Phylogeny, systematics and ecology of free living protists: case study: family Hyalospheniidae

Despite the fact that free-living protists compose the major part of Earth’s eukaryotic biodiversity and play numerous essential roles in ecosystems, knowledge on their true diversity, evolution and ecology remain highly limited. <br> In this thesis I choose testate amoebae as a model group to address several key questions on the diversity, evolution and ecology of free-living protists. Family Hyalospheniidae is one of the most species-rich and conspicuous families of testate amoebae combining species with a broad range of test shapes and ultra-structures. Some species are easily identifiable but several species complexes are composed of an unknown number of cryptic and pseudocryptic forms. Thus this is an excellent model group to address open questions on the concept of the species in free-living protists, to assess the validity of characters used to define “true species” and to reveal the true diversity and ecology of these organisms. The studies presented in this thesis are based on the combination of morphological (light and scanning-electron microscopy) and molecular approaches (mitochondrial cytochrome oxidase subunit 1 - COI - sequence data). <br> We used COI to assess the phylogenetic relationships and taxonomy of the family Hyalospheniidae in order to assess the validity of morphological characters within this group (Chapter 1). The COI data successfully separated all studied morphospecies and revealed the existence of several cryptic species. The phylogenetic analysis shows that genus <i>Nebela</i> was paraphyletic and could be split into genus <i>Nebela s.str. </i> and a newly defined genus, <i>Padaungiella</i>. Family Hyalospheniidae Schulze was redefined: Genus <i>Quadrulella</i>, one of the few arcellinid genera building its shell from self-secreted siliceous elements, the mixotrophic <i>Hyalosphenia papilio</i>, and six other genera of true Nebelids (<i>Apodera</i>, <i>Alocodera</i>, <i>Certesella</i>, <i>Nebela</i>, <i>Porosia</i>, <i>Padaungiella</i>) were included in this family, while genera <i>Argynnia</i> and <i>Physochila</i> did not. Thus we redefined the family as Hyalospheniidae Kosakyan et Lara, which now includes <i>Hyalosphenia</i>, <i>Quadrulella</i> (previously in the Lesquereusiidae) and all true Nebelids. We defined the general morphology of the shell and the presence of an organic rim around the aperture as synapomorphies for Hyalospheniidae. <br> Our next task was to analyse in more depth the complex group of species in the Hyalospheniids. <br> We examined the relationship between the morphological and genetic diversity within two species complexes, <i>Nebela collaris s.l. </i> and <i>Quadrulella symmetrica s.l. </i> We combined analyses of light microscopy imaging and COI sequence data obtained from the same individual single cell (Chapters 2 and 3). We showed that small variations in test morphology easily overlooked by traditional taxonomy corresponded to separate, sometimes quite divergent, genotypes. The position of each taxa within species complex was revised, and overall the taxonomy of these two species complexes was redefined. <br> We developed a method to estimate the qualitative and quantitative community structure of <i>Nebela collaris</i> species complex from environmental samples, and validated this approach through microscopic observations (Chapter 4). We assessed the relative biomass and density of species using cloning-sequencing of the mitochondrial cytochrome oxydase (COI) gene amplified from environmental DNA and from artificial communities. Comparisons with direct microscopy counts showed that the COI clone library data were correlated to community counts corrected for biovolume, which allowed making inferences about individual taxon abundance and biomass in a community. We then used this approach to define the ecological niches of closely related /cryptic species in the different microhabitats that compose a peatland complex (Appendix I, contribution as a second author). Our sequence analysis revealed four of the seven barcoded <i>Nebela collaris s.l. </i> species, plus two new genotypes of yet unknown morphology. Species ranged from generalists found in most habitats (e.g. <i>N. collaris</i>) to specialists, encountered only but pervasively in particular habitats (e.g. <i>N. rotunda</i> in forested bogs). Experimental approaches would be needed to assess whether the observed niches correspond to the ecological optimum of the different species or if some at least are pushed towards less favourable habitats by competition. Our study suggests that speciation should have occurred sympatrically by specialization towards divergent niches instead of through geographical isolation. <br> My direct contribution to the taxonomy of this group was the introduction of one new genus <i>Padaungiella</i> Lara et Todorov, and four novel species of <i>Nebela</i>: <i>N. aliciae</i> Mitchell et Lara, <i>N. guttata</i> Kosakyan et Lara, <i>N. meisterfeldi</i> Heger et Mitchell, <i>N. pechorensis</i> Kosakyan et Mitchell. Additionally at least 3 potentially new <i>Quadrulella species</i> will be described (work in progress). <br> Finally we compiled all known taxonomic, molecular and ecological data on hyalospheniid testate amoeba in a monograph entitled “Family Hyalospheniidae” (Chapter 5). Based on a careful revision of historical data combined with recent molecular data, this work aims at establishing a clear state of the art of current knowledge on the diversity of this family, providing improved species descriptions of hyalospheniid testate amoebae and hopefully to familiarize a broader audience with these beautiful protists. <br> Overall my thesis illustrates how traditional taxonomy often underestimates the true diversity of microorganisms, and calls for a renewed research effort on the taxonomy of free-living protists. My work contributes of understanding of a tiny piece of microbial diversity and shows how a combination of morphological and molecular approached can help improving our knowledge on the evolution, systematics and ecology of these organisms.

Thèse de doctorat : Université de Neuchâtel, 2014