Ulrich G. Mueller - US grants

9983879
Mueller

Fungus-growing ants comprise a group of about 200 species, all obligately dependent on the cultivation of fungus for food. Fungal cultivation by ants involves complex manuring regimes and use of antibiotic "herbicides" to control alien fungi and garden parasites. Recent genetic analyses indicate that fungus-growing ants domesticated fungal cultivars multiple times during their evolutionary history and frequently exchange these cultivars between ant species. The evolutionary ecology of cultivar exchange between ants will be investigated by testing a series of hypotheses on the ecological factors that govern exchanges. Field experiments will be conducted in Panama and the southwestern US, complemented by genetic analyses of the diversity of ants, their cultivars, and associated pathogens. Model ant-fungus systems will be developed to test specific hypotheses on the evolutionary and ecological relationships between ant farmers, their cultivars, associated garden parasites, and the ecological conditions modulating these relationships.

The attine ant-fungus symbiosis represents a unique case of evolutionary and ecological complexity. This complexity derives from the interaction of pathogenic and mutualistic microbes that evolved to form integrated parts of the ecological fabric of a social animal, the ant farmers. The research will yield a synthetic understanding of this 50-million-year antfarmer-cultivar association.

Ant agriculture, exemplified by the symbiosis between fungus-growing (attine) ants and their fungal cultivars, is intensively studied as a model system for understanding the general ecological and coevolutionary mechanisms that shape highly integrated symbioses. The attine ant agricultural symbiosis encompasses both mutualistic and parasitic interactions between (1) ant farmers, (2) fungal cultivars, (3) parasitic fungal "weeds" that infest ant gardens, and (4) antibiotic-producing bacteria that control the garden parasites and are cultured on the bodies of ants. This project specifically explores the ecological complexity of the attine ant-fungus-bacterium symbiosis and elucidates its 50-million-year coevolutionary history by testing hypotheses posed at multiple ecological and evolutionary levels, including: (1) the local ecological level within ant colonies; (2) the population and community levels; (3) the biogeographic level; and the phylogenetic level, including (4) coevolutionary processes and historical ecology; (5) symbiont-mediated speciation; and (6) the effects of symbiotic life histories on rates of evolutionary change. Because symbiosis is a major recurring theme in the history of life, this research is expected to generate general insights into the broad mechanisms that drive biological complexity and diversity. Such insights could in some cases impact issues directly relevant to human welfare, including the evolution of antibiotic resistance and the practice of sustainable agriculture.

Speciation is the basic process that generates biodiversity. In the tropics, leaf-cutter ants and their sister species the lower attine ants comprise a significant proportion of tropical insects, but little is known about their diversification. Patterns of speciation by switching fungal cultivar types between ant species will be examined in three Central American species complexes of lower attine ants (Cyphomyrmex longiscapus, Mycocepurus smithi, and Apterostigma pilosum). The goals of this study are to: split each species complex into valid species using molecular genetic techniques; assess genetic diversity of each species; examine biogeographic patterns of each species complex in Central America; and differentiate between allopatric and sympatric modes of speciation.

This research examines the fundamental process of forming new species. By investigating patterns of diversification in cryptic attine ant species, the attine-fungus mutualism may emerge as a new model system for speciation studies, thus contributing to our understanding of biodiversity. This research will provide training for a graduate student in molecular genetic, entomological, and field techniques, and the graduate student will train and supervise several undergraduate assistants. These results will add new information to the already astonishing level of complexity that has evolved within the attine system over the fifty million years that these ancient farming ants have been cultivating fungi.

This study uses fungus-growing ant species to address host specialization by a virulent pathogen of the ants' fungus gardens. Molecular analyses of host-parasite phylogenetic relationships will be combined with analyses of population differences to determine if parasites specialize on particular host fungal species. Subsequent experiments will be initiated to determine the consequences of host specialization, and analyses of the chemical mechanisms underlying resistance will explain how specialization is maintained. The project will show how mechanisms of resistance and infectivity shape parasite-host specialization and the dynamics of these interacting populations. The research includes collaboration with scientists at domestic and foreign institutions and deposition of collections at museums in Latin America. Undergraduate students will be trained in molecular and experimental techniques.

This study will test three hypotheses concerning the processes that drive diversification of tropical organisms. These hypotheses have been widely discussed and have specific predictions that should be broadly applicable to tropical organisms. Dr. Ulrich Mueller and Scott Solomon will use three species of leafcutter ants in the genus Atta for this project. Specimens will be collected throughout the geographic range of each species, and genetic analyses, conducted in the United States and in Brazil, will test each prediction in turn.

This project will be the first to test these hypotheses with invertebrates, a group that comprises the majority of diversity in many tropical ecosystems. Furthermore, leafcutter ants are a fundamental component of many tropical habitats, and some species are major agricultural pests. By documenting genetic diversity within three such species, this analysis that could reveal the presence of new, cryptic species. Finally, the elucidation of the patterns by which new species are formed is critical for the establishment of effective conservation strategies, since the goal of conservation is to preserve not only existing diversity but also the processes by which diversity is generated.

To a large extent, the history of life is a history of symbiotic interactions, where organisms exist together, either competing or collaborating. The extent to which selfish or cooperative behavior dominates a symbiosis depends on whether the interacting partners' genes are co-transmitted from generation to generation. If strict co-inheritance does not occur, cheating and other forms of virulent behavior can evolve. Using the well-studied intimately co-evolved attine ant-fungal cultivar symbiosis, this study will examine conflict and cooperation between the ants and the cultivar fungi from the perspective of mutualistic and antagonistic evolution theory. In particular, this study will focus on the outcome of the ancient conflict over cultivar escape through fruiting.

Results will be broadly applicable to other symbioses, such as evolution of disease virulence. Additionally, as fungus-gardening ants (the leaf-cutters) are the most important plant pests of the Neotropics, the economic consequences of the research permit collaboration with a Brazilian research group. This collaboration will foster international academic exchange, building a framework for field research and lab visits. Students from both the US and Brazil will participate in the project, receiving training and opportunities for conducting independent research. These international activities are supported by funds from the Office of International Science and Engineering.

Social parasitism, the exploitation of a society by other social organisms, is a
widespread phenomenon that has evolved independently numerous times within
many social systems. The monophyletic Megalomyrmex genus is an excellent
system to investigate how parasitism evolves in a social society. Using an integrative
approach, phylogenic analysis will reveal the origins of social parasitism, while
behavioral, ecological, and venom alkaloid information will help distinguish the
characteristics necessary for social parasitism to evolve and be maintained. The
following questions will be investigated: 1) Did social parasitism evolve once in
the Megalomyrmex genus? 2) Did social parasitism arise from behaviors
related to a primarily predatory lifestyle?

Merit:
By gathering these data the Megalomyrmex system will be comparable with other
parasite systems, and will thus broaden our understanding of the evolutionary
processes associated with the onset and maintenance of host-parasite associations.

Impacts:
This project will continue to incorporate undergraduate mentorship and training.
Ongoing collaborations will strengthen ties between Brazilian and American
institutions while expediting and developing this research. In addition, voucher
specimens have and will be deposited at one domestic and five foreign museums.
These collaborations and associations will not only make the proposed research
feasible but will effectively disseminate scientific knowledge.

Mueller, Ulrich
Klein, Barrett Anthony
NSF DDIG proposal number IOS-0710142


SLEEP IN A SOCIETY: the behavioral ecology of sleep within colonies of insects

Sleep is a phenomenon that greatly impacts the lives of organisms, yet aspects of it are little understood, or greatly ignored by biologists. The very functions of sleep are still in question and few studies have explored how social organization and individual need for sleep may interact. This research will address questions relating to uniquely social aspects of sleep within colonies of honey bees (Apis mellifera). Objectives of this research include mapping sleep, spatially and through the day and night, and testing possible functions of sleep related to communication and acquisition of food. Researchers will individually mark and examine bees in glass observation hives and train bees to food sources, recording behaviors resulting from different experimental regimes within the colony. The expectation is that maps of sleep will reveal differences in sleep, depending on bees' age or function. Further, the researchers will test the hypothesis that sleep-deprived bees will show reduced communication and food collecting performance, suggestive of sleep's specific importance within a social context. The proposed research will contribute to the demystification of sleep and to promote integration across behavioral, societal, ecological, physiological, and medical disciplines. Many undergraduate students will be trained in various aspects of this research and scientific progress will be presented to a wide range of audiences. Sleep is a widespread phenomenon and the proposed research direction will strive to address its relevance across all audiences.

A major unresolved problem in biology is explaining the maintenance of cooperation and mutualism. One unstudied mechanism that stabilizes mutualisms between species is partner choice, where one of the mutualists discriminates between superior versus inferior partners, and rewards the superior types through preferential association. This proposal develops the attine ant-fungus mutualism as a model for the study of symbiont choice. Attine ants cultivate monocultures of fungal clones as their major food source, whereas the fungus receives from the ants both nourishment and protection from pathogens. The proposal develops several ant-fungus systems, including leafcutter ants from the southwestern USA, to study the genetic consequences of symbiont choice exerted by the ants on their fungi. In essence, the proposed experiments ask whether cultivar diversity in ant gardens, arising through genetic mutation, may evolve under an analog of ant-driven "artificial selection" (symbiont choice).

Because symbiosis is a major recurring theme in the history of life, this research is expected to generate insights into the mechanisms that drive biological complexity and diversity. Such insights could impact issues directly relevant to human welfare, including disease evolution and the practice of sustainable agriculture. The case study of the attine ant-fungus mutualism will also be used to promote education of students and the public on the importance of ecological, symbiotic, and ultimately all biological processes.

SYSTEMATICS, PHYLOGENETICS AND THE EVOLUTION OF ASEXUALITY IN
THE FUNGUS-GARDENING ANT GENUS MYCOCEPURUS

The fungus-gardening ant Mycocepurus smithii reproduces exclusively via unfertilized eggs. Strict asexual reproduction makes M. smithii almost unique among insects. Studying M. smithii contributes to our understanding of why sexuality is so common among animals, and why asexuality is rare. The proposed research will test theoretical predictions of obligate and long-term asexuality in an evolutionary context. First, a modern taxonomic revision of the genus will identify existing species and describe new species. Second, a molecular phylogenetic analysis will infer the evolutionary transition from sexuality to asexuality and identify the sexual sister species of M. smithii. Third, M. smithii will be tested for the presence (or absence) of genetic signatures indicative of obligate, long-term asexuality.

The presence of sexual reproduction among most animals is a longstanding puzzle in evolutionary biology. If asexual organisms are able to persist over evolutionary time, they may have evolved a different solution insuring the same benefits of sexuality with two sexes. The proposed research will foster the exchange of information and technology through collaboration with scientists in Latin America. Several undergraduate research assistants will be trained in molecular genetic techniques. Scientific progress will be presented in public outreach programs and scientific meetings. In addition, a taxonomy & phylogeny workshop will be held at the partner Institute in Rio Claro, Brazil.

Certain ants cultivate fungi as their major food source. The ants collect and transport vegetable substrate to a "garden," usually a sheltered chamber excavated in the ground, then plant fungus on this new substrate. Gardens of fungus-growing ants are foci of interactions within a community of ants, the cultivated fungus, and a great diversity of additional microbes. Specifically, pathogens attacking the ants or the fungal gardens can cause the death of the nest community, but a variety of auxiliary microbes contribute disease-suppressing properties that control such pathogens. Disease resistance to pathogens is shaped by the genetic identity of both the ant and the fungus and the interaction of these two dominant community members with the auxiliary microbes. This research will elucidate how these nest community members interact and co-evolve in response to pathogen presence. To understand disease dynamics, the research will (a) characterize the inheritance of assemblages of auxiliary microbes from maternal to offspring ant nest; (b) determine the importance of co-inheritance of ant-fungus-microbe combinations to disease resistance of the community; and (c) evaluate the relative contribution to disease-resistance by ant, fungus, and auxiliary microbes. The research integrates a variety of experimental approaches within an ecological-genetics and quantitative-genetics framework, contributing towards a unification of ecology and evolutionary biology.

Inheritance of auxiliary microbes between generations occurs in diverse hosts, including humans, but the importance of communities of auxiliary microbes in their contribution to the health of a host is incompletely understood. This research develops novel experimental approaches to elucidate the role of auxiliary microbe communities in disease suppression. Because microbial communities also confer disease resistance for humans (e.g., the microbiome of human skin or gut) and for crops (e.g., the microbiome of roots), this research on fungus-growing ants will contribute to the understanding of general microbial principles with applications to human and agricultural disease management. Fungus-growing ants and their microbes will also be used to promote education of students and the public on the importance of ecological, evolutionary, and all biological processes. A postdoctoral researcher and multiple undergraduates will be trained and mentored in this research project.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).


Mutualistic alliances between different organisms generate common goods for the interacting organisms that often exceed the sum of the parts. For example, attine fungus-growing ants provide their fungal cultivars with growth material, shelter, and protection from pathogens, while the fungus in turn serves as the ants' main food source. The fungus also provides the ants with digestive enzymes. Fungal cultivars are transferred to offspring ant nests from parental nests, but on rare occasions cultivars are also passed from one ant species to another ant species, creating new ant-fungus combinations with novel properties and novel common goods. This proposal aims to elucidate the novel common goods that are generated in natural and experimentally induced ant-fungus associations, focusing on enzyme efficiency, growth rate, and health (pathogen resistance) of different ant-fungus combinations. The project therefore elucidates general principles governing mutualism and cooperation in a social, symbiotic organism. Because the project focuses specifically on several ant-fungus mutualisms occurring in the USA, the research also presents rich opportunities in teaching and outreach (e.g., workshops at public schools and nature centers) to promote education of students and the public on the importance of symbiosis in local biodiversity and local environments.

This dissertation research seeks to understand how brain anatomy correlates with task-specialization within ant societies. The research develops a model experimental system of acacia ants, which nest symbiotically in hollow spines of acacia plants, feed on food produced by the plant, and in return protect the plant. Acacia ants are ideal for this study because workers are similar in shape but exhibit distinct behavioral groups that are specialized on different tasks critical for the coordinated functioning of the society. The research specifically tests (1) whether task-specialization correlates with different brain structures; and (2) how society size affects the behavioral and neuro-anatomical specialization among workers. The research contributes to the understanding of animal sociality. The research will be the first to use multiple behavioral groups of an ant species to explore the links between brain and behavior, while controlling for worker morphology. The study will also provide insights into the poorly understood brain anatomy of ants that rely heavily on visual stimuli and visual communication. The investigations involve collaborating researchers in Panama, Costa Rica, and the USA, as well as field sites in Costa Rica and Panama. As part of the research, the doctoral trainee is supervising the Master's thesis of a female student from the Universidad de Costa Rica. Undergraduate students from the University of Texas and a high school student are digitally reconstructing brain anatomy. The trainee also disseminates research results through regular presentations to students, schoolteachers, park rangers, and naturalist guides.

This research will elucidate how the ecological and evolutionary processes of interacting species will respond to climate change at their range limits, with implications for conservation of species in marginal habitat. Climate change alters species distributions, with major consequences for species embedded in complex ecological interactions, such as mutualistic symbioses (e.g., plant-pollinator or host-microbe mutualisms). This project focuses on an insect-fungus mutualism, leafcutter ants cultivating fungi for food, which are agricultural pests in the southwestern USA and throughout the New World. Leafcutter mutualisms are ideal to study how symbioses respond to climate change because leafcutter ants are dominant components in ecosystems, and because experimental ant-fungus combinations can be manipulated under laboratory conditions simulating the altered temperature stresses expected under climate change. This research adapts techniques developed for the study of gene-by-gene interactions within an organism to test whether ant-by-fungus synergy enhances temperature-stress adaptations and thus determines range-limits at the northern (USA) and southern (Uruguay/Argentina) distributional limits of leafcutter ants.

Improved understanding of how interacting species respond to climate change has scientific and societal benefits, contributing to development of models for conservation of species in marginal habitat, and to models predicting whether mutualistic pest species may become more problematic under climate change. Collaborations with researchers in Uruguay and Argentina will provide training of US researchers in an international setting. This project is a collaboration among a major research institution, a regional institution, and an institution serving underrepresented students where some of the field research will be conducted, and will strengthen long term interactions and help foster enhanced STEM education initiatives. Furthermore, collaborations with researchers in Uruguay and Argentina will provide training of US researchers in an international setting. Workshops will be conducted at public schools, museums, and nature centers to foster understanding of local biodiversity, conservation, and challenges under environmental change.

What are the rules to engineer microbiomes to improve health of animals and plants? What organisms use microbiome engineering to improve their own health? Do these organisms use specific mechanisms to assemble beneficial microbes into their microbiomes? Addressing these questions will elucidate general principles of microbiome engineering, with implications for medicine and agriculture. The proposed work will synthesize research on one group of organisms that have practiced microbiome engineering for millions of years, the fungus-growing insects, specifically fungus-growing ants. Fungus-growing insects are farmers. They cultivate gardens of fungal food embedded in complex communities of beneficial microbes that serve nutritive and health functions, but can also include microbial pathogens. Research during the last three decades vastly increased genomic, microbial, physiological, and biochemical understanding of such insect-microbe interactions. There exists therefore now an interest in a comparative synthesis of these diverse host-microbiome associations and the underlying rules of microbiome engineering.

The proposed work will generate two syntheses to elucidate general principles of microbiome assembly and microbiome interactions with the host. The first synthesis will summarize recent microbial, genomic, and biochemical insights on fungus-growing insects to (i) generate an integrative understanding of ecological and evolutionary processes shaping such complex mutualisms; (ii) elucidate unresolved controversies; (iii) evaluate alternate hypotheses underlying these controversies; (iv) outline experimental work to test these hypotheses; and thus (v) identify promising novel research directions. The second synthesis will summarize the biology of fungus-growing ants occurring in the US to enable researchers to address the promising research directions that emerge from the first synthesis. The second synthesis will also integrate 25 years of unpublished observations and photography by the collaborating investigators on the fungus-growing ants of the USA. The second synthesis will therefore enable US researchers to take advantage of their local biodiversity and develop local study systems. The project will also generate visual media on all species of fungus-growing ants in the US and disseminate commons-use images to the public.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.