARA A COMUNIDADE: VARIAÇÃO FITOQUÍMICA MOLDA A ESTRUTURA DA COMUNIDADE DE HERBÍVOROS E A HERBIVORIA
DENTRO E ENTRE INDIVÍDUOS DE PLANTAS
CAMPINAS 2019
LEANDRO GIACOBELLI COSMO
DE DENTRO DA FOLHA PARA A COMUNIDADE: VARIAÇÃO FITOQUÍMICA MOLDA A ESTRUTURA DA COMUNIDADE DE HERBÍVOROS E A HERBIVORIA
DENTRO E ENTRE INDIVÍDUOS DE PLANTAS
Dissertação apresentada ao Instituto de Biologia da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Mestre em Ecologia.
Este arquivo digital corresponde à versão final da Dissertação defendida pelo aluno Leandro Giacobelli Cosmo e orientada pelo Prof. Dr. Martín Francisco Pareja Piaggio e co-orientada pelo Prof. Dr. Rodrigo Cogni
Orientador: Prof. Dr. Martín Francisco Pareja Piaggio Co-orientador: Prof. Dr. Rodrigo Cogni
CAMPINAS 2019
Mara Janaina de Oliveira - CRB 8/6972
Cosmo, Leandro Giacobelli,
C821d CosDe dentro da folha para a comunidade : variação fitoquímica molda a estrutura da comunidade de herbívoros e a herbivoria dentro e entre indivíduos de plantas / Leandro Giacobelli Cosmo. – Campinas, SP : [s.n.], 2019.
CosOrientador: Martín Francisco Pareja Piaggio. CosCoorientador: Rodrigo Cogni.
CosDissertação (mestrado) – Universidade Estadual de Campinas, Instituto de Biologia.
Cos1. Ecologia de comunidades. 2. Relação inseto-planta. 3. Defesas de plantas. I. Pareja, Martín Francisco, 1976-. II. Cogni, Rodrigo. III. Universidade Estadual de Campinas. Instituto de Biologia. IV. Título.
Informações para Biblioteca Digital
Título em outro idioma: From within the leaf to the community : phytochemical variation
drives herbivore community structure and herbivory within and among individual plants
Palavras-chave em inglês:
Community ecology Insect-plant relationships Plant defenses
Área de concentração: Ecologia Titulação: Mestre em Ecologia Banca examinadora:
Martín Francisco Pareja Piaggio [Orientador] Mariana Alves Stanton
Mathias Mistretta Pires
Data de defesa: 29-05-2019
Programa de Pós-Graduação: Ecologia
Identificação e informações acadêmicas do(a) aluno(a)
- ORCID do autor: https://orcid.org/0000-0003-2541-2645 - Currículo Lattes do autor: http://lattes.cnpq.br/8022223997680224
COMISSÃO EXAMINADORA
Prof. Dr. Martín Francisco Pareja Piaggio Profa. Dra. Mariana Alves Stanton
Prof. Dr. Mathias Mistretta Pires
Os membros da Comissão Examinadora acima assinaram a Ata de Defesa, que se encontra no processo de vida acadêmica do aluno.
AGRADECIMENTOS
O presente trabalho foi financiado pela Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – código de financiamento 1688672; e pela Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, auxílio 14/50316-7).
Agradeço ao Instituto de Biologia e ao Programa de Pós-Graduação em Ecologia da Unicamp pelas condições que permitiram a realização deste trabalho. Agradeço também ao Instituto de Química da Universidade Estadual de São Paulo pela colaboração nas análises químicas. Agradeço à Fundação Serra do Japi e à Prefeitura do município de Jundiaí (SP) por viabilizarem a realização deste trabalho.
Agradeço ao meu orientador, Martin Pareja e ao meu co-orientador, Rodrigo Cogni, por todo o tempo dedicado ao trabalho e à minha formação. Ter tido a oportunidade de discutir ecologia com vocês me motivou muito ao longo de todo o desenvolvimento do nosso trabalho. Agradeço ao Massuo J. Kato, à Lydia F. Yamaguchi, e todos os colaboradores do projeto Dimensions-BIOTA FAPESP. Muito obrigado pela colaboração, amizade e por toda a paciência e ajuda nas análises químicas.
Agradeço ao André V. L. Freitas por ter me apresentado às interações inseto-planta, às lagartas, e às Piperáceas. Obrigado por ter sido (e continuar sendo) meu mentor por tanto tempo, por ter me apresentado aos meus orientadores neste trabalho e por ter me mostrado o quão bonita a história natural pode ser.
Agradeço ao Sérgio F. Reis, cuja disciplina e conversas motivaram grande parte da escrita e do contexto desse trabalho. O título do meu ensaio para a sua disciplina, “Padrões de diversidade como consequência de mecanismos em níveis de complexidade inferiores”, não me deixa mentir.
Agradeço ao Rafael S. Oliveira e toda a equipe de seu laboratório por gentilmente cederem o uso de seus equipamentos e me ajudarem no processamento de minhas amostras.
Agradeço a todos os membros do antigo LEEDP e atual LEIA, e dos laboratórios vizinhos. Obrigado pelas conversas, conselhos, discussões e ajuda no campo e no laboratório. Sem vocês tudo teria sido muito menos interessante.
Agradeço à minha mãe, Mara, por ter sempre me apoiado em minhas escolhas. Ao meu irmão, Alexandre, e à Fabiana Gropelo por estarem sempre disponíveis para me escutar e para me ajudar.
Agradeço à Juliana Rink por ter sido minha companheira ao longo de toda essa jornada. Você me inspira todos os dias e, sem você ao meu lado, nada disso teria valido a pena.
RESUMO
Atributos químicos de plantas (i.e. fitoquímica) conduzem muitos processos ecológicos e evolutivos que moldam interações inseto-planta e estruturam comunidades de insetos herbívoros. Quando a fitoquímica varia ao longo do espaço e do tempo, isso dá origem a um mosaico heterogêneo de atributos químicos, definido como paisagem fitoquímica, que molda padrões de diversidade de insetos herbívoros. Isso inevitavelmente levanta uma questão-chave: em que escalas organizacionais e espaciais a fitoquímica varia, é percebida pelos insetos herbívoros e, portanto, estrutura suas comunidades? Nesta dissertação, investigamos essa questão para os níveis de organização intraespecíficos e sub-individuais. Quantificamos a variação fitoquímica no campo entre e dentro de indivíduos de Piper amalago L. e avaliamos como ela molda a herbivoria e a comunidade de seus principais herbívoros - larvas de Lepidoptera. Nosso estudo obteve três resultados principais. Primeiro, mostramos que mesmo dentro de indivíduos de plantas a variação fitoquímica pode influenciar a estrutura da comunidade de herbívoros e a herbivoria. Em segundo lugar, verificamos que a fitoquímica difere entre indivíduos de plantas (ou seja, intraespecificamente) em escalas espaciais muito pequenas, quando um gradiente ambiental claro e / ou quimiotipos de plantas distintos estão ausentes, e esta variação fitoquímica também pode estruturar a comunidade de herbívoros e a herbivoria nessas plantas. Por fim, mostramos que o dano e a estrutura da comunidade de insetos herbívoros são influenciados por menores quantidades de variação fitoquímica dentro de plantas do que entre plantas. Juntos, nossos resultados sugerem: 1) que os efeitos da variação fitoquímica podem cascatear através de seus efeitos no comportamento de insetos dentro das plantas hospedeiras para ter consequências em toda a comunidade de herbívoros; 2) que, entre plantas, não são necessários fortes gradientes ambientais para que a fitoquímica varie e estruture a comunidade de herbívoros e a herbivoria; e 3) através de escalas espaciais e níveis de organização, pode haver várias camadas distintas da paisagem fitoquímica que são percebidas e têm diferentes efeitos nos padrões de diversidade de insetos herbívoros.
PALAVRAS-CHAVE: diversidade fitoquímica, paisagem fitoquímica, variação fitoquímica sub-individual, variação fitoquímica intraespecífica, comunidade de herbívoros, herbivoria
ABSTRACT
Phytochemistry drives many ecological and evolutionary processes that shape insect-plant interactions and structure herbivore communities. Phytochemical variation through space and time gives rise to a heterogeneous mosaic of chemical traits, formally known as phytochemical landscape, that affects herbivorous insect communities. This inevitably raises one key question: in what scales does phytochemistry vary, is perceived by herbivorous insects and thus, structures herbivore communities? In this dissertation, we pursued this question for the intraspecific and sub-individual organizational levels. We quantified phytochemical variation in the field among and within individuals of the neotropical shrub Piper amalago L. and assessed how it affects herbivory and its main herbivores – caterpillars. Our study has three main findings. First, we found that even within individual plants phytochemical variation can drive herbivore community structure and herbivory. Second, chemical traits can differ among individual plants (i.e. intraspecifically) at very small spatial scales, when a clear environmental gradient and/or distinct plant chemotypes are absent, and this phytochemical variation can also drive herbivore community and herbivory in these plants. Third, we found that within-plants herbivores respond to finer-scale variation when compared to among plants. Together, our results suggests: 1) that phytochemical variation effects can cascade through individual insect behavior within host-plants to have community-wide consequences; 2) that among-plants, strong environmental gradients are not necessary for phytochemistry to vary and drive herbivore community and herbivory; and 3) across spatial scales and levels of organization there may be several distinct layers of the phytochemical landscape which are perceived by and have different effects on diversity patterns of herbivorous insects.
KEY-WORDS: phytochemical diversity, phytochemical landscape, sub-individual phytochemical variation, intraspecific phytochemical variation, herbivore community, herbivory
Sumário
INTRODUÇÃO ... 11
From within the leaf to the community: phytochemical variation drives herbivore community structure and herbivory within and among individual plants ... 16
Abstract ... 17 Introduction ... 17 Figure 1 ... 20 Methods ... 20 Study site ... 20 Study system ... 21
Plant sampling and study design ... 21
Data collection and processing ... 22
Caterpillar community ... 22
Leaf area and leaf damage measurements ... 22
Leaf chemical analysis... 23
Phytochemical data pre-processing and statistical analysis ... 23
Within-individual variation ... 25
Among-individual variation ... 26
Results ... 26
Phytochemical variation drives caterpillar community structure and herbivory within-plants ... 27
Figure 2 ... 28
Phytochemical variation drives caterpillar community structure and herbivory among plants ... 29
Figure 3 ... 30
Different amounts of phytochemical variation drive caterpillar community structure and herbivory within and among plants ... 30
Table 1 ... 31 Figure 4 ... 32 Discussion ... 32 Conclusion ... 35 Acknowledgements ... 36 CONCLUSÃO ... 37 REFERÊNCIAS ... 38 APÊNDICES ... 45
Table S1 ... 45
Table S2 ... 46
Table S3 ... 47
Table S4 ... 48
ANEXOS ... 49
Anexo I – Declaração de Bioética e Biossegurança ... 49
INTRODUÇÃO
As interações ecológicas que envolvem insetos fitófagos e plantas vasculares estão entre as mais complexas e diversas encontradas na natureza (Price et al. 2011). Além de, juntos, contemplarem mais da metade de toda a riqueza de espécies conhecida (Grimaldi & Engel, 2005; Schoonhoven et al., 2005), insetos herbívoros e suas plantas hospedeiras exercem forte influência um no outro, conduzindo muitos processos ecológicos e evolutivos em comunidades naturais. Por exemplo, insetos podem estar relacionados com a manutenção da diversidade de árvores em florestas tropicais ricas em espécies vegetais (Janzen, 1970; Connell, 1971; Bagchi
et al. 2014); com alterações na composição de espécies em sucessões ecológicas (Brown &
Gange, 1992); com a promoção da especialização de habitat em espécies arbóreas (Fine et al. 2004); ou ainda influenciar a estrutura genética, capacidade competitiva e defesas químicas em populações de plantas em pequenos intervalos de tempo (Agrawal et al. 2012).
Do mesmo modo, alterações em comunidades e populações vegetais também podem ocasionar mudanças na composição e abundância de herbívoros. Muitos insetos têm uma dieta com amplitude limitada (Dyer et al. 2007; Bodner et al. 2012; Price et al. 2011) que pode, inclusive, ser restrita a um único gênero ou a uma única espécie vegetal (Novotny & Basset 2005; Connahs et al. 2009; Forister et al. 2015). Assim, mesmo pequenas perturbações ambientais podem ter como consequência alterações na biodiversidade em comunidades desses insetos.
Essas alterações nas dinâmicas de comunidades e populações são um reflexo do impacto dos insetos no desempenho de indivíduos vegetais (revisado por Maron & Crone 2006) e vice-versa. Plantas apresentam uma série de estratégias defensivas que atuam na diminuição do dano por herbívoros (Fincher et al. 2008; Howe & Jander 2008). Do mesmo modo, insetos fitófagos também apresentam atributos que os permitem ultrapassar as barreiras impostas pelas plantas. Essas pressões seletivas podem resultar em processos mútuos de especialização ecológica, levando, em última instância, à coevolução das partes envolvidas (Ehrlich & Raven 1968, Forister et al. 2012). Tal padrão de evolução recíproca pode estar relacionado e ser direcionado pela presença de metabólitos secundários (i.e. que não participam do metabolismo primário de plantas) em estruturas e tecidos vegetais (Ehrlich & Raven 1968; Becerra 2007; Janz 2011), uma das classes de defesa mais comuns em plantas (Howe & Jander, 2008). Esses metabólitos, por sua vez, podem ser voláteis ou não voláteis, estar presentes independentemente da ocorrência de dano por herbívoros, ou seja, constitutivamente; ou podem ser produzidos como
resposta a estresse sofrido pela planta, como o dano por insetos fitófagos, caracterizando uma indução de defesas químicas (revisado por Kaplan et al. 2008). Ou seja, o resultado das interações entre insetos herbívoros e suas plantas hospedeiras dependem de processos ecológicos e evolutivos, os quais, em grande parte, são mediados por atributos químicos de plantas (i.e. fitoquímica). Assim, para melhor compreendermos o papel da fitoquímica em mediar interações inseto-planta, é essencial estudarmos como ela varia ao longo do tempo e espaço, e quais os efeitos dessa variação em padrões de diversidade de insetos herbívoros.
Entretanto, entender padrões de variação fitoquímica é um grande desafio pois há dois componentes chaves que precisam ser desemaranhados. Em primeiro, a fitoquímica consiste de um conjunto multivariado de compostos químicos. Cada composto, no entanto, raramente varia de forma isolada. Ao invés disso, combinações de compostos variam de forma conjunta, sendo que essas combinações podem influenciar insetos herbívoros de forma isolada ou sinergística (Dyer et al. 2003, Richards et al. 2010, 2012, 2016). A variação de tais combinações ao longo do tempo e espaço pode ser visualizada como um mosaico fitoquímico, o qual foi definido como paisagem fitoquímica (Hunter 2016). Em segundo, variações ecológicas podem resultar em padrões distintos quando concebidas em diferentes escalas espaciais e níveis de organização (Levin 1992). Assim, essa paisagem fitoquímica pode variar em diferentes escalas de espaço e tempo, de modo que em cada uma dessas escalas tal variação pode mediar padrões distintos de diversidade de herbívoros. Nesse sentido, o conjunto desses dois componentes torna essencial que a variação fitoquímica e suas consequências sejam mensuradas através de diferentes escalas espaciais e níveis de organização.
Tradicionalmente, porém, estudos de ecologia química abordam apenas compostos individuais, denominados compostos majoritários, e quantificam sua variação e seus efeitos apenas no nível de organização de espécies (Richards et al. 2016). No que se refere às escalas espaciais, o foco tem sido direcionado para variação fitoquímica e suas consequências ao longo de grandes gradientes ambientais, como por exemplo gradientes de altitude na ordem de quilômetros, ou em sistemas agrícolas. Embora repetidamente suas relevâncias tenham sido demonstradas, os níveis de organização abaixo de espécies (e.g. indivíduos), pequenas escalas espaciais (i.e. na ordem de poucos metros) e sistemas naturais têm sido negligenciados (Bolnick
et al. 2011, Des Roches et al. 2018, Lamke & Unsicker 2018). Apenas recentemente foram
publicados trabalhos que mensuraram como variação fitoquímica no nível do indivíduo (i.e. intraespecífica) molda padrões de diversidade e dano de insetos herbívoros. Por exemplo, Poelman et al. (2009) demonstraram que combinações de compostos que variam entre
quimiotipos de indivíduos de Brassica oleracea moldam a estrutura da comunidade de herbívoros associada a esses indivíduos. De forma parecida, Balín et al. (2011) estudaram indivíduos de diferentes quimiotipos de Tanacetum vulgare e verificaram que variação fitoquímica entre esses influenciou a estrutura de uma rede trófica de artrópodes inteira. Distanciando-se de perspectiva dos quimiotipos, Glassmire et al. (2016, 2019) demonstraram que gradientes altitudinais e a proximidade do dossel influenciam a fitoquímica de indivíduos de Piper kelleyi, o que, por sua vez, correlaciona-se com a estrutura da comunidade e até a estrutura genética de populações de insetos herbívoros especialistas. Esses trabalhos demonstram que variação fitoquímica entre indivíduos de uma mesma espécie, causada por gradientes ambientais ou determinações genéticas (i.e. quimiotipos), também são relevantes e podem moldar padrões de diversidade de insetos herbívoros.
Contudo, em pequenas escalas espaciais e níveis de organização menos complexos também pode ocorrer variação fitoquímica. Por exemplo, em florestas tropicais, diversas variáveis ambientais que potencialmente alteram atributos químicos podem mudar até mesmo entre plantas vizinhas (Townsend et al. 2008). As consequências dessa variação para a comunidade dos herbívoros associados à essas plantas, porém, permanece inexplorada. Além disso, dentro de indivíduos de plantas, diversos atributos também podem variar, como por exemplo o conteúdo de néctar de flores, tamanho de sementes e conteúdo de compostos fenólicos de folhas (Herrera 2009, 2015, 2017). Porém, embora essa variação dentro dos indivíduos já tenha sido demonstrada até mesmo superar a variação entre indivíduos, apenas recentemente ela passou a ser considerada como relevante ecologicamente ao invés de apenas um ruído estatístico (Herrera 2017, Wetzel & Meek 2018). No caso da variação fitoquímica, mesmo quando pequena ela pode influenciar insetos de uma forma geral, especialmente os herbívoros cuja performance depende da variabilidade na qualidade da planta hospedeira (Pareja et al. 2009, Wetzel et al. 2016). Assim, é provável que a variação fitoquímica dentro de indivíduos de plantas influencie padrões de diversidade de insetos herbívoros tanto quanto a variação entre indivíduos. Ainda, é possível que as combinações de compostos dentro dos indivíduos variem e influenciem insetos herbívoros de forma diferente das que variam entre indivíduos. Ou seja, do ponto de vista de insetos herbívoros, a paisagem fitoquímica pode ser composta de mais de uma camada ao longo de níveis de organização e escalas espaciais: um único indivíduo de planta pode representar uma camada inferior, mais refinada da paisagem fitoquímica, a qual está contida em uma camada superior representada pela variação entre indivíduos. Nesse sentido, mensurar como a fitoquímica varia e seus efeitos nos padrões de
dano e diversidade de herbívoros em diferentes escalas espaciais e de organização pode auxiliar na nossa compreensão de grandes questões ecológicas, como, por exemplo, o por quê de variação fenotípica ser mantida em sistemas naturais e quais as consequências para biodiversidade da perda dessa variação.
Entre os diferentes insetos que apresentam hábito fitófago em uma ou mais etapas do seu desenvolvimento, os estágios imaturos de Lepidópteros (lagartas) são frequentes, quando não dominantes, em comunidades de herbívoros (Novotny et al. 2006). A ordem Lepidoptera é a segunda mais rica em espécies da classe Insecta. São cerca de 155.000 espécies descritas, distribuídas em 121 famílias de borboletas e mariposas (Pogue 2009; Aguiar et al. 2010). Apesar de esse grupo ser relativamente bem conhecido, a maioria dos estudos publicados trata dos indivíduos adultos. Trabalhos que envolvem lagartas são menos abundantes e ainda existem grandes lacunas no conhecimento taxonômico, morfológico e ecológico desses organismos. Controversamente, nas últimas décadas a utilização de lagartas tem se mostrado de alto valor na obtenção de informações que podem auxiliar na compreensão de uma série de questões ecológicas. Um exemplo clássico consiste na proposição da hipótese da coevolução por Ehrlich & Raven (1964) a partir da utilização de lepidópteros como organismos modelo, a qual representou importantes desdobramentos para a explicação de processos de diversificação. Dessa forma, estudos que envolvem imaturos de lepidópteros não só produzem informações empíricas acerca desses organismos, como também auxiliam na compreensão de processos e padrões gerais ecológicos.
Dentre os grupos vegetais que apresentam interações com insetos, o gênero Piper (Piperales: Piperaceae) é considerado um modelo em estudos fitoquímicos, ecológicos e evolutivos (Dyer & Palmer 2004). Este gênero é um dos dois mais ricos em espécies da família Piperaceae, com cerca de dois terços das espécies descritas sendo encontradas na região neotropical. As plantas pertencentes a este gênero são caracterizadas pela presença de metabólitos secundários em todas as suas estruturas, os quais podem ser tóxicos para alguns insetos fitófagos (Dyer et al. 2004). No que se refere a suas interações com insetos herbívoros,
Piper é um gênero especialmente rico, o que, em partes, pode estar associada direta e
positivamente com a grande diversidade fitoquímica apresentada por este grupo (Richards et
al. 2015). Com base nessas características, esse gênero tem sido alvo de estudos que abordam
desde os efeitos dos seus metabólitos secundários, individualmente e sinergisticamente (e.g. Dyer et al. 2003; Richards et al. 2010); até estudos que avaliam qual a relação entre diversidade fitoquímica, diversidade de insetos herbívoros e especialização trófica (Richards et al. 2015).
Entre as espécies do gênero Piper, a planta Piper amalago L. é um arbusto perene que varia de 1m a 7m de altura, é encontrado comumente em áreas de Mata Atlântica e interage com muitas espécies de Lepidoptera, em especial às da família Geometridae (Cosmo et al. 2019). Portanto, tanto Piper amalago quanto seus principais herbívoros são ótimos modelos para avaliação de questões ecológicas sobre padrões de diversidade e dano por insetos herbívoros mediados quimicamente.
Nesse contexto, essa dissertação é composta por um capítulo único em que abordamos as seguintes questões: (1) como a variação fitoquímica dentro de indivíduos de plantas influencia a comunidade e o dano por herbívoros? (2) Como a variação fitoquímica entre indivíduos de plantas, na ausência de gradientes ambientais e em pequenas escalas espaciais, molda a estrutura da comunidade e o dano por herbívoros? (3) Entre essas escalas de organização, há diferenças em como a fitoquímica varia e influencia a estrutura da comunidade e o dano por insetos herbívoros? Para isso, utilizamos como modelo a planta Piper amalago L. e seus principais insetos herbívoros, larvas de Lepidoptera (Dyer & Palmer 2004, Cosmo et al. 2019). Nosso estudo obteve três resultados principais. Primeiro, mostramos que mesmo dentro de indivíduos de plantas de uma mesma espécie a variação fitoquímica pode influenciar a estrutura da comunidade de herbívoros e a herbivoria. Em segundo lugar, verificamos que a fitoquímica difere entre indivíduos de plantas em escalas espaciais muito pequenas, quando um gradiente ambiental claro e / ou quimiotipos de plantas distintos estão ausentes, e essa variação fitoquímica também influencia a comunidade de herbívoros e a herbivoria. Por fim, mostramos que dentro de plantas uma menor quantidade de variação fitoquímica é necessária para influenciar o dano e a comunidade de herbívoros do que entre plantas. Juntos, nossos resultados sugerem que variação fitoquímica em escalas espaciais pequenas e de organização menos complexas também é ecologicamente relevante. Além disso, através dessas escalas a paisagem fitoquímica pode ser composta por camadas distintas que são percebidas e têm diferentes efeitos nos padrões de diversidade de insetos herbívoros.
From within the leaf to the community: phytochemical variation drives herbivore community structure and herbivory within and among individual plants
Leandro G. Cosmo¹, Lydia F. Yamaguchi², Massuo J. Kato², Rodrigo Cogni³, Martín Pareja4* Author information: ¹Programa de Pós-Graduação em Ecologia, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, 13083-970, Brazil; ²Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, 05508-000, São Paulo-SP, Brazil; ³Department of Ecology, University of São Paulo, São Paulo, 05508-900, Brazil; 4 Departamento de Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, 13083-970, Brazil *Corresponding author: Martin Pareja, E-Mail:
mpareja@unicamp.br, Phone: +5519-3521-6324, CP 6109.
Running title: Phytochemical variation across scales
Keywords: phytochemical diversity, phytochemical landscape, sub-individual phytochemical variation, intraspecific phytochemical variation, herbivore community, herbivory
Abstract word count: 149; Main text word count: 4997 Number of figures: 4 Number of tables: 1
Author contributions: LGC, MP and RC designed the study. LGC and MP collected field data. LGC, LFY and MJK performed chemical analysis. LGC, MP and RC conducted statistical analysis. LGC wrote the first draft of the manuscript. All authors contributed substantially to further revisions.
Data accessibility statement: Experimental data supporting the results will be archived in the Dryad Digital Repository and the data DOI will be made available should this manuscript be accepted.
Abstract
Phytochemistry may structure herbivorous insects’ communities across different ecological scales. Recent studies suggest that intraspecific phytochemical mosaics along environmental gradients, known as phytochemical landscape, affect herbivorous insects. However, phytochemistry can vary among individual plants at small spatial scales and even within individuals. Here we quantified phytochemical variation among and within individuals of the neotropical shrub Piper amalago L. and its effects on herbivory and its main herbivores - caterpillars. We found that 1) even within individual plants phytochemical variation can drive herbivore community structure and herbivory; 2) phytochemistry can vary among individual plants at small spatial scales and affect herbivore community and herbivory; and 3) within-plants, herbivores respond to finer-scale variation when compared to among plants. Together our results suggest that the effects of phytochemical variation on herbivores can differ across scales and that even a single individual plant can represent an entire "fine-grained" phytochemical landscape for herbivorous insects.
Introduction
How does phytochemical variation shape insect-plant interactions and drive herbivorous insect communities and their leaf damage levels? This is a long-standing question that for the past 50 years has guided research on how phytochemistry affects the ecological and evolutionary processes that drive insect-plant interactions (Ehrlich & Raven 1964, Becerra 2015, Richards et al. 2015). However, answering it remains a challenge because it involves disentangling two major components of phytochemical variation. First, plant chemistry is not a single trait. Instead, phytochemical blends are complex mixtures in which combinations of compounds can vary by different amounts and synergistically affect insect herbivores (Dyer et
al. 2003, Richards et al. 2010, 2012, 2016). Second, ecological variation can mediate distinct
patterns when viewed across different spatial and organizational scales (Levin 1992). The interplay between these two components can give rise to a phytochemical landscape across space and time (Hunter 2016, Glassmire et al. 2019), in which insects are embedded. Thus, phytochemistry may vary across scales in which each one can lead to different patterns of herbivore diversity. This make it essential to address how combinations of compounds vary at different spatial and organizational scales, and which patterns of herbivore diversity and herbivory result from this variation.
Only recently, however, the focus on individual compounds started to shift towards an integrative and synergistic approach, and different spatial and organizational scales of phytochemical variation started to be considered. For instance, Richards et al. (2015) found that at the plant species level, phytochemical diversity (a metric that incorporates both inter and intra molecular complexity of phytochemical blends) increases herbivore diversity and decreases herbivory. Descending one level of organization, Poelman et al. (2009) used principal component analysis to show that at the individual level, variation in the foliar glucosinolate profiles of Brassica oleracea chemotypes affects biodiversity of insect herbivores. Exploring this same level of organization, though across larger spatial scales, Glassmire et al. (2016, 2019) found that phytochemistry can also vary in response to elevational gradients and plant individual proximity to the canopy, which, in turn, affects the herbivore community and even specialist insect population genetic structure. Therefore, environmental gradients and genetic differences can both lead to a highly heterogeneous and unpredictable intraspecific phytochemical landscape which can impact herbivorous insect performance and have community-wide consequences (Hunter 2016, Glassmire et al. 2019).
However, such phytochemical heterogeneity can also emerge at finer spatial and organizational scales. In tropical forests, for instance, multiple variables can affect plant chemistry via subtle microhabitat differences at very small spatial scales, even within a single square meter (Townsend et al. 2008). Therefore, even neighbor individual plants could significantly differ in their foliar chemical blends and harbor different herbivore communities and levels of leaf damage. Although there is evidence for the effects of this type of small-scale phytochemical variation in controlled experiments (Bustos-Segura et al. 2017), we still do not know if they hold true in a field setting, especially in tropical forests. Furthermore, several plant traits can vary at a sub-individual level of organization. For instance, within-plants, leaf morphology, phenolic and flavonoid content, flower nectar content, fruit and seed mass, all have previously been shown to vary (Herrera et al. 2015, Herrera 2017). The ecological consequences of this variation for the interactions of plants with insects, though, still is not well understood (Herrera 2017, Wetzel & Meek 2018). Nevertheless, because insects in general can respond even to very small amounts of phytochemical variation (Pareja et al. 2009) and herbivore performance strongly depends on the variability of their host plant quality (Wetzel et
al. 2016), when phytochemistry varies within-plants this could also emerge as an entire,
“fine-grained” (Herrera 2009) phytochemical landscape. This would suggest that the phytochemical landscape is comprised of several layers across scales which can be distinctly perceived by
herbivorous insects and give rise to complex feedbacks through different spatial and organizational scales. Thus, it is essential to investigate at what organizational and spatial scales phytochemical variation shapes herbivore communities and leaf damage levels, which in turn, can help us understand why such variation is maintained.
In order to address the levels of organization and spatial scales within which phytochemical variation can structure herbivore communities we performed a field study in a Brazilian tropical forest with the neotropical shrub Piper amalago L. and its main insect herbivores – caterpillars. We quantified phytochemical variation in combinations of compounds within and among-plants through principal component analysis, and its effects on the caterpillar community structure and herbivory in both scales. Specifically, we tested the hypothesis that (1) phytochemistry can vary at sub-individual levels and significantly affect herbivore community and herbivory levels within plants, (2) among-individuals, phytochemistry can vary at small spatial scales (e.g. among neighbor plants), and drive herbivore community structure and herbivory, even in the absence of clear environmental gradients and/or distinct chemotypes and (3) the necessary amount of phytochemical variation to affect herbivore communities and herbivory is smaller at the sub-individual scale, i.e., within plants insects respond to finer-scale phytochemical variation that may constitute an entire phytochemical landscape layer. (Figure 1)
Figure 1 – Hypothesized layers of the phytochemical landscape. Variation among-plants give rises to a higher layer, intraspecific, phytochemical landscape. Phytochemical variation within-plants, in turn, emerges as a lower layer, individual phytochemical landscape. The sub-individual layer is nested within the intraspecific layer and may be distinctly perceived by herbivore insects.
Methods
Study site
This study was carried out at the Serra do Japi Municipal Biological Reserve (Jundiaí, São Paulo, 23°14'S 46°58'W). The site covers an area of approximately 354km², and its altitude varies from 900 to 1300m (Pinto 1992). Serra do Japi is an area of remnant Atlantic Forest vegetation and is mostly composed of semi-deciduous mesophyll forests and high-altitude mesophyll forests (Leitão-Filho 1992). Following Koppen (1948), the climate in the reserve is of the Cwa type, characterized as hot and humid, with a dry season from April to September and rainy season from October to March. Average annual temperatures range from 15.7 ° C to
19.2 ° C, with July being the coldest month and January the warmest. The average annual precipitation is approximately 1,600 m, and it peaks in the months of December and January.
Study system
To address our hypothesis, we used the neotropical shrub Piper amalago L. (Piperales: Piperaceae) and its herbivorous caterpillars as a model system. Piper is a species-rich tropical genus of plants that contains about 1,000 species and is specially abundant in the neotropics (Dyer & Palmer, 2004). Among these species, P. amalago is a perennial shrub that ranges from 1m to 7m in height and is abundantly found in Atlantic forest sites (Guimarães & Valente, 2001). Although it is a perennial shrub, this species loses most of its leaves at the end of the dry season, which is followed by a flush of new leaves during the wet season (Cosmo et al. 2019).
Piper species leaves are rich in chemical compounds and this phytochemical diversity has
recently been found to decrease herbivory and increase herbivore species and genetic diversity (Richards et al., 2015; Glassmire et al., 2016; Salazar et al., 2016). Caterpillars (especially of the family Geometridae) are the main Piper herbivorous insects (Dyer and Palmer 2004, Cosmo
et al. 2019). They are common, often dominant in herbivore communities, have low dispersal
capabilities among individual plants, however, can change their feeding behavior within individual plants (Novotny et al. 2006; Pogue 2009, Singer 2016). Therefore, Piper species and their herbivorous caterpillars are excellent models for phytochemical and ecological studies in general (Dyer & Palmer 2004), and in particular for addressing intraspecific and sub-individual variation of both herbivore community structure and herbivory in relation to plant traits.
Plant sampling and study design
To assess how among and within-individual phytochemical variation drives caterpillar community structure and leaf damage levels, we randomly sampled individual P. amalago plants every three months at the study site for one year. Sampling started in July 2017 and ended in April 2018. This approach allowed us to collect data during two months of the dry season (July 2017 and April 2018) and two months of the rainy season (October 2017 and January 2018)at the study site and control for seasonal variation. We sampled 62 plants during each season (124 for the entire study period) and recorded the sampling locations within the study site to control for unmeasured variation among locations. Because Piper plants share several herbivore species, we drew circular plots of 2m radius using each sampled plant as a central point and recorded the total number of individuals of all Piper species. This allowed us to
control for density-dependent effects on caterpillar community structure and leaf damage levels. Moreover, because plant age, total leaf count and proximity to canopy can affect herbivore communities and herbivory (Bowers & Stamp 1993, Glassmire et al. 2019) we measured each plant total height, stem diameter (to estimate plant age) and total number of leaves to control for the potential independent effects of these traits. Each sampled plant was divided in three strata of equal height, starting at the first branch with leaves. Each strata height was defined as: 𝑆𝑡𝑟𝑎𝑡𝑎𝐻= (𝑇𝑜𝑡𝑎𝑙𝐻− 𝐼𝑛𝑖𝑡𝑖𝑎𝑙𝐻)/3, in which 𝑆𝑡𝑟𝑎𝑡𝑎𝐻 is the height of each strata, 𝑇𝑜𝑡𝑎𝑙𝐻 is the total height of the plant and 𝐼𝑛𝑖𝑡𝑖𝑎𝑙𝐻 is the height of the stem from the ground to the first branch with leaves. Categorically, each strata along the plant vertical axis was defined, respectively, as low, medium and high strata. Then, we randomly collected 10 leaves in each strata of the sampled plants (30 leaves per individual plant). Furthermore, we used these strata within individual plants as sampling units to assess within-plant variation in caterpillar community structure and herbivory (n=372), while the individual plants themselves were used as sampling units to assess among-plant variation in these variables (n=124).
Data collection and processing Caterpillar community
To assess caterpillar community structure among and within plants, we manually collected all caterpillars found within each height of the sampled plants. In the field, caterpillars were initially identified as morphospecies and the total count of each morphospecies in each height level were recorded. The caterpillars collected were taken to the laboratory and reared in plastic pots following standard techniques (Cosmo et al. 2014). We characterized caterpillar community structure with three metrics: abundance, here defined as total caterpillar counts, species richness and species diversity, calculated as the Hill’s number of the Shannon-Weiner diversity index (Jost 2006).
Leaf area and leaf damage measurements
To estimate leaf area and leaf damage levels among and within plants we photographed all collected leaves against a white background with a calibrating scale of known measure and used the software ImageJ to measure leaf area (in cm²) from the obtained images. This same software was used to reconstruct leaves that contained chewing herbivore damage and estimate the total leaf area before herbivore damage. We then used the total leaf area before and after
herbivore damage to quantify the leaf area lost for each leaf, here defined as the proportion of total leaf area damaged by chewing herbivores. Finally, to estimate herbivory within and among individual plants we used, respectively, the average loss of leaf area of the 10 collected leaves for each height level, and the 30 collected levels for each individual plant.
Leaf chemical analysis
To estimate phytochemical variation among and within individual plants, we used the same leaves sampled to assess herbivory. After photographing, we placed the 10 leaves collected in each strata of each sampled plant to dry in an oven at 45 ºC for 48h. The dry leaves of each strata, were pooled, weighted and ground to a fine powder in a ball mill (SPEX SamplePrep 2010, Geno, USA). To extract the secondary metabolites: (a) 200 mg of the ground samples were extracted with 100% MeOH; (b) the MeOH brute extracts were centrifuged at 12000 rpm for 10 minutes; (c) the obtained supernatants were separated and step (b) was repeated; and (d) the final supernatants were prepared for HPLC-MS analysis.
The samples were analyzed by liquid chromatography coupled to mass spectrometry (LC/MS). We used a Shimadzu (Kyoto, Japan) liquid chromatograph, which consisted of a LC-20AD pump, SIL-20AHT automatic injector, a UV/Vis SPD-20A detector, CTO-20A column oven and CBM-20A controller. The mobile phase consisted of MeOH (+ 0.1% formic acid) and water with 0.1% formic acid, while the gradient employed was initially 35% MeOH, remaining at this concentration for 2 min. From 2 min to 10 min, the MeOH concentration rose to 60%, from 10 to 20 min the percentage of MeOH rose from 60 to 80%, reaching 100% MeOH in 35 min and remaining for 5 min. The column used was a reverse phase, Kinetex 2.6u PFP 100A, 100 x 1 mm (Phenomenex). The wavelengths monitored were 254 and 330 nm, the furnace for columns was programmed at 40° C. The flow of the mobile phase was 200 μl/min which was infused directly to the mass spectrometer. The mass spectra were acquired in a MicroTOF-QII equipment (Bruker), with an acquisition window between 100 and 1000 m/z. In the positive mode, the energy of the capillary was of 4500 V and in the negative mode of 3500 V. The nebulization and drying gas, N2, were programmed to 4 bar and 8 l/ml, respectively. The energies of the quadrupole and collision were 8 and 10 eV, respectively. Collision RF: 200 Vpp.
Phytochemical data pre-processing and statistical analysis
The HPLC-MS analysis results were pre-processed through the R environment package XCMS (Smith et al. 2006, Tautenhahn et al. 2008, Benton et al. 2010, R Core Team 2018). We
used this package to detect, align and correct the retention time of the chromatographic peaks of our samples. XCMS parameters settings were optimized using the R package IPO (Libiseller
et al. 2015). The integrated peak area for each detected feature in the resulting data was
normalized into proportions, clr-transformed (following Brückner and Heethoff 2017) and submitted to principal component analysis (PCA). Because each phytochemical multivariate sample consisted of our within-plant sampling units, we performed an additional principal component analysis to assess among-plant variation with the mean of each feature peak area for each individual plant. We then used the scores of the sampling units of each PCA analysis as a measure of phytochemical variation. Furthermore, the scaled values of the sum of the integrated peak areas of all features were used as a measure of total secondary metabolite content for each sampling unit. Insects can be affected in different ways by distinct combinations of compounds and by how much these compounds vary (Pareja et al. 2009, Richards et al. 2016, Glassmire et al. 2019), and this approach allowed us to assess how combinations of compounds and their degree of variation (captured by the PCA scores and the proportion of the variation explained by each principal component) affects caterpillar community structure within and among plants. However, in both PCA analysis multiple features contributed equally to the principal components (PCs) loadings which limited our mechanistic interpretations. Furthermore, almost all principal components (i.e. >300 PCs for the within-plants PCA and >100 PCs for the among-plants PCA) were needed to cumulatively explain more than 90% of the total phytochemical variation. For this reason, we limited the principal components included in our statistical analysis to PCs 1-10, which cumulatively explained approximately 50% of the total variation.
To test the effect of phytochemical variation on caterpillar community structure and herbivory within and among plants, we modelled our hypothesized relationships through Linear Mixed Models (LMMs) and Generalized Linear Mixed Models (GLMMs). All statistical analyses were performed in the R environment (R Core Team, 2018). Our modelling approach consisted of four steps. First, we constructed and fitted a global model with an error distribution adequate to the response variable (R package lme4 – Bates et al, 2015). For each response variable the initial fixed and random effects structure consisted of the most complex and biologically reasonable ones that reflected our hypothesis. However, because plant total height and stem diameter were highly correlated with plant leaf numbers, we only included the later in our models to avoid collinearity. Second, to avoid overfitting and reduce model complexity we sequentially removed fixed effect and interaction terms and refitted the models. The
resulting models were compared via their AICc score (R package AICcmodelavg – Mazerolle, 2017) and the ones that had a AICc score < 2 were excluded until we obtained a minimal adequate model. Third, we verified the minimal adequate model assumptions through the normality of the residuals, presence/absence of zero inflation, and temporal and spatial autocorrelation (R package DHARMa – Hartig, 2018). Finally, we assessed the significance of the fixed effects on the minimal adequate model through likelihood-ratio tests (R package afex – Singmann et al, 2018), and constructed partial residual plots of the fixed effects that had statistically significant effects on the response variables (R package visreg – Breheny and Burchett, 2017). This approach allowed us to graphically present our results because partial residual plots show how a dependent variable is affected by a given fixed effect when all others in the model are controlled. Detailed modelling description for each response variables can be found below.
Within-individual variation
To test the effects of within-individual phytochemical variation our initial model fixed effects structure for all response variables consisted of the principal components 1-10 of the within-individual PCA analysis, the scaled values of the total secondary metabolite content of the HPLC-MS analysis, the within-plant strata, the total Piper species density within a 2m radius of the individual plant, and season. To control for variation among sampling locations and because within-individual samples were not independent, we included plant ID nested within location ID as a random intercept for all response variables.
Effects of phytochemical variation on caterpillar community structure
To test the effects of phytochemical variation on the caterpillar community structure, we fitted two GLMMs and one LMM with, respectively, caterpillar counts, caterpillar species richness and caterpillar diversity as response variables. In the first two models we used a Poisson error distribution and because we expected that caterpillar counts and species richness would correlate with leaf numbers, we included the logarithm of the number of leaves as an offset in these models. Likewise, we initially included the number of leaves as a covariate in the LMM with caterpillar diversity as a response variable.
Effects of phytochemical variation on herbivory levels
To test our hypothesis that herbivory is driven by within-plant phytochemical variation, we fitted a linear mixed model with the arcsine square root transformation of herbivory percentages (Crawley, 2012) as a response variable.
Among-individual variation
To test the effects of among-individual phytochemical variation our initial global model fixed effects structure was the same as the within-individual models, with the exception that the plant height levels were not included. As the within-individual models, we included sampling location as a random intercept for all response variables.
Effects of phytochemical variation on caterpillar community structure
The effects of among-individual phytochemical variation on caterpillar abundance, richness and diversity was tested with, respectively, two poisson GLMMs and one LMM. We also included the logarithm of the number of leaves as an offset in the poisson GLMMs and initially the number of leaves as a covariate in the LMM.
Effects of phytochemical variation on herbivory levels
To test the hypothesis that among-plant phytochemical variation drives herbivory, we fitted a linear mixed model with the arcsine square root transformation of herbivory percentages (Crawley, 2012) as a response variable.
Results
Our study has three main findings. First, we found that plant chemical traits can vary even within individual plants and that this variation can drive their associated herbivore community structure and herbivory (Figure 2). Second, among-plants, phytochemical variation at small spatial scales can also drive herbivore community structure and herbivory (Figures 3). Third, herbivore community structure and herbivory may respond to finer scale variation within plants than among plants: within-plants, the principal components that significantly affect herbivore community structure and herbivory explains smaller amounts of phytochemical variation when compared to the among-plant ones. (Figure 4).
Phytochemical variation drives caterpillar community structure and herbivory within-plants
After controlling for the effects of plant strata and season (Table S1 and S3), within-plants caterpillar abundance, species richness, species diversity, and herbivory, were all driven by phytochemical variation as captured by the PCA scores (Figure 2).
Caterpillar abundance significantly decreased with the scores for principal components 2 ,3 and 7 (Figure 2A, 2B, 2C, Table S1 and S3). Species richness also decreased with PC 2 scores (Figure 2D, Table S1 and S3), however, it increased with PC 10 scores (Figure 2E, Table S1 and S3). Caterpillar species diversity was affected by several principal components. Scores of PCs 2, 4, 5 and 8 decreased diversity (Figure 2F, 2G, 2H and 2I, Table S1 and S3), while PC 9 scores increased diversity (Figure 3J, Table S1 and S3). Total secondary metabolite content, alongside PC 10 scores (although marginally), significantly decreased herbivory (Figures 2K and 2L, Table S1 and S3).
Figure 2 - Within-plant phytochemical variation drives (A)-(C) caterpillar abundance; (D)-(E) caterpillar richness; (F)-(J) caterpillar diversity and (K)-(L) herbivory. Points represent partial residuals and shaded bands the 95% confidence intervals. For caterpillar abundance and richness the y axis is in the model link scale and thus, represents log(caterpillar
abundance/number of leaves) and log(caterpillar species richness/number of leaves) respectively.
Phytochemical variation drives caterpillar community structure and herbivory among plants
We found that among-plants, after controlling for seasonal effects (Table S2 and S4), phytochemical variation also drove caterpillar abundance, species diversity, and herbivory. Species richness, however, was not affected by any PC scores (Figure 3).
Among-plants, caterpillar abundance significantly decreased with PCs 1 and 3 scores, and increased with PC 4 scores (Figures 3A, 3B and 3C, Tables S2 and S4). Caterpillar species richness, however, was not significantly affected by any principal component, while caterpillar diversity decreased with PC 1 scores but increased with PC 2 scores (Figure 3D and 3E, Tables S2 and S4). Finally, herbivory increased with PC 5 scores, but decreased with PC 8 scores and plant total secondary metabolite (Figure 3F, 3G and 3H, Tables S2 and S4).
Figure 3 - Among-plant phytochemical variation drives (A)-(C) caterpillar abundance; (D)-(E) caterpillar species diversity and (F)-(H) herbivory. Points represent the partial residuals and shaded bands the 95% confidence intervals. For caterpillar abundance and richness the y axis is in the model link scale and thus, represents log(caterpillar abundance/number of leaves) and log(caterpillar richness/number of leaves) respectively.
Different amounts of phytochemical variation drive caterpillar community structure and herbivory within and among plants
Different principal components that explains varying amounts of phytochemical variation significantly drove caterpillar community structure within and among individual plants.
Within-plants, caterpillar abundance, species richness, species diversity and herbivory were significantly affected by PCs 2-10, which overall explained smaller amounts of phytochemical variation than the PCs 1-4 that significantly affected these variables
among-plants (Figure 4, Table 1). Interestingly, the principal component that explains most of the within-plant phytochemical variation (PC 1, 23,4%), affected neither caterpillar community structure nor herbivory.
Table 1 - Principal components that significantly affect herbivore community and herbivory within and among plants, and their proportional explained variation
Principal components
Variables affected Explained variation
Within-plants Among-plants Within-plant PCA Among-plant PCA PC1 n.s. Abundance and diversity 0.234 0.238 PC2 Abundance, richness and diversity Diversity 0.072 0.060 PC3 Abundance Abundance 0.032 0.051 PC4 Diversity Abundance 0.025 0.041 PC5 Diversity Herbivory 0.017 0.029 PC6 n.s. n.s. 0.015 0.024 PC7 Abundance n.s. 0.013 0.022 PC8 Diversity Herbivory 0.012 0.020 PC9 Diversity n.s. 0.011 0.017 PC10 Richness and herbivory n.s. 0.010 0.017
Figure 4 - Mean proportion of the variation explained of the principal components that significantly affect caterpillar community structure and herbivory within and among-plants. Among-plants caterpillar richness was not affected by any principal component, and thus, was not included in the figure.
Discussion
At what levels of organization and spatial scales does phytochemistry structure herbivore communities? Recently phytochemical variation at plants species and individual levels has been shown to affect herbivore insect communities (Poelman et al. 2009, Bálint et
al. 2015, Richards et al. 2015, Glassmire et al. 2016, 2019). However, how plant chemical traits
vary at sub-individual levels and the consequences of this variation for insect-plant interactions when compared to intraspecific phytochemical variation remains an open question. Here, for the tropical shrub P. amalago and its associated caterpillars, we showed that (1) even within individual plants phytochemical variation can drive herbivore community structure and herbivory; (2) phytochemistry differs among individual plants in the absence of explicit environmental gradients and/or plant chemotypes, which in turn also drives herbivore community and herbivory; and (3) herbivore community and herbivory is driven by finer-scale phytochemical variation within plants when compared to among-plants. Taken together, these results contribute with three key points that advance our understanding of how phytochemistry structures herbivore communities.
First, even within individual plants phytochemical variation can have ecological and evolutionary implications. Our results show that plant chemical traits can vary even within plants and affect both community structure of the herbivorous insects and the resulting leaf damage levels. This suggests that ecological processes that occur at very small spatial scales can cascade through changes that affect individual herbivore insects and have community-wide consequences. Such cascade could be mediated by several mechanisms. For instance, herbivores can plastically change their behavior to feed or oviposit on less chemically defended plant parts (Singer 2016). These behavioral changes are likely to differ between generalist and specialist herbivores because they diverge in their diet breadths and responses to distinct combinations of chemical compounds (Herrera 2009, Richards et al. 2010, 2012, 2016). Insect herbivore mobility can also affect these feeding and oviposition responses. They are more likely to be driven by feeding behavior in insects that can easily disperse within hosts (e.g. most caterpillar species), while insects that spend their entire development within a single plant part (e.g. gall makers and leaf miners) are more likely to differ in their oviposition choices (Herrera 2009, Singer 2016). Thus, within plants both herbivore community and herbivory may be shaped by individual herbivore responses to phytochemical variation.
Furthermore, all these mechanisms can have a genetic basis and affect both insect and plant fitness. Therefore, our results agree with previous suggestions that within-plant trait variation can trigger evolutionary responses in herbivore insects, which, in turn, can affect how trait variation within individual plants evolve (Herrera 2009, 2017). For instance, when herbivores feed on different plant parts and they are not able to distinguish among the ones that are less toxic, they can have longer development times and exposure to predators, lower pupal mass and reduced offspring (Herrera 2009). These fitness costs can select for herbivores to discriminate and selectively feed on plant parts that provide them an optimal resource (Herrera 2009, 2017). Likewise, this variability-caused fitness costs on herbivores can select for unpredictability and thus, heterogeneity and modularity in plant chemical defences, whether these are constitutive or induced (Haukioja et al. 1991, Karban et al. 1997, Shelton 2004, De Kroon et al. 2005, Shelton 2005, Herrera 2017).
Second, our results show that strong environmental gradients and/or distinct chemotypes are not necessary for plant chemistry to vary among individuals (i.e. intraspecifically) and drive herbivore community and herbivory in plants. However, in contrast with similar studies that explicitly included strong environmental gradients and/or different plant chemotypes, we did not find a correlation between any principal component and caterpillar species richness. For
instance, Poelman et al. (2009) found that differences in the glucosinolate profiles among different Brassica oleracea chemotypes affects herbivore abundance, richness and diversity. Conversely, Bálint et al. (2015) showed that different chemotypes of the tansy plant Tanacetum
vulgare affect the structure of an entire arthropod food web. Deviating from the chemotype
perspective, Glassmire et al. (2016, 2019) found that a strong elevational gradient and proximity to the canopy affects phytochemical diversity among Piper kelleyi individuals, which in turn can shape species diversity and population genetic structure of the specialist caterpillar genus
Eois (Geometridae: Larentiinae). Together with our results, this suggests that while small-scale
phytochemical variation due to subtle microhabitat differences can affect the relative abundance of species, and therefore both abundance and diversity of herbivores among individual plants, a higher degree of variation (e.g. due to stronger environmental gradients and/or distinct chemotypes) may be necessary to influence species richness. It is exciting, however, that such small-scale phytochemical variation affects several components of herbivore communities because it opens the question of whether this variation is enough to drive the genetic structure of herbivore populations. Alternatively, although we did not explicitly test this hypothesis, among-plant variation effects could also be a result of within-plant variability. As Herrera (2009, 2017) previously suggested, this could happen, for instance, if insect herbivores have variance sensitive behavior and their among-plant foraging or oviposition decisions depend on within-plant trait variability. Therefore, the effect of phytochemical variation on insect herbivores can be a much more complex process than previously thought, involving ecological and evolutionary feedbacks through different spatial and organizational scales.
Finally, both within and among-individual phytochemical variation occurs at different, yet potentially interlinked spatial scales. However, although both can be interlinked, we found that within-plants herbivore community and herbivory is driven by smaller amounts of phytochemical variation than among-plants. This variation results from multiple chemical features that contributes almost equally to principal component loadings. Although this limits mechanistic interpretations, it provides novel insights into the ecological and evolutionary processes that structure herbivore communities: at very small scales combinations of compounds that vary by small amounts may affect insect herbivores more than the ones that vary the most. Thus, the processes that act upon each one and structure herbivore communities are different, which may result in complex feedbacks across levels of organization. Such feedbacks inevitably raise the question of what scales and variation of combinations of chemical compounds drives herbivore diversity patterns at higher spatial scales. Or, paraphrasing Levin
(1992) words, how do we scale from within the leaf to the community to the landscape and beyond? This landscape, as envisioned by Hunter (2016) and first shown by Glassmire et al. (2019), may be a phytochemical one. However, our results show that for herbivorous insects, even a single individual host plant may represent an entire, “fine-grained” (Herrera 2009) phytochemical landscape. Thus, we expand upon Hunter’s (2016) hypothesis and propose that the phytochemical landscape is comprised of several, potentially interconnected layers across levels of organization and spatial scales. Whether there is information transfer that is perceived by herbivorous insects across these layers, though, remains an open question. Therefore, we argue that to indeed scale from the leaf to the ecosystem (Levins 1992), future studies should focus on how phytochemical variation across different spatial and organizational scales collectively drives diversity patterns of herbivore insects.
Conclusion
Interspecific and intraspecific differences in phytochemistry have recently been show to structure populations and communities of herbivorous insects. We took one step further and showed that sub-individual differences in phytochemistry can structure herbivore communities, likely through ecological and evolutionary processes that cascade through individual insect behavior to have community wide consequences. Furthermore, we showed that strong environmental gradients are not necessary for phytochemistry to vary and drive herbivore community and herbivory. These processes may help us understand how phytochemical diversity is maintained in plants and what are the consequences of very small spatial scale variation in plant chemical traits for both, plant and their insect herbivores species diversity patterns. Together, such implications move us one step forward to answer the long-standing question of why the tropics harbor such a high biodiversity. However, to what extent within-individual phytochemistry drives intraspecific and even interspecific differences in insect herbivore communities is a question that remains unexplored. Therefore, future work should explore how phytochemical variation across different scales synergistically drive herbivore communities so that we can indeed scale from the leaf to the ecosystem and across the phytochemical landscapes.
Acknowledgements
LGC thanks CAPES for partially financing this research and for the MSc fellowship (1688672). LGC, LFY RC, MJK and MP thanks São Paulo Research Foundation (FAPESP). This publication is part of the Biota Fapesp Program through the collaborative grant “Chemically mediated multi-trophic interaction diversity across tropical gradients” (2014/50316-7).
CONCLUSÃO
A variação em atributos químicos de plantas é uma das principais forças condutoras de interações inseto-planta. Porém, os efeitos dessa variação estão sujeitos às escalas espaciais e níveis de organização em que ela ocorre. Neste trabalho nós mostramos que a fitoquímica varia entre indivíduos de plantas em pequenas escalas espaciais e até mesmo dentro de plantas. Em ambas as escalas essa variação influenciou o dano e a comunidade de insetos herbívoros. Porém, essa influência ocorreu de forma distinta: dentro dos indivíduos de plantas uma menor quantidade de variação fitoquímica foi necessária quando comparada à escala intraespecífica. Assim, insetos herbívoros respondem a variações fitoquímica mais refinadas em níveis de organização menos complexos, o que sugere que os efeitos da paisagem fitoquímica dependem da escala espacial e do nível de organização. Ou seja, a paisagem fitoquímica pode ser formada por diferentes camadas através de escalas diferentes, as quais têm efeitos distintos e potencialmente interconectados em padrões de diversidade de insetos herbívoros. Nesse sentido, diferentes escalas da paisagem fitoquímica, suas conexões e seus padrões resultantes de diversidade de insetos herbívoros precisam ser considerados para que então possamos aumentar nossa compreensão dos processos que conduzem interações inseto-planta. De dentro da folha para a comunidade.
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