Mutualistic interactions between species are balanced on a delicate scale of net benefits to both interacting partners. The dynamics of such interactions could change depending on the context in which these interactions occur. One of the most well-studied models for interspecies mutualisms are myrmecophytic systems, also known as ant-plant systems, where the host plant (myrmecophyte) provides shelter (domatia), solely or along with food resources, for ant partners, while the domatia-resident ants intensively patrol and protect the host plant from herbivory. In some cases, nutrient flux has also been reported from the ant-derived debris in the domatia to the host plant. Such mutualisms are often vulnerable to exploitation by non-mutualist organisms or interlopers such as non-protective ants and other invertebrates that use the plant rewards without any returns. Since provision of domatia and food imposes costs on the host plants, the trajectory of evolution in such cases where protection is partial or absent needs investigation to understand the evolution of myrmecophytism. In this thesis, we investigate the possibility of evolution of myrmecophytism in the absence of universal protection by partner ants, using the unique semi-myrmecophyte (domatia are not expressed in all individuals of the species) Humboldtia brunonis as the study model. H. brunonis is endemic to the tropical wet evergreen forests of the Western Ghats of India. Being locally abundant in its distribution range, this plant species has also been used in characterising forest types in the Western Ghats. H. brunonis provides domatia (modified stem internodes) and food for ants in the form of extrafloral nectar (EFN) on leaves and bracts of floral buds in all individuals. Each domatium has a self-opening slit, which could have led to the domatia being accessed and inhabited by numerous ants and other non-ant invertebrates throughout its distribution range. Of these, only one ant species, Technomyrmex albipes, has been reported to be significantly protective against herbivores, and the protection received by the plant is reported to be restricted only to one site where T. albipes is most abundant. In the light of the above, the possible explanation for continued expression of rewards (domatia as well as EFN) in the absence of universal protection was investigated. Chapter 1: Introduction This chapter starts with a brief history of the concept of mutualism, evolution and maintenance of mutualism, and trophic mutualism amongst organisms in general. This is followed by a description of ant–plant mutualisms, and the various interactions that drive the interaction in such systems, with a more detailed emphasis on trophic mutualism in ant-plants, and stable isotope analysis as a technique that is used to study trophic mutualism in ant-plants. The study system, Humboldtia brunonis is introduced, and all the studies on this system preceding this current thesis are discussed in the light of findings in other ant-plant systems. Lastly, the objectives of the thesis are briefly introduced as separate chapters. Chapter 2: Context dependency of rewards and services in an Indian ant–plant interaction: southern sites favour the mutualism between plants and ants (published in Journal of Tropical Ecology) Earlier studies on the H. brunonis system have shown that there is geographic variation in the occupancy of the domatia, with domatia in the northern part of the H. brunonis range being dominantly occupied by an arboreal earthworm species, while domatia in the south are mostly occupied by ants, especially the sole protective ant T. albipes. Further, it has been reported that herbivory is significantly reduced in the presence of ants in the south. In the present study, conducted at 5 sites spanning the distribution range of H. brunonis, we observed that there is a geographic variation in various ant-related plant traits such as abundance of domatia-bearing individuals, number of nectaries per leaf, size of nectaries, and volume and composition of the EFN, with a clear north–south increasing gradient. However, strong protection mutualism was observed only at one site in the south where herbivory pressure was highest. By comparing our results with earlier findings, we show that in addition to geographic variation, there is also temporal variation in the strength of protection mutualism, and that protection mutualism in this system is context-dependent. These results provide new perspectives on the evolution of myrmecophytism. Chapter 3: Leaf expansion and foliar extrafloral nectar as defence strategies in a paleotropical ant-plant Humboldtia brunonis (Fabaceae) (a section of this chapter is submitted to Biotropica) Despite the absence of universal protection against herbivory, H. brunonis plants constitutively secrete EFN and domatia. We therefore explored other non-chemical defences in this system, and investigated possible explanations for the continued reward production. We observed rapid rate of leaf expansion during the early and most vulnerable phase of leaf phenology, and propose this as a strategy to escape herbivory. The young leaves are also subject to being infested by phloem-feeding Hemiptera, but there was seldom any case of ants tending Hemiptera for honeydew (sugary material excreted by the Hemiptera) on the plant. We analysed the sugar and amino acids compositions of EFN, honeydew and phloem sap, and found that EFN composition was much richer and more attractive (to ants) than honeydew, thereby suggesting that EFN could possibly function to distract ants from tending Hemiptera on the plant, thereby avoiding further damage to the plant. We also observed that EFN composition was much richer than phloem sap, and thereby confirmed that EFN is not mere phloem exudate; rather, our results suggests that EFN could possibly be synthesized actively in the secretory cells of the extrafloral nectary. Anatomical observations of the foliar nectaries further support the synthesis of EFN in the secretory cells of the nectary. Chapter 4: Nutritional benefits from domatia-inhabitants in an ant–plant interaction: interlopers do pay the rent (published in Functional Ecology) In this chapter, we explore how a myrmecophytic system could evolve in the absence of protection benefits from the partner ants. We investigate non-protective benefits, specifically trophic (nutrient) benefits, from the protective and non-protective ants and other invertebrates to the host plant, using stable isotope techniques. We selected three representative inhabitant species for our analysis, viz., the protective ant T. albipes, a non-protective ant Crematogaster dorhni, and the arboreal earthworm Perionyx pullus. We observed that earthworms contributed approximately 9% while protective or non-protective ants contributed approximately 17% of the nitrogen to the plant tissues nearest to the domatium. We also observed from 15N labelling experiments that that nutrients from the domatia are not restricted solely to the domatia-bearing branch but could travel to distant non-domatia bearing branches as well. This study demonstrated for the first time that non-protective ants and non-ant invertebrates that inhabit the domatia, and hitherto referred to as interlopers, could be in a trophic mutualism with the host plant, thereby proposing the possibility of trophic mutualism as a factor for the evolution and maintenance of the domatia trait in addition to or in the absence of protection mutualism. It is also possible that fitness benefits of bearing domatia, acquired via trophic mutualism, could later facilitate the establishment of a specialised ant–plant protection mutualism. Chapter 5: Structure and development of the caulinary domatia of Humboldtia brunonis In this chapter, we investigate the morphology of domatia at different ontogenetic stages in order to understand the mode of development of the domatia. Our observations show that the domatium of H. brunonis is formed spontaneously near the terminal end of a growing branch, next to the young apical shoot. It appears as a young swollen internode which is soft and fleshy with the pith tissue still present. As the domatia grows and expands, the collective effect of both schizogeny as well as lysogeny, act on on the pith region. We also observed acropetal lignification of the pith cells around the hollow chamber. We investigate micro-scale anatomy of the inner wall of the domatia using scanning electron microscopy, and observed that the inner lining of the domatia cavity have canaliculated, lignified sclerenchyma with numerous plasmodesmata (intercellular pits) that could facilitate the flow of occupant-derived nutrients supporting trophic interaction between the plant and its domatia inhabitants. We also observed fungal mycelia-like structures in ant-occupied domatia that suggests the possibility of a fungus as a third party in the ant–plant trophic mutualism, as is observed in some other myrmecophytic systems. This aspect however needs further investigation. Chapter 6: Conclusion In this chapter, the main findings of the preceding chapters are summarised. A general conclusion of the thesis is provided, and future directions leading from the present thesis are also listed. The present thesis has explored the dynamics of interactions between a unique semi-myrmecophyte and its domatia-inhabitants; while the unprotected host plant resorts to an escape strategy to evade foliar herbivory, the ants (and other invertebrates) seem to have bid “a farewell to arms” and yet maintain a mutualism with its host via nutrient exchange. The results of this thesis contribute to furthering our current understanding of the evolution and stability of inter-species mutualisms.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第18359号 / 人博第672号 / 新制||人||162(附属図書館) / 25||人博||672(吉田南総合図書館) / 31217 / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 市岡 孝朗, 教授 加藤 眞, 教授 松井 正文 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Ecological interactions between organisms are complex and can present dynamic results, which are dependent on environmental context. Plants with extrafloral nectaries attract a wide variety of ants species, in associations commonly considered mutualistic. In tropical environments the combination of plants and ants play an important role in communities. Turnera subulata is a ruderal shrub widely distributed in disturbed areas, which has a pair of extrafloral nectaries in the base of each leaf. In this study, we evaluated whether the ants associated with T. subulata: (i) vary spatially (between locals) and temporally (over day period); (ii) respond to simulate presence of herbivores and the signs of damage caused in different structures of the host plant (stem and leaf); (iii) reduce herbivory rates; (iv) influencing the vegetative growth and reproductive success of the host plant, along its phenology. The experiments were conducted in São Cristóvão-SE. For the initial understanding of the system studied, T. subulata individuals were selected, in which we performed simulations of the presence of herbivorous and realization of injuries in the stem and leaves of the host plants. The total number of individuals and species associated ants was quantified during three periods of the day. It was quantitated the percentage of leaf area lost and leaves attacked by sucking. They also plot experiments were performed in which were established with and without ants. In each plot were transplanted T. subulata seedlings that were followed throughout their phenological stages. Observations were made in order to quantify the vegetative growth and reproductive investment measures the host plant as well as the number of sheets to damage by sucking insects and chewing; and the number of herbivores. The number of visitors associated with T. subulata was quantified during three periods of the day, being held 5min observation / share. In all cases, data were analyzed using general linear models. 21 species of ants were found. The composition of the ant meeting showed significant variation between locations and time of day. Visitation rates and predation by ants was higher in stems than in the leaves of plants. In general, herbivory rates were not correlated with the association / activity of ants, with the exception of leaf area proportion consumed, which showed a significant reduction in plants where the ants defended the leaves. The results showed that the maturation stage there was a trade-off between growth x propagation of plants; which is favorable for plants (ex.: higher reproduction) who were in plots with ants. Plants on treatment with ants had fewer visitors, including predators and parasitoids. The number of sucking herbivores was significantly reduced in the presence of ants, during the flowering stage. Our results suggest that the benefits of the association may be dependent on context. Moreover, they can contribute to the understanding of the mechanisms involved in facultative interactions between ants and plants and to the understanding of the communities in interaction nets. / Interações ecológicas entre organismos são complexas, podendo apresentar resultados dinâmicos, os quais são dependentes do contexto ambiental. Plantas com nectários extraflorais atraem uma ampla variedade de espécies de formigas, em associações comumente consideradas mutualísticas. Nos ambientes tropicais a associação de plantas e formigas desempenha importante papel nas comunidades. Turnera subulata é um arbusto ruderal, amplamente distribuído em áreas antropizadas, que apresenta um par de nectários extraflorais na base de cada folha. Neste estudo, avaliamos se as formigas associadas a T. subulata: (i) variam espacialmente (entre locais) e temporalmente (ao longo do período do dia); (ii) respondem à simulação da presença de herbívoros e aos sinais de danos ocasionados em diferentes estruturas da planta hospedeira (caule e folha); (iii) reduzem as taxas de herbivoria; (iv) influenciam o crescimento vegetativo e sucesso reprodutivo da planta hospedeira, ao longo da sua fenologia. Os experimentos foram realizados em São Cristóvão-SE. Para o entendimento inicial do sistema estudado, foram selecionados indivíduos de T. subulata, nos quais realizamos simulações da presença de herbívoro e realização de injúrias no caule e folhas das plantas hospedeiras. O número total de indivíduos e de espécies de formigas associadas foi quantificado durante três períodos do dia. Foi quantificada a proporção de área foliar perdida e de folhas atacadas por sugador. Também foram realizados experimentos em que foram estabelecidas parcelas com e sem formigas. Em cada parcela foram transplantadas mudas de T. subulata que foram acompanhadas ao longo de seus estágios fenológicos. Foram feitas observações a fim de quantificar as medidas de crescimento vegetativo e investimento reprodutivo da planta hospedeira, assim como o número de folhas com danos por insetos sugadores e mastigadores; e o número de herbívoros. O número de visitantes associados a T. subulata foi quantificado durante três períodos do dia, durante 5min de observação/parcela. Em todos os casos, os dados foram analisados através de modelos lineares generalizados. Foram encontradas 21 espécies de formigas. A composição da assembleia de formigas apresentou variação significativa no espacialmente e temporalmente. As taxas de visitação e de predação pelas formigas foi maior no caule do que nas folhas das plantas. De forma geral, as taxas de herbivoria não foram correlacionadas com a associação/atividade das formigas, com exceção da proporção de área foliar consumida, que mostrou redução significativa em plantas onde as formigas defenderam as folhas. Os resultados mostraram que no estágio de maturação houve um trade-off entre crescimento x reprodução das plantas; sendo este favorável (ex.: maior reprodução) para as plantas que estavam em parcelas com formigas. Plantas sobre tratamento com formigas tiveram menor número de visitantes, incluindo predadores e parasitoides. O número de herbívoros sugadores foi significativamente reduzido na presença de formigas, durante o estágio de floração. Nossos resultados sugerem que os benefícios da associação podem ser dependentes de contexto. Além disso, podem contribuir para a compreensão dos mecanismos envolvidos nas interações facultativas entre formigas e plantas, e para o entendimento das redes de interações em comunidades.

Orientadores: Paulo Sergio Moreira Carvalho de Oliveira, Sebastian Felipe Sendoya Echeverry / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T06:11:49Z (GMT). No. of bitstreams: 1 Vidal_MayraCadorin_M.pdf: 1986442 bytes, checksum: da9ed52c0a73527b0590731f5ee8d10b (MD5) Previous issue date: 2013 / Resumo: Exploradores do mutualismo - indivíduos que utilizam recursos/serviços produzidos pelos mutualistas sem recompensá-lo - podem trazer sérios danos aos mutualistas explorados, principalmente quando acaba matando um dos parceiros mutualistas. Plantas portadoras de NEFs podem manter mutualismos com formigas visitantes, que defendem a planta contra insetos herbívoros. No cerrado de Itirapina (SP), encontramos larvas de uma nova espécie de díptero do gênero Rhinoleucophenga (Drosophilidae) que constroem abrigos de consistência pegajosa em cima dos NEFs de Q. grandiflora. Assim, larvas de Rhinoleucophenga podem interferir no mutualismo formiga-planta, agindo potencialmente como exploradora dessa interação. O presente estudo teve como objetivos principais investigar a história natural dessas larvas, principalmente aspectos do comportamento e interação com Qualea e formigas, e analisar seu possível efeito sobre o mutualismo formiga-Q.grandiflora. Durante observações de campo comprovamos que formigas e outros insetos visitantes dos NEFs podem ficar presos ao abrigo larval e servir de alimento para o díptero. Larvas de Rhinoleucophenga sp. nov. ocorrem em 85% dos indivíduos de Q. grandiflora, principalmente na época chuvosa ocupando preferencialmente nectários ativos, perto do ápice e na face abaxial dos ramos. No levantamento da mirmecofauna visitante de Q. grandiflora encontramos 27 morfoespécies de formigas, sendo as duas mais frequentes Camponotus crassus, e uma espécie do gênero Brachymyrmex, as mesmas que foram mais comumente encontradas presas aos abrigos das larvas mirmecófagas. Vimos que as larvas expõem uma substância líquida na abertura de seu abrigo, que comprovamos possuir composição química muito similar a do néctar extrafloral de Q. grandiflora, o que sugere que as larvas utilizam o néctar da própria planta para atrair suas presas. Na presença de larvas de Rhinoleucophenga, menos formigas visitam as plantas e também por menos tempo. Esse forrageamento diferenciado resultou em menor ataque de formigas a cupins vivos (herbívoros simulados). Além disso, na presença das larvas mirmecófagas houve maior abundância de herbívoros mastigadores e maior área foliar removida por herbívoros. Podemos afirmar que as larvas de Rhinoleucophenga sp. nov. utilizam o recurso da planta sem beneficiá-la. Além disso, as larvas do díptero também prejudicam a planta e suas formigas mutualísticas, uma vez que alimentando-se delas, aumentam a incidência de herbívoros e a herbivoria foliar na planta. Dessa forma, as larvas de Rhinoleucophenga sp. nov. estão agindo como exploradoras e do mutualismo formiga-Qualea grandiflora e predadoras de topo, causando efeito cascata nesse sistema / Abstract: Exploiters of mutualism - individuals that use resources/services offered by mutualists giving nothing in return - can cause serious damages to mutualists, especially when it involves the death of one of the partners. Plants bearing EFNs usually maintain mutualism with aggressive ants, which defend the plant against herbivores. In a cerrado area at Itirapina (SP), we found a new dipteran species of the genus Rhinoleucophenga (Drosophilidae) whose larvae construct sticky shelters on top of active EFNs of Q. grandiflora. Field observations revealed those ants and others insects that visit the EFNs can get trapped at the sticky larval shelters, and are consumed by the larvae. We hypothesized that Rhinoleucophenga larvae could be interfering with the ant-Qualea mutualism, and thus be acting as an exploiter of this interaction. Here, we investigate the natural history of Rhinoleucophenga larvae, mainly its behavior and association with ants and Qualea, and their possible effect on the ant-Qualea mutualism. Larvae of Rhinoleucophenga sp. nov. occur in 85% of the individuals of Qualea grandiflora inspected at Itirapina. Rhinoleucophenga larvae occur mostly during the rainy season, mainly at the apex and abaxial surface of the branches. We found 27 ant species visiting Qualea. The two most frequent visiting species, Brachymyrmex sp. 1 and Camponotus crassus, were most common insects trapped at larval shelters. Chemical analyses revealed that Rhinoleucophenga larvae use Qualea's extrafloral nectar to attract insect prey to their shelters. Qualea branches infested by ant-preying Rhinoleucophenga larvae had ant visitors for less time and in lower numbers than dipteran-free branches. This negative effect on ant foraging activity resulted in decreased levels of ant aggression to live termite-baits (i.e., simulated herbivores) on leaves of dipteran-infested compared to dipteran-free branches. Controlled field experiments demonstrated that branches hosting Rhinoleucophenga larvae had higher numbers of chewing herbivores and higher levels of foliar herbivory than dipteran-free branches. By using Qualea's EFNs as larval shelters and as attractants to ant prey, larvae of Rhinoleucophenga sp. nov. negatively affect both the plant and ant visitors, with cascading effects ultimately resulting in increased herbivore damage to leaves. Thus we can conclude that ant-eating Rhinoleucophenga larvae are acting as exploiters of the mutualism between ants and Q. grandiflora and also as top predator, causing cascade effect on this system / Mestrado / Ecologia / Mestra em Ecologia

Various aspects of myrmecochory were investigated. Effect of different storage methods on atractiveness of seeds with elaiosomes for ants was examined. Viable seed bank of refuse piles (places where ants deposit unused objects from ant nests, e.g. seeds after the elaiosome was eaten) and places outside them were compared. Chemical content of five major chemical groups, specifically amino acids, free fatty acids, organic acids, polyols and sugars, of elaiosomes and seeds of selected species was determined, and compared with respect to their taxonomic relatedness.

Various aspects of myrmecochory were investigated. Effect of different storage methods on atractiveness of seeds with elaiosomes for ants was examined. Viable seed bank of refuse piles (places where ants deposit unused objects from ant nests, e.g. seeds after the elaiosome was eaten) and places outside them were compared. Chemical content of five major chemical groups, specifically amino acids, free fatty acids, organic acids, polyols and sugars, of elaiosomes and seeds of selected species was determined, and compared with respect to their taxonomic relatedness.