Mitchell, Edward

2014, Kosakyan, Anush, Mitchell, Edward

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.
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).
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 Nebela was paraphyletic and could be split into genus Nebela s.str. and a newly defined genus, Padaungiella. Family Hyalospheniidae Schulze was redefined: Genus Quadrulella, one of the few arcellinid genera building its shell from self-secreted siliceous elements, the mixotrophic Hyalosphenia papilio, and six other genera of true Nebelids (Apodera, Alocodera, Certesella, Nebela, Porosia, Padaungiella) were included in this family, while genera Argynnia and Physochila did not. Thus we redefined the family as Hyalospheniidae Kosakyan et Lara, which now includes Hyalosphenia, Quadrulella (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.
Our next task was to analyse in more depth the complex group of species in the Hyalospheniids.
We examined the relationship between the morphological and genetic diversity within two species complexes, Nebela collaris s.l. and Quadrulella symmetrica s.l. 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.
We developed a method to estimate the qualitative and quantitative community structure of Nebela collaris 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 Nebela collaris s.l. species, plus two new genotypes of yet unknown morphology. Species ranged from generalists found in most habitats (e.g. N. collaris) to specialists, encountered only but pervasively in particular habitats (e.g. N. rotunda 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.
My direct contribution to the taxonomy of this group was the introduction of one new genus Padaungiella Lara et Todorov, and four novel species of Nebela: N. aliciae Mitchell et Lara, N. guttata Kosakyan et Lara, N. meisterfeldi Heger et Mitchell, N. pechorensis Kosakyan et Mitchell. Additionally at least 3 potentially new Quadrulella species will be described (work in progress).
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.
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.

2008, Lara, Enrique, Heger, Thierry J., Ekelund, Flemming, Lamentowicz, Mariucz, Mitchell, Edward

To date only five partial and two complete SSU rRNA gene sequences are available for the lobose testate amoebae (Arcellinida). Consequently, the phylogenetic relationships among taxa and the definition of species are still largely dependant on morphological characters of uncertain value, which causes confusion in the phylogeny, taxonomy and the debate on cosmopolitanism of free-living protists. Here we present a SSU rRNA-based phylogeny of the Hyalospheniidae including the most common species. Similar to the filose testate amoebae of the order Euglyphida the most basal clades have a terminal aperture; the ventral position of the pseudostome appears to be a derived character. Family Hyalospheniidae appears paraphyletic and is separated into three clades: (1) Heleopera sphagni, (2) Heleopera rosea and Argynnia dentistoma and (3) the rest of the species from genera Apodera, Hyalosphenia, Porosia and Nebela. Our data support the validity of morphological characters used to define species among the Hyalospheniidae and even suggest that taxa described as varieties may deserve the rank of species (e.g. N. penardiana var. minor). Finally our results suggest that the genera Hyalosphenia and Nebela are paraphyletic, and that Porosia bigibbosa branches inside the main Nebela clade.

2005, Nikolaev, Sergey I, Mitchell, Edward, Petrov, Nikolay B, Berney, Cédric, Fahrni, José, Pawlowski, Jan

Testate lobose amoebae (order Arcellinida Kent, 1880) are common in all aquatic and terrestrial habitats, yet they are one of the last higher taxa of unicellular eukaryotes that has not found its place in the tree of life. The morphological approach did not allow to ascertain the evolutionary origin of the group or to prove its monophyly. To solve these challenging problems, we analyzed partial small-subunit ribosomal RNA (SSU rRNA) genes of seven testate lobose amoebae from two out of the three suborders and seven out of the 13 families belonging to the Arcellinida. Our data support the monophyly of the order and clearly establish its position among Amoebozoa, as a sister-group to the clade comprising families Amoebidae and Hartmannellidae. Complete SSU rRNA gene sequences from two species and a partial actin sequence from one species confirm this position. Our phylogenetic analyses including representatives of all sequenced lineages of lobose amoebae suggest that a rigid test appeared only once during the evolution of the Amoebozoa, and allow reinterpretation of some morphological characters used in the systematics of Arcellinida. (c) 2005 Published by Elsevier GmbH.

2012, Gomaa, Fatma, Todorov, Milcho, Heger, Thierry J., Mitchell, Edward, Lara, Enrique

The systematics of lobose testate amoebae (Arcellinida), a diverse group of shelled free-living unicellular eukaryotes, is still mostly based on morphological criteria such as shell shape and composition. Few molecular phylogenetic studies have been performed on these organisms to date, and their phylogeny suffers from typical under-sampling artefacts, resulting in a still mostly unresolved tree. In order to clarify the phylogenetic relationships among arcellinid testate amoebae at the inter-generic and inter-specific level, and to evaluate the validity of the criteria used for taxonomy, we amplified and sequenced the SSU rRNA gene of nine taxa - Difflugia bacillariarum, D. hiraethogii, D. acuminata, D. lanceolata, D. achlora, Bullinularia gracilis, Netzelia oviformis, Physochila griseola and Cryptodifflugia oviformis. Our results, combined with existing data demonstrate the following: 1) Most arcellinids are divided into two major clades, 2) the genus Difflugia is not monophyletic, and the genera Netzelia and Arcella are closely related, and 3) Cryptodifflugia branches at the base of the Arcellinida clade. These results contradict the traditional taxonomy based on shell composition, and emphasize the importance of general shell shape in the taxonomy of arcellinid testate amoebae.

2007, Lara, Enrique, Heger, Thierry J, Mitchell, Edward, Meisterfeld, Ralf, Ekelund, Flemming

The existing data on the molecular phylogeny of filose testate amoebae from order Euglyphida has revealed contradictions between traditional morphological classification and SSU rRNA phylogeny and, moreover, the position of several important genera remained unknown. We therefore carried out a study aiming to fill several important gaps and better understand the relationships among the main euglyphid testate amoebae and the evolutionary steps that led to the present diversity at a higher level. We obtained new SSU rRNA sequences from five genera and seven species. This new phylogeny obtained shows that (1) the clade formed by species of genera Assulina and Placocista branches unambiguously at the base of the subclade of Euglyphida comprising all members of the family Trinematidae and genus Euglypha, (2) family Trinematidae (Trachelocorythion, Trinema, and Corythion) branches as a sister group to genus Euglypha, (3) three newly sequenced Euglypha species (E. cf. ciliata, E. penardi, and E. compressa) form a new clade within the genus. Since our results show that Assulina and Placocista do not belong to the Euglyphidae (unless the Trinematidae are also included in this family), we propose the creation of a new family named Assulinidae. Consequently, we give a family status to the genera Euglypha and (tentatively) Scutiglypha, which become the new family Euglyphidae. The evolutionary pattern suggested by SSU rRNA phylogeny shows a clear tendency towards increasing morphological complexity of the shell characterised by changes in the symmetry (migration of the aperture to a ventral position and/or compression of the shell) and the appearance of specialised scales at the aperture (in families Trinematidae and Euglyphidae). (C) 2007 Elsevier GmbH. All rights reserved.

2005, Nikolaev, Sergey I., Mitchell, Edward, Petrov, Nikolay B., Berney, Cédric, Fahrni, José, Pawlowski, Jan

Testate lobose amoebae (order Arcellinida Kent, 1880) are common in all aquatic and terrestrial habitats, yet they are one of the last higher taxa of unicellular eukaryotes that has not found its place in the tree of life. The morphological approach did not allow to ascertain the evolutionary origin of the group or to prove its monophyly. To solve these challenging problems, we analyzed partial small-subunit ribosomal RNA (SSU rRNA) genes of seven testate lobose amoebae from two out of the three suborders and seven out of the 13 families belonging to the Arcellinida. Our data support the monophyly of the order and clearly establish its position among Amoebozoa, as a sister-group to the clade comprising families Amoebidae and Hartmannellidae. Complete SSU rRNA gene sequences from two species and a partial actin sequence from one species confirm this position. Our phylogenetic analyses including representatives of all sequenced lineages of lobose amoebae suggest that a rigid test appeared only once during the evolution of the Amoebozoa, and allow reinterpretation of some morphological characters used in the systematics of Arcellinida.

2008, Lara, Enrique, Heger, Thierry J, Ekelund, Flemming, Lamentowicz, Mariusz, Mitchell, Edward

To date only five partial and two complete SSU rRNA gene sequences are available for the lobose testate amoebae (Arcellinida). Consequently, the phylogenetic relationships among taxa and the definition of species are still largely dependant on morphological characters of uncertain value, which causes confusion in the phylogeny, taxonomy and the debate on cosmopolitanism of free-living protists. Here we present a SSU rRNA-based phylogeny of the Hyalospheniidae including the most common species. Similar to the filose testate amoebae of the order Euglyphida the most basal clades have a terminal aperture; the ventral position of the pseudostome appears to be a derived character. Family Hyalospheniidae appears paraphyletic and is separated into three clades: (1) Heleopera sphagni, (2) Heleopera rosea and Argynnia dentistoma and (3) the rest of the species from genera Apodera, Hyalosphenia, Porosia and Nebela. Our data support the validity of morphological characters used to define species among the Hyalospheniidae and even suggest that taxa described as varieties may deserve the rank of species (e.g. N. penardiana var. minor). Finally our results suggest that the genera Hyalosphenia and Nebela are paraphyletic, and that Porosia bigibbosa branches inside the main Nebela clade. (c) 2007 Elsevier GmbH. All rights reserved.

2007, Lara, Enrique, Heger, Thierry J., Mitchell, Edward, Meisterfeld, Ralf, Ekelund, Flemming

The existing data on the molecular phylogeny of filose testate amoebae from order Euglyphida has revealed contradictions between traditional morphological classification and SSU rRNA phylogeny and, moreover, the position of several important genera remained unknown. We therefore carried out a study aiming to fill several important gaps and better understand the relationships among the main euglyphid testate amoebae and the evolutionary steps that led to the present diversity at a higher level.
We obtained new SSU rRNA sequences from five genera and seven species. This new phylogeny obtained shows that (1) the clade formed by species of genera Assulina and Placocista branches unambiguously at the base of the subclade of Euglyphida comprising all members of the family Trinematidae and genus Euglypha, (2) family Trinematidae (Trachelocorythion, Trinema, and Corythion) branches as a sister group to genus Euglypha, (3) three newly sequenced Euglypha species (E. cf. ciliata, E. penardi, and E. compressa) form a new clade within the genus.
Since our results show that Assulina and Placocista do not belong to the Euglyphidae (unless the Trinematidae are also included in this family), we propose the creation of a new family named Assulinidae. Consequently, we give a family status to the genera Euglypha and (tentatively) Scutiglypha, which become the new family Euglyphidae.
The evolutionary pattern suggested by SSU rRNA phylogeny shows a clear tendency towards increasing morphological complexity of the shell characterised by changes in the symmetry (migration of the aperture to a ventral position and/or compression of the shell) and the appearance of specialised scales at the aperture (in families Trinematidae and Euglyphidae).