Theme 1: Adaptation
The major features of the living world reflect how organisms have adapted to global changes in environmental conditions. In the past, these have included events such as oxygenic photosynthesis and glaciation. In the immediate future, one of the most pervasive and important global changes that will occur is an increase in atmospheric CO2. This will have very important consequences for plant and algal communities throughout the world, and therefore for the animals, including ourselves, that depend on them. One response to CO2 elevation is likely to be the fertilization of the open oceans with iron, to provide a carbon sink by increasing phytoplankton production. Changes in climate are also expected to increase supply of aerosols and dust to the sea thereby fertilizing the phytoplankton community with iron and providing a carbon sink by increasing autotrophic production. Although the physiological effects of CO2 and Fe have been intensively studied, the evolutionary response of populations over periods of a few hundred generations is essentially unknown. We shall use LE3 to investigate an ongoing process of great scientific interest and deep social concern, to provide a blueprint for the next hundred years of Earth history.
(a) Adaptation to elevated CO2. We aim to identify the physiological and changes likely to occur in lineages exposed to increasing levels of CO2 over 100-1000 generations. Our model system is the unicellular green alga Chlamydomonas reinhardtii, which has been used extensively to investigate the physiology and molecular biology of photosynthesis. We have used this organism for ten years in selection experiments and are familiar with all relevant techniques, including recent developments such as cryopreservation, GFP markers and a Rubisco knockout. The basic concept is to culture lines at increasing concentrations of CO2 on a schedule that mimics the anticipated increase in atmospheric CO2 over the next 100-200 years, scaled for the short generation time of C. reinhardtii (6-8 hours) relative to that of annual plants. Longer-term experiments of up to 1000 generations will predict changes in phytoplankton populations over the same period. We are presently conducting pilot experiments in large-volume liquid batch culture to evaluate buffering, enzyme assays and competition trials. These will be scaled up in LE3. We shall also use solid culture on microwell plates to estimate the entire spectrum of response among hundreds of lines. The laboratory work will be linked to the field projects directed by C. Potvin, who has conducted extensive greenhouse and field investigations of the physiological effects of high CO2.
(b) Adaptation to low Fe. We propose to investigate the evolution of low Fe tolerance in phytoplankton by conducting long-term selection experiments in Fe-limited cultures. As described above, the model organism will be Chlamydomonas reinhardtii, but our initial work, which will be expanded under LE3 as we examine a wider range of species, will focus on coastal and oceanic diatoms (Thalassiosira sp) that differ greatly in their Fe requirements for growth. We have considerable experience in cultivating these and other aquatic microbes in Fe deplete media and in quantifying relevant biochemical and physiological rates. The experimental approach will be to maintain chemostat cultures at low Fe for 100-1000 generations and to use growth assays to document changes in fitness among wild-type and evolved populations. Preliminary results show a doubling in growth rate of an evolved strain of coastal diatom concomitant with a 3-fold reduction in cellular Fe. Iron content, elemental composition, and the rates of Fe acquisition and abundances of the dominant Fe-containing proteins will be determined in each of the evolved strains. Photosynthetic physiology will be evaluated using PAM fluorometry and by measuring rates of C fixation. Complementary experiments will examine adaptation to high Fe concentrations of Chlamydomonas and of species freshly isolated from Fe-deficient waters of the Pacific Ocean. These LE3 experiments will be coordinated with our on going field research program in the subarctic Pacific.
(c) Genetic basis of adaptation in yeast. It is usually very difficult to identify the genetic changes underlying adaptation because target loci are not known. We shall use reverse genetics to approach this issue. A set of 5000 yeast strains (Saccharomyces cerevisiae) has been constructing by deleting a single known gene from an isogenic ancestor. Each strain is viable under laboratory conditions using complete medium. We shall measure the growth of each strain in a wide range of more stressful environmental conditions, identifying the genes essential for growth in each. This experiment will be coupled with a field program designed to discover the natural habitat of wild yeast. In this way we shall be able to study interactions between known genes and defined environmental factors at an unprecedented level of detail.
Theme 2: Diversity
The loss of biodiversity caused by industrial and agricultural development has become a matter of increasing concern over the last decade, and is now not only an area of intensive scientific investigator but also a motor for legal and political initiatives. The contribution of LE3 to this debate will be a deeper understanding of the maintenance and generation of diversity at the levels of genotypes and species.
(a) Maintenance of diversity. We have used Pseudomonas fluorescens to investigate ecological conditions favouring the maintenance of diversity. This bacterium spontaneously generates a range of ecologically differentiated genotypes with distinctive colony morphology, through mutations at structural and regulatory loci affecting cellulose synthesis. It is an excellent model for studying low-level diversity with a simple genetic and phenotypic basis. We have used it to show that diversity is maximized at intermediate levels of productivity and disturbance, provided that the environment is heterogeneous. This provides an attractive synthetic theory of diversity, and we shall continue to develop it within this simple and genetically well-understood context. We shall extend this work to much more complex communities of eukaryotic protists, however, by using LE3 to construct artificial landscapes where patterns of productivity and disturbance can be manipulated at will. This will allow us to perform large-scale tests capable of evaluating and refining the synthetic theory, providing a general platform for interpreting patterns of diversity in natural communities. Appropriate organisms will be identified by our large-scale screens (see below, under Stability). These experiments will use robotic programmable units to construct spatially heterogeneous environments on microwell plates and temporally varying environments in chemostats.
(i) Levels of biodiversity in spatially structured environments should depend on the quantity of environmental variance, its patchiness and the rate of dispersal among sites. Constructing artificial landscapes on microwell plates with complicated spatial structure and transfer patterns is impracticable except with the highly automated procedures that we shall employ, and has not hitherto been attempted.
(ii) Although the potential for temporal coexistence of species populations under non-equilibrium conditions has been recognized for some time, it has generally been considered to be less important than spatial heterogeneity. A microcosm experiment utilizing chemostats to which nutrients are supplied by a robotic programmable unit can be carried out over a time interval of months to years.
The laboratory work will be linked to the biological survey of old-growth forest at Mont St-Hilaire, directed by M. Lechowicz and G. Bell, where we have very detailed information on plant diversity in relation to environmental factors.
(b) Adaptive radiation. Pseudomonas fluorescens can also be used to study adaptive radiation on qualitatively different substrates. We are developing the use of Biolog plates as a device for selecting in many environments simultaneously. These are commercially available microwell plates on which every well is supplied with a different carbon source. Metabolism of a substrate is detected by reduction of a tetrazolium indicator, which can be measured on a plate reader. By assaying a line selected on a given substrate on all other substrates, a constellation of correlated responses can be obtained, defining the consequences of an experimental adaptive radiation. Coupling Biolog plates with an automated plate reader communicating directly with a computer expands the scale of experiments that can be attempted by at least an order of magnitude over previous systems. We shall use LE3 to run highly replicated experiments of this sort. This research will be linked to the study of the adaptive radiation of Carex (sedges) conducted by M. Waterway, where we now have both a detailed nuclear and chloroplast phylogeny of the clade and very detailed information about the distribution, ecology and ecophysiology of about fifty species.
Moreover, we shall use the robotic pipetting facilities of LE3 to create new kinds of multiple-environment plates that will evaluate the correlated response to selection on toxins of different kinds: antibiotics, herbicides, organic pollutants, metals and so forth. An important aspect of global change is the increasing burden of pollutants that natural systems will continue to receive: not only higher concentrations, but a greater diversity of substances. The output of these experiments will be a comprehensive account of the unforeseen long-term consequences of exposure to new substrates or toxins. Using proteomic approaches, we will determine the proteins whose expression is affected and relate this to the observed genetic diversity so that patterns of genetically-programmed responses to these changing environments can be obtained.
(c) Speciation. A new species, ecologically distinct and sexually isolated from its relatives, has never yet been produced in the laboratory. Until this is done, we have no direct means of evaluating the mechanisms responsible for producing species diversity. We believe that this failure can be attributed to using inappropriate organisms, ineffective selection procedures and inadequate replication. We shall use Chlamydomonas reinhardtii, where sex expression is controlled by a single region that has been completely sequenced, and where sexual behaviour is mediated by flagellar agglutinins whose structure is known. We shall apply sexual and natural selection in parallel by selecting in environments that affect flagellar expression. Above all, we shall accommodate the fact that speciation may be a rare event by the massive replication that LE3 will make possible. This project will be strongly linked to the research on incipient speciation in salmonids directed by A. Hendry.
Theme 3. Interaction.
The ocean participates in the global carbon cycle by releasing and absorbing large quantities of CO2. Part of the absorption is mediated by the production of phytoplankton, which consumes CO2 during photosynthesis to make organic C. Heterotrophic bacteria and protozoa respire organic C during growth and are thus completely dependent on phytoplankton for its production. In the contemporary ocean, supply of C to heterotrophs is reduced because production of phytoplankton is limited by the availability of iron. [The efficiency of CO2 absorption by biological uptake in the sea is also greatly reduced for the same reason]. Bacteria, which respire organic C, also require large quantities of Fe for metabolism because their respiration is so highly Fe-dependent and must compete with phytoplankton for this limiting resource. High surface area to volume ratio and production of iron-binding siderophore molecules, as part of a high affinity Fe uptake system, make bacteria superior competitors for Fe in the sea. Low C supply by phytoplankton however may minimize this competitive advantage by reducing the bacteria demand for Fe. We can thus envisage a number of scenarios in which bacteria and phytoplankton develop antagonistic or mutualistic relationships depending on the environments in which they evolve. Model systems, such as those proposed for LE3, are ideally suited to test hypotheses and to develop general principles by which such interactions between organisms may arise.
Our model system will use Pseudomonas and Chlamydomonas in mixed cultures on minimal medium with and without an exogenous organic C source. Our choice of C substrate will depend on the metabolic capabilities of Chlamydomonas, since we want to avoid in the first experiments competition for C between the bacteria and the algae. Iron concentration will then be varied to obtain a range of Fe-limited environments with appropriate replication. Selection will proceed through 100-1000 of generations and the evolved populations will be compared to the wild-type. Pseudomonas is particularly well suited for these experiments in producing fluorescent siderophores that can be quantitatively detected. Measurement of stability constants for Fe complex formation shall enable us to establish whether changes have occurred in the molecular structure of the siderophores.
Theme 4. Stability.
The growing pressure of pollution and biodiversity loss, with other processes such as invasion by exotic species, threatens the integrity of ecosystems, and therefore their ability to deliver the clean air, water and other resources on which society depends. The disruption of foodwebs by eradicating resident species, or by introducing exotic species, may destabilize the system as a whole and thereby cause large and unexpected shifts in its overall properties. Progress in understanding the behaviour of whole ecosystems has been retarded, however, by the lack of the appropriate laboratory model - a trophically complex, materially closed, self-sustaining microcosm, with defined chemistry and biology, that will sit on a lab bench. The difficulties in developing this technology are obvious, but we think that it is important to try because a successful device would have the potential to revolutionize ecology, by making it possible to conduct large-scale, replicated experiments on defined complex ecosystems. The research team includes M. Lechowicz, who has directed the McGill Phytotron for the last ten years, and we can also call on M. Romer, the chief technician for expert advice on the design of controlled environments. We intend to proceed in four steps. First, we shall design a shell that will enable us to monitor community composition, activity and metabolism in sufficient detail to estimate the flows of matter and energy among the components of the system. Secondly, we shall design the interior of the microcosm, at first as a spatially structured aqueous system based on a simple minimal medium. Thirdly, we shall conduct large-scale screens of natural communities of microbes, fungi and micrometazoans for candidate species, which must have desirable ecological properties and be viable as pure cultures in the lab. Finally, we would assemble communities from these candidates by setting up large numbers of microcosms and selecting those with persistent complex structure. The successful microcosms would then provide the platform for a new program in experimental ecosystem ecology.
(a) Species invasions, biotic resistance, and food web assembly. The increasing rate at which human activities are contributing to the invasions of ecosystems by exotic species, and the frequency with which such invasions can lead to fundamental functional changes points to the need to examine this problem in greater detail, and with more systematic approaches than the purely reactive measures that we have used in the past. We are presently developing a set of community assembly models for food webs that are built within an ecosystem framework, and emphasize the importance of functional trade-offs between generalists and specialists along trophic dimensions such as prey size, and tolerance to defence mechanisms and abiotic environmental factors. These fundamental trade-offs affect food web structure, which in turn affects the ecosystem as a whole. The ability to study species invasions, and their effects on communities and ecosystems, through the interplay of theory and microcosm experimentation will greatly clarify our understanding of this increasingly important subject. The experimental work is linked to field programs on the characteristics of invasive aquatic organisms in the St Lawrence Valley (A. Ricciardi).
(b) Dynamics of the detrital system. The importance of detritus processing rates to nutrient cycling, and its effect on the stable functioning of the primary producer community, and the whole ecosystem. In most natural ecosystems, primary producers obtain at least a portion of their nutrient supply from internal cycling--that is nutrients released from the decomposition of detritus. Most ecosystems contain a gradation of detritus pools ranging from rapidly turning over pools of labile organic matter to extremely slow humic matter pools. While the existence of slow pools can lock up a significant portion of the nutrient supply in the system, and thus reduce productivity, it can also enhance stability by damping out oscillations within the food web. The detrital pool has been very difficult to study because of its extremely complicated composition, both chemical and biological. It is arguably the most important single "missing link" in ecosystem ecology. The resources of LE3 will enable us to use defined microbial communities and advanced analytical chemistry to elucidate the impact of detrital processes on the system as a whole.
The development of the laboratory components of all four themes will proceed alongside the development of new theoretical techniques. To understand long-term adaptation, we need theories in which new and unexpected properties can emerge as the result of the cumulation of novel mutations. To understand high diversity, we need theories in which large numbers of species compete in environments with defined spatial and temporal structure. Most difficult of all, understanding the structure of whole ecosystems requires a general theory of foodweb structure, which has so far proven very difficult to obtain. The crucial point is that because species interact within the framework of a complex ecosystem, species populations, and indeed the whole biotic community, must be considered as components or subsystems of the ecosystem as a whole. Studies that address coexistence within a holistic ecosystem framework either theoretically, experimentally, or in empirical field studies are rare, although there is a growing concern in many applied areas, such as fisheries management and conservation, that important environmental factors are being overlooked by traditional population-based approaches. There is at present little consensus about how this synthesis can be achieved. While there have been many successful heuristic models developed to explore relatively simple ecological interactions and evolutionary scenarios, many phenomena are not amenable to simple, analytical solutions. In these cases, individual-based modelling approaches (e.g., models that keep track of the fate individual genes and organisms in populations) could aid in furthering our understanding of complex systems. We shall use the new opportunities provided by the availability of large-capacity high-speed computation to develop individual-based models to address each of these problems. Such models follow the fate of individuals whose behaviour is governed by a few simple rules. The properties of the system then arise naturally from the properties of individuals, rather than being imposed by equations. Individual-based models are the most promising way of handling the emergent properties of highly complex systems such as evolving genomes, evolving quantitative genetic traits, environmental heterogeneity and dispersal, community structure and succession, and community interactions in relation to material transfer, and energy flow. The new experimental systems and the new computer-based theoretical tools could thus advance hand-in-hand.