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.

Myrmecophytic plant species form associations where the ant colony inhabits structures in the plant and offers protection against herbivory in exchange for food and shelter. Widely distributed across the tropics, myrmecophytic mutualisms are particularly diverse in the Neotropics, a region characterized by the rapid and recent uplift of the Andean mountain range. It has been suggested that the abrupt change in terrain triggered the emergence of new niches, new barriers to gene ow and speciation. Studying ant-plant associations in the Neotropics not only provides insight into how associations evolve in time but also the impact that external factors, such as geographic changes, have in the evolution of mutualisms. Because of its wide distribution on both sides of the Andes, The Tococa guianensis- Azteca system is useful to explore the effects the Andean uplift had on the evolution of mutualisms. This thesis aims to 1. Identify the ants associating with T. guianensis and the lineages of ants and plants involved in the mutualisms in different populations on both sides of the Andes, 2. generate genomic data for both ants and plants to increase sampling of loci, and 3. estimate and calibrate the species trees to compare patterns of phylogenetics and temporal congruence between ants, plants and the Andean uplift. Most ant-plant studies focus on only one partner or study both partners by using already collected data for one of them. This project is the first study inferring the evolutionary history of both partners associated at that point in time and across a large area. This thesis identifies two main Azteca lineages associated with T. guianensis, each one distributed on different sides of the Andes. It addresses the monophyly of T. guianensis (and related species) and why such monophyly cannot be confirmed. Results show how both plants and ants were geographically structured congruent with timing of a split of populations coinciding with the Andean uplift. Moreover, four plants and fifteen ant genomes were assembled and used to estimate gene and species trees. For Tococa, candidate markers were selected for future resolution of the plant's phylogeny. Different histories but similar divergence times between ants and plants suggest that the mutualism has evolved in response to geographic changes rather than through codiversication, but that the mutualism persists thanks to the availability of the host. The information generated during this study provides the basis to understand the evolution of mutualisms, the genomic features of ants and plants and opens the possibility for Tococa and Azteca to become a model system.

Despite a long history of investigations on protective ant-plant interactions, since the late 19century (Thomas Belt 1874), a comprehensive quantitative understanding of the adaptations that facilitate these associations between plants and ants and the differential importance of these adaptations in predicting the benefits and costs to each partner and in shedding light on the evolutionary trajectories of this ecologically widespread interaction is yet to be realized. In the present study we have experimentally shown that the identity of the ant species (T. albipes), the abundance of this ant species and the composition of EFN produced by floral buds and young leaves of H. brunonis plant populations contribute to facilitating protection of floral bud inflorescences and young leaves of H. brunonis populations in a particular site. Of all the 16 ant species that inhabit the domatia of H. brunonis at several sites, the plant has found its protective ant partner only in the dolichoderine ant T. albipes and that too, only in the southernmost site In our experimental set up of three populations of H. brunonis at three different latitudes, where each site is separated from another site by at least 200 km, we have shown that only the southernmost population (Solaikolli) in protected by the ant species T. albipes. Although T. albipes is found in two of the three populations used in the present study (Sampaji and Solaikolli), it is present in a relatively larger abundance both within the domatia of H. brunonis and in the terrestrial stratum only in the southernmost site Solaikolli and is hence able to protect the EFN producing structures of H. brunonis at this site. In comparison, T. albipes is found in low abundance both in the domatia and in the terrestrial stratum in the site Sampaji and does not protect EFN producing plant parts of H. brunonis at this site. We have also experimentally shown that the young leaves of H. brunonis at this southernmost site Solaikolli are protected to a greater extent than the floral bud inflorescences, since T. albipes preferentially utilized EFN produced by young leaves compared to EFN produced by floral buds at this site. Moreover, we have also shown that the floral buds of H. brunonis in the northernmost site are unprotected for two reasons: i. the absence of the protective ant species T.albipes at this site. ii. The EPN produced by some of floral buds at this site is differentially utilized to a much lower extent than floral buds from other sites due to EFN from Agumbe being > 400-fold more viscous than the EFN produced by floral buds at the other two sites (sampaji and Solaikolli) (at 30º C). Although previous studies have independently shown that the identity of the ant partner (Janzen 1966, Schemske 1980, Horvitz and Schemske 1984, Heads 1986, Oliveira et al. 1987a, b; Jaffe et al. 1989, RicoGray and Thien 1989, Davidson et al. 1991) and the abundance of the protective ant species (Koptur 1984, Rocha and Bergallo 1992, Di Giusto et al. 2001) are important in predicting the protective outcome of the interaction between a plant species and its interacting ants, few studies have examined these two factors along with an examination of the EFN volume and composition (Inouye and Inouye 1980, Rudgers and Gardener 2004) and differential utilization of these qualitatively different EFNs produced by different populations of the same ant plant. Humboldtia brunonis and its interacting ant species provides a unique system to address questions about the evolution of ecological specialization and the evolution of preadapted plant traits that facilitate interactions between plants and ants (especially domatia), due to its unique polymorphism for the presence of caulinary domatia and its widespread abundance in the low-elevation wet-evergreen forests of the Western Ghats.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18113号 / 理博第3991号 / 新制||理||1576(附属図書館) / 30971 / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 酒井 章子, 教授 山内 淳, 准教授 永益 英敏 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

<p>Although we often study mutualisms (interactions in which both species benefit) at the level of the individual partners, mutualistic interactions take place in the context of populations and communities. Sharing mutualists with others in a population could result in indirect interactions in the form of mutualist-mediated competition or facilitation. In my dissertation work I asked whether intraspecific competition or facilitation for ants might occur in an extrafloral nectary-bearing (EFN) plant, and what the consequences would be for long-term population dynamics of the plant. My focal species was <italic>Colubrina spinosa</italic> (Rhamnaceae), a neotropical treelet on which I observed 69 ant species at La Selva Biological Station, Costa Rica. </p><p> Demonstrating intraspecific competition for mutualists requires that 1) neighbor densities affect mutualist visits to an individual, and 2) change in mutualist visits results in reduced benefit. To determine how mutualist density affects plant benefit, I experimentally manipulated ant abundances on plants over two years and measured growth and survival. To assess competition for mutualists, I excluded ants from conspecific neighbors and followed ant abundance on focal plants. To consider long-term facilitation, in which greater local nectar resources increase local ant abundance, I manipulated nectar resources in a two-year field experiment and estimated ant abundance on <italic>C. spinosa</italic> plants and on baits. </p><p> Considering local neighbor density both within a 1m radius and in 5x5 m plots, ant densities on <italic>C. spinosa</italic> plants showed evidence for a small-scale competition effect and a contrasting plot-level facilitation effect. The small-scale competition was sized-based; smaller plants lost ants to larger plants. Ant benefit to plants also depended on plant size. For larger plants, those with greater size-adjusted ant density had higher growth and survival than those with fewer ants than expected for their size. </p><p> To determine whether these contrasting competition and facilitation effects could impact population growth or densities, I modeled population dynamics with an integral projection model (IPM). Growth and survival were functions of ant density, which in turn depended on conspecific neighbors, plant size, and mean background ants. Results suggest that larger-scale facilitation of mutualists impacts long-term population growth more than small-scale competition. Population growth rate increased with increasing background ant density, which depended on facilitation at the 5x5m plot scale. In contrast, small-scale competition caused a redistribution of mutualist ants among plants of different sizes, but had very little effect on long-term population growth. </p><p> I thus conclude that on the scale of individuals there is evidence of intraspecific competition for ants as well as facilitation in the EFN plant <italic>C. spinosa</italic>, but only facilitation effects lead to appreciable changes in population dynamics. If mutualist-mediated facilitation effects tend to occur over long time scales in other systems as well, facilitation might prove to be more important than competition in other mutualisms.</p> / Dissertation

<p>Plants commonly produce multiple, seemingly redundant defenses, but the reasons for this are poorly understood. The specificity of defenses to particular herbivores could drive investment in multiple defenses. Alternatively, genetic correlations between defenses could lead to their joint expression, even if possessing both defenses is non-adaptive. Plants may produce multiple defenses if putative resistance traits do not reduce damage, forcing plants to rely on tolerance of damage instead. Furthermore, resource shortages caused by herbivore damage could lead to compensatory changes in expression and selection on non-defense traits, such as floral traits. Natural selection could favor producing multiple defenses if synergism between defenses increases the benefits or decrease the costs of producing multiple defenses. Non-linear relationships between the costs and benefits of defense trait investment could also favor multiple defenses.</p><p>Passiflora incarnata (`maypop') is a perennial vine native to the southeast United States that produces both direct, physical traits (leaf toughness and trichomes) and rewards thought to function in indirect defense (extrafloral nectar in a defense mutualism with ants), along with tolerance of herbivore damage. I performed two year-long common garden experiments with clonal replicates of plants originating from two populations. I measured plant fitness, herbivore damage, and defense traits. I ran a genotypic selection analysis to determine if manipulating herbivore damage through a pesticide exclusion treatment presence mediated selection on floral traits, and if herbivore damage led to plastic changes in floral trait expression. To evaluate the role of selection in maintaining multiple defenses, I estimated fitness surfaces for pairwise combinations of defense traits and evaluated where the fitness optima were on each surface. </p><p>I found that resistance traits did not reduce herbivore damage, but plants demonstrated specific tolerance to different classes of herbivore damage. Tolerance was negatively correlated with resistance, raising the possibility that tolerance of herbivore damage instead of resistance may be the key defense in this plant, and that production of the two type of defense is constrained by underlying genetic architecture. Plants with higher levels of generalist beetle damage flowered earlier and produced proportionally more male flowers. I found linear selection for both earlier flowering and a lower proportion of male flowers in the herbivore exclusion treatment. I found that selection favored investment in multiple resistance traits. However, for two tolerance traits or one resistance and one tolerance trait, investment in only one trait was favored. </p><p>These results highlight the possibility of several mechanisms selecting for the expression of multiple traits, including non-defense traits. Resistance traits may have a non-defensive primary function in this plant, and tolerance may instead be a key defense strategy. These results also emphasize the need to consider the type of trait--resistance or tolerance--when making broad predictions about their joint expression.</p> / Dissertation

Mutualism is an often-complex positive interaction between species, each of which responds independently to varying biotic and abiotic conditions. Temperature is an important factor that can affect species both directly (e.g., physiologically) and indirectly (e.g., via its effects on interactions with consumers, competitors, and mutualists). Although much research has investigated the consequences of temperature for individual organisms, the effects of temperature on the formation, dissolution, and success of species interactions remain minimally understood. The unique ways in which species respond to temperature likely play a role in structuring communities. Environmental heterogeneity, including the thermal environment, can promote coexistence when species exploit resources in different ways, such as by occupying different thermal niches. This dissertation examines the consequences of temperature for participants in an ant-plant protection mutualism, and investigates how the thermal ecology of individual species affects the interaction. Many mutualisms involve multiple species, or interacting guilds. In these mutualisms, species interact with partner species that vary in multiple characteristics. Mutualists are quite sensitive to both partner quantity and partner quality (e.g., their effectiveness at performing a beneficial task). Mutualisms between ants and plants are common across a variety of habitats worldwide, which differ in thermal range, fluctuation, and seasonality. In light of ants’ well-studied and predictable responses to temperature, ant-plant interaction networks provide excellent systems for studying the thermal ecology of mutualisms. In ant-plant protection mutualisms, each of the participants (ants, plants, and enemies) likely differs in its response to temperature. In addition to the direct effects of temperature on ant species, temperature may affect the magnitude of mutualistic interactions among species by affecting the quantity and quality of the reward offered to partners, and the activity of the partners themselves and the plant’s enemies (i.e., herbivores). If herbivores are more thermally tolerant than the mutualistic ant defenders, the consequences for plants may well be severe; however, if herbivores are less thermally tolerant than are the ants, the effects of rising temperatures might be mitigated: although less-effective ants might be more frequent in a warmer world, herbivores would be less abundant there. This dissertation describes the thermal ecology of the participants in a mutualism between the cactus Ferocactus wislizeni and four of its common ant defenders (Forelius pruinosus, Crematogaster opuntiae, Solenopsis aurea, and Solenopsis xyloni) in the extreme environment of the Sonoran Desert, USA. The ants are attracted to extrafloral nectar produced by the plant, and in exchange protect the plants from herbivores, including a common phytophagous cactus bug, Narnia pallidicornis (Hemiptera: Coreidae). Specifically, it investigates how thermal ecology of the individual species affects the interactions among those species. Also, it considers the impact of a tradeoff between behavioral dominance and thermal tolerance among ants.

abstract: For interspecific mutualisms, the behavior of one partner can influence the fitness of the other, especially in the case of symbiotic mutualisms where partners live in close physical association for much of their lives. Behavioral effects on fitness may be particularly important if either species in these long-term relationships displays personality. Animal personality is defined as repeatable individual differences in behavior, and how correlations among these consistent traits are structured is termed behavioral syndromes. Animal personality has been broadly documented across the animal kingdom but is poorly understood in the context of mutualisms. My dissertation focuses on the structure, causes, and consequences of collective personality in Azteca constructor colonies that live in Cecropia trees, one of the most successful and prominent mutualisms of the neotropics. These pioneer plants provide hollow internodes for nesting and nutrient-rich food bodies; in return, the ants provide protection from herbivores and encroaching vines. I first explored the structure of the behavioral syndrome by testing the consistency and correlation of colony-level behavioral traits under natural conditions in the field. Traits were both consistent within colonies and correlated among colonies revealing a behavioral syndrome along a docile-aggressive axis. Host plants of more active, aggressive colonies had less leaf damage, suggesting a link between a colony personality and host plant health. I then studied how aspects of colony sociometry are intertwined with their host plants by assessing the relationship among plant growth, colony growth, colony structure, ant morphology, and colony personality. Colony personality was independent of host plant measures like tree size, age, volume. Finally, I tested how colony personality influenced by soil nutrients by assessing personality in the field and transferring colonies to plants the greenhouse under different soil nutrient treatments. Personality was correlated with soil nutrients in the field but was not influenced by soil nutrient treatment in the greenhouse. This suggests that soil nutrients interact with other factors in the environment to structure personality. This dissertation demonstrates that colony personality is an ecologically relevant phenomenon and an important consideration for mutualism dynamics. / Dissertation/Thesis / Doctoral Dissertation Biology 2018

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