Biogeography, Evolution and Diversity of Free-Living Protists

Although widely recognized as essential actors of ecosystem processes and representing a significant part of the Earth's biodiversity, free-living protists are poorly known. Major questions concerning their diversity, ecology, and evolution remain completely unresolved, partly because our knowledge of their taxonomy, at each different levels, is still extremely limited. In this thesis, I used testate amoebae as model organisms to get insights into the diversity and biogeography of protists. Testate amoebae are a polyphyletic group of free-living protists that are characterized by a shell composed of proteinaceous, calcareous, or siliceous material. The shells are differentiated by their shapes and ultrastructures, allowing for the identification of morphospecies more easily than in the vast majority of other protists. However, the taxonomic validity of the characters used to define morphospecies and the true diversity of testate amoebae are largely unknown. We studied the taxonomy of cyphoderiid testate amoebae (Rhizaria: Euglyphida), by combining morphological and molecular genetic approaches (chapters 2 and 3). We used light- and scanning electron microscopy to analyze the shell ultrastructure and biometry of several Cyphoderia morphospecies and sequenced gene portions of the Cytochrome Oxidase Subunit 1 (COI) and nuclear small subunit rDNA (SSU). These analyses revealed that while several described species are morphologically distinct and well-supported by the genetic data, other species, such as Cyphoderia ampulla, represent a complex of cryptic or pseudo-cryptic species. The presence of cryptic or pseudo-cryptic Cyphoderia species illustrates the limits of a morphology-based approach for assessing protist diversity using only characters visible by light microscopy. DNA sequence analyses may allow for a better resolution for both species identification and diversity estimates, provided that appropriate genetic markers can be developed. Genetic marker development in protists is particularly challenging because their representation in the international genomic databases is still very limited. The SSU marker also allowed us to build an extensive phylogeny of eulgyhid testate amoebae. We then used this phylogeny as a framework to analyze macro-evolutionary processes in testate amoebae, in particular, how easily a species can cross an ecological barrier and colonize new habitats. The taxa included in our phylogeny occurred either in marine, marine supralittoral or freshwater habitats, and species of each habitat segregated into distinct sub-clades. This indicates that transitions between these environments occur only rarely in the course of evolution (chapter 4). A reliable taxonomy for microorganisms is not only an essential prerequisite for estimating species diversities, it would also be a key component to resolve the long-lasting debate of whether free-living microorganisms are strictly cosmopolitan, or whether at least some species have limited geographic distributions (chapter 1). We analyzed Arcellinida and Euglyphida testate amoeba communities from Amsterdam Island, one of the most remote islands in the world located in the Indian Ocean, to search for endemic species with limited geographic distributions (chapter 5). In macroorganisms, the degree of endemism is generally high in such remote islands. Our extensive inventory of testate amoeba morphospecies provided no clear evidence for endemism. However, given that the morphology-based species identification gives only a limited taxonomic resolution, we cannot exclude the presence of morphologically non-distinctive endemic species (cryptic species). Thus, such taxonomic uncertainties undermine biogeographical studies of testate amoebae and other protists. To circumvent taxonomical uncertainties in biogeographical studies, we analyzed the distribution of a morphologically highly distinctive testate amoeba, Nebela ansata (chapter 6). We report the presence of this species in Nova Scotia, Canada and at sites in New Jersey where it was initially described. An extensive literature survey further confirmed the absence of this species from both, the remaining North American continent as well as other continents. Together, these data provide an unusually convincing case of a free-living microorganism with a very limited distribution range along the temperate East American coast. Overall, my thesis illustrates how, in most cases, the current morphology-based species identification greatly underestimates testate amoeba diversity and calls for an improvement of the taxonomic resolution and reliability. The extension of the current taxonomy using molecular-genetic tools should greatly contribute to this task and also shed light on the debate on the distribution of protists and other microorganisms. Together, such studies will provide a first step towards a better understanding of the diversity and evolution of microorganisms and improve their utility as model organisms in both fundamental and applied questions.

The curse of taxonomic uncertainty in biogeographical studies of free-living terrestrial protists: a case study of testate amoebae from Amsterdam Island

Thierry Jean Heger, Edward Mitchell

Aim A current debate in microbial biogeography contrasts two views concerning the distribution of free-living microorganisms. The first view assumes a ubiquitous distribution, while the second view assumes that at least some species have limited geographical distributions. We tested for limited geographical distributions by identifying testate amoebae morphospecies from an extremely remote oceanic island where the potential for endemism is high. Location Amsterdam Island, Indian Ocean. Methods Sixty moss and water samples collected from the top of the volcano to the lowland were investigated for their testate amoeba content. Due to taxonomical uncertainties among the Argynnia (Nebela) dentistoma species complex (including A. antarctica), we also performed light- and scanning electron microscopy investigations on the shell ultrastructure and biometric analysis on several specimens of this taxon. Results We identified a total of 43 testate amoeba taxa belonging to 15 genera. Only 4 testate amoeba taxa had previously been recorded on this island. Testate amoeba communities of Amsterdam Island are dominated by cosmopolitan ubiquitous euglyphid taxa such as Trinema lineare, Assulina muscorum and Corythion dubium. The length and width ranges for Argynnia dentistoma on Amsterdam Island overlap with other records of this species and of A. antarctica, suggesting that A. antarctica is not a distinct taxon. Main conclusions Although Amsterdam Island is among the most remote islands in the world, an extensive inventory of testate amoebae morphospecies provided no clear evidence for endemism. On the one hand, our detailed morphometic analysis of the Argynnia dentistoma complex revealed that A. antarctica, a morphospecies previously suggested to display endemism, cannot be confidently distinguished from the cosmopolitan morphospecies A. dentistoma. On the other hand, five morphotaxa could not be identified with certainty and might represent new species, potentially with limited distribution. These examples illustrate how taxonomical uncertainties undermine biogeographic studies of testate amoebae. In order to allow better interpretation of morphology-based testate amoeba distribution data, an assessment of genetic diversity among and within morphotaxa in relation to geographic distance for some common testate amoebae should be given high priority.

2009

Molecular analysis of the biodiversity of protists in hydrocarbon-polluted and pristine soils

Enrique Lara

Eukaryotic microorganisms are an essential component of the Earth's global diversity. It is believed that the number of protistan taxa far outnumbers the one of viruses and bacteria. The taxonomic classification of these organisms, which also represent the major part of the total eukaryotic diversity, is still not very well known. It is only lately that the main phylogenetically consistent eukaryotic super assemblages (also called "kingdoms") have been defined. Inside these kingdoms, lateral branches represent the main multicellular taxa, the plants, animals and fungi. Protists are present in virtually all environments; in this thesis, attention was focused on the soil as habitat for the eukaryotic microorganisms. The communities inhabiting soil are characterized by adaptations to drought (the capacity to form cysts). Moreover, certain groups are found exclusively in this environment, to which they are morphologically adapted, whereas others are absent and exclusively found in aquatic systems. Detailed studies on the ecology of certain groups have been performed, and it is possible to define amongst them typical fast reproducing, early colonizers for new environments (r-specialists), whereas others can be found only in stable and highly productive habitats (K-specialists). All these organisms are distributed in a heterogeneous manner in the soil, being usually more frequent in the top horizon and around hotspots such as, for instance, root tips. Prokaryotes have drawn most of the attention of microbial ecologists, who almost neglected the eukaryotic microbes with the exception of fungi. Ecologists usually define four categories ("guilds") of protists in soil: Flagellates, naked amoebae, testate amoebae and ciliates. The importance of protistan activity in soil ecosystems has been shown with the concept of microbial loop: nutrients, produced by autotrophic organisms, are taken up by decomposers (bacteria and fungi) to produce their own biomass. Heterotrophic protists, by preying on them, release these compounds in a form which can be directly used by the autotrophs ("mineralization"), thus closing the loop. This phenomenon is of major significance, because protistan grazing controls the composition and activity of the prokaryotic community, and also the growth of the autotrophs. In soil, this phenomenon takes mainly place at the tip of the roots of vascular plants, where exudates are released. When soils are exposed to an organic pollution, the protistan communities and thus also their impact on the microbial loop, are influenced in two different ways: either by the direct toxic effects of the pollutant and or the indirect effects on their bacterial preys. A detailed study on the negative effects of environmental pollution, however, requires reliable methods to study protists. Molecular approaches offer the possibility to study protists in situ without the limitations inherent to cultivation dependent approaches. The aim of this thesis was to develop molecular methods and to apply these in combination with classical approaches for the characterization of protistan communities in a polycyclic aromatic hydrocarbon contamined and a non-contaminated control soil (PAH). The eukaryotic communities in a PAH polluted and a non-polluted control soil were first compared by amplifying total soil DNA with eukaryotic domain-specific primers targeting the 18S rRNA genes, cloning and sequencing the resulting PCR products. Using non-specific eukaryotic primers proved to be non-applicable for a reliable analysis of the 18S rRNA gene pools in soil, since protistan 18S rRNA gene sequences were (with the exception of the genus Acanthamoeba) underrepresented in the clone libraries obtained.. The biased picture obtained in the first chapter of the thesis was clearly shown when protist were cultivated from both soils: enrichment and cultivation of protists confirmed that members of the genus Acanthamoeba were indeed the most common protists in polluted soils, whereas they were completely absent from the control soil. Additionally, a diverse protistan community was recovered, which could not be detected by the molecular 18S rRNA-based approach. Twenty-two isolates of flagellates and naked amoebas were obtained, and characterised morphologically as well as by 18S rRNA gene sequence analysis. The diversity of cultivated organisms from the polluted soil was lower as compared to the control soil; however, the total number of protists was higher. Some of the cultures obtained in this cultivation dependant approach were used for further detailed studies; a jakobid isolate was described using light and electron microscopy, thus providing new insights into the knowledge of a relatively unknown group of excavates, i.e. the group of Jakoba incarcerata. Other cultures belonging to the kineoplastea were used to design a set of 18S rRNA-targeting fluorescently labelled oligonucleotide probes for fluorescence in situ hybridization. These probes are specific for the detection of the class Kinetoplastea and four genera within the class, i.e. Bodo, Parabodo, Rhynchomonas and Dimastigella and were applied to pure cultures and to environmental samples obtained by the so-called coverslip method. The effect of the PAH-pollution on a selected soil protistan guild, the ciliates, was further analyzed by applying ciliate-specific PCR based techniques. A specific primer set was used to amplify ciliate 18S rRNA genes from the environment and to conduct comparative sequence analysis of environmental clone libraries. This study indicated that the ciliate 18S rRNA gene pools present in the polluted soil were less diverse than those of the control soil. They contained mainly representatives of the class Colpodea, a clade of r-selected ciliates, typical for unstable environments. The control soil was characterized by a variety of trophic specialists, including carnivores, cyanobacteria and diatom feeders, in contrast to the polluted soil, which harboured mainly sequences affiliating to bacterivores and generalists. Based on these results, we could deduce that the trophic webs are less complex in a PAH polluted soil in comparison to a pristine control. This observation was further confirmed when we studied a larger number of samples using a fingerprinting technique, i.e. denaturing gradient gel electrophoresis (DGGE) with ciliate specific PCR primers. The profiles obtained showed again a dominance of the members of the class Colpodea in polluted soils.

EPFL2005

Theoretical and Experimental Studies of Dendritic Metacommunities

Francesco Carrara

The present thesis deals with the understanding of the origins and the mechanisms of maintenance of biodiversity in natural landscapes, in particular by identifying key processes that define large-scale patterns of abundance and diversity. Biological communities often occur in spatially structured habitats where connectivity directly affects dispersal and metacommunity processes. Recent theoretical work suggests that dispersal constrained by the connectivity of specific habitat structures affects diversity patterns and species interactions. This is particularly relevant in dendritic networks epitomized by fluvial ecological corridors. This thesis addresses whether connectivity alone can explain observed features of biodiversity and selectively promote different components of community composition in river-like landscapes, such as local species richness or the among-community similarity. The relevance of this thesis lies in the major ecological challenges posed by the topic, and its fundamental importance to conservation biology. The studies pursued herein are also deemed relevant because of the influence of the spatial connectivity and dispersal on population dynamics and of the relevance of biodiversity to ecosystem functioning. Mechanisms of species coexistence were investigated with a blend of theoretical tools (broadly related to statistical mechanics and the theory of stochastic processes) and experimental work using laboratory microbial communities. The research tools ranged from aspects of modern coexistence theory in a local perspective to the recent concept of the metacommunity in spatial ecology, within a unified framework. The study of biodiversity in riverine ecosystems guided by observational data has been addressed by combining theoretical metacommunity models with laboratory experiments. The results are diverse. First, they show experimentally that connectivity per se shapes key components of biodiversity in metacommunities. Local dispersal in isotropic lattice landscapes homogenizes local species richness and leads to pronounced spatial persistence. By contrast, dispersal along dendritic landscapes leads to higher variability in local diversity and among-community composition. Although headwaters exhibit relatively lower species richness, they are crucial for the maintenance of regional biodiversity. By suitably arranging patch sizes within river-like networks the effect of local habitat capacity (i.e., the patch size) and dendritic connectivity on biodiversity can be experimentally disentangled in aquatic microcosm metacommunities. It is shown in this thesis that species coexistence and community assembly depend on intricate, non-trivial interactions of local community capacity and network positioning. Furthermore, an interaction of spatial arrangement of habitat capacity and dispersal along river-like networks also affects a key ecosystem descriptor, namely regional evenness. High regional evenness in community composition is found only in landscapes preserving geomorphological scaling properties of patch sizes. In riverine environments some of the rarer species sustained regionally more abundant populations and were better able to track their own niche requirements compared to landscapes with homogeneous patch size or landscapes with spatially uncorrelated patch size. All the experimental results were supported and extended by a theoretical analysis where the above mechanisms have been generalized. This thesis provides the first direct experimental evidence that spatially constrained dendritic connectivity is a key factor for community composition and population persistence in riverine landscapes. As such, this thesis assesses a longstanding issue in spatial community ecology. It offers unique insights into the ecological forces structuring natural communities in a key ecosytem, and demonstrates principles that can be further tested in theoretical metacommunity models possibly to be extended to real riverine ecosystems. Taken together, the analyses show how the structure of ecological networks interacts with the spatial environmental matrix in determining biodiversity patterns and the functioning of biological communities. The analyses also suggest that altering the natural linkage between dendritic connectivity and patch size strongly affects community properties at multiple scales. The first part of this thesis (chapters 2 and 3) addresses key aspects of biodiversity-ecosystem functioning research where the combination of theory-guided experiments and theoretical investigations shows how a stochastic implementation of population dynamics proves fundamental for key community properties such as species persistence and community stability. The diversity-productivity and diversity-stability relationships are explored. Both experimental findings and the results of a stochastic model fitted to the experimental interaction matrix, suggest the emergence of strong stabilizing forces when species from different functional groups interact in the same environment, increasing species coexistence and community biomass production. The last part (chapter 6) provides a synthesis of this thesis work, in that it aims at unifying aspects from niche-theory, usually adopted in spatially implicit models, with those characteristic of a spatially explicit context from a typical real-life mountainous regions. It is dedicated to the possible explanation for a macroecological pattern routinely observed from organisms in different domains of life, that is, the mid-elevational peak in local species richness. Guided by empirical observations on diversity of macroinvertebrates in Swiss river basins, a theoretical ansatz is provided which is deemed to capture the essential geomorphological drivers and controls relating species-fitness to altitude. A set of overarching conclusions and perspectives for future research are discussed in the concluding chapter.

EPFL2013