Assessing morphological congruence in Dinobryon species and their stomatocysts, including a newly established Dinobryon pediforme-stomatocyst connection.

The chrysophyte genus Dinobryon Ehrenberg consists of 44 taxa, which occur in freshwaters, rarely marine waters, mostly in temperate regions of the world. The taxa of Dinobryon produce characteristic solitary or dendroid colonies and resting stages called stomatocysts. Only 20 Dinobryon taxa have information on produced stomatocysts and only four stomatocysts are reliably linked with vegetative stages using modern identification standards employing scanning electron microscopy (SEM) analyses. In this study, an encysted material of Dinobryon pediforme (Lemmermann) Steinecke was collected in two lakes in contrasting regions of Poland. Light microscopy (LM) and scanning electron microscopy (SEM) analyses revealed that Dinobryon pediforme produces stomatocyst #61, Piatek J. that is described here as new morphotype following the International Statospore Working Group (ISWG) guidelines. This raises to five the number of reliable links between vegetative stages of Dinobryon species and corresponding stomatocysts. Phenotypic similarities between Dinobryon species and their stomatocysts, analysed for five reliably established links, showed no relationships in size and shape between loricas and stomatocysts belonging to the same species. The morphological characters of loricas and stomatocysts mapped onto the phylogenetic tree of the five Dinobryon species revealed only little congruence between their morphology and phylogenetic relationships.

Remarkably preserved cysts of the extinct synurophyte, Mallomonas ampla, uncovered from a 48 Ma freshwater Eocene lake.

Chrysophyte algae produce a siliceous stage in their life cycle, through either asexual or sexual reproduction, known as a cyst. Cysts form in response to shifts in environmental conditions, population density, or predation pressure, and upon germination provide a seed source for future populations. Cysts are morphologically distinct for each species, and since their remains become part of the sediment or fossil record cysts are valuable tools in ecological and paleolimnological investigations. However, their value as biological indicators is limited because the vast majority of cyst morphotypes have not been linked to specific vegetative species. In the current work, an exquisitely preserved and morphologically complex cyst type is described from a 48 million year old early Eocene fossil site. This finding is remarkable since many of the cysts were still associated with components of the living vegetative cells that produced them, enabling the morphotype to be immediately linked to the synurophyte, Mallomonas ampla. Fusion of identifiable components of the living cell post cyst formation is unknown in modern investigations. The identification of the cyst structure for M. ampla could be valuable in determining cyst morphotypes for other species in the lineage.

Mineralized scale patterns on the cell periphery of the chrysophyte Mallomonas determined by comparative 3D Cryo-FIB SEM data processing.

Unicellular protists can biomineralize spatially complex and functional shells. A typical cell of the photosynthetic synurophyte Mallomonas is covered by about 60-100 silica scales. Their geometric arrangement, the so-called scale case, mainly depends on the species and on the cell cycle. In this study, the scale case of the synurophyte Mallomonas was preserved in aqueous suspension using high-pressure freezing (HPF). From this specimen, a three-dimensional (3D) data set spanning a volume of about 25.6 µm × 19.2 µm × 4.2 µm with a voxel size of 12.5 nm × 12.5 nm × 25.0 nm was collected by Cryo-FIB SEM in 3 h and 24 min. SEM imaging using In-lens SE detection allowed to clearly differentiate between mineralized, curved scales of less than 0.2 µm thickness and organic cellular ultrastructure or vitrified ice. The three-dimensional spatial orientations and shapes of a minimum set of scales (N = 13) were identified by visual inspection, and manually segmented. Manual and automated segmentation approaches were comparatively applied to one arbitrarily selected reference scale using the differences in grey level between scales and other constituents. Computational automated routines and principal component analysis of the experimentally extracted data created a realistic mathematical model based on the Fibonacci pattern theory. A complete in silico scale case of Mallomonas was reconstructed showing an optimized scale coverage on the cell surface, similarly as it was observed experimentally. The minimum time requirements from harvesting the living cells to the final scale case determination by Cryo-FIB SEM and computational image processing are discussed.

Small but Manifold - Hidden Diversity in "Spumella-like Flagellates".

Colourless, nonscaled chrysophytes comprise morphologically similar or even indistinguishable flagellates which are important bacterivors in water and soil crucial for ecosystem functioning. However, phylogenetic analyses indicate a multiple origin of such colourless, nonscaled flagellate lineages. These flagellates are often referred to as "Spumella-like flagellates" in ecological and biogeographic studies. Although this denomination reflects an assumed polyphyly, it obscures the phylogenetic and taxonomic diversity of this important flagellate group and, thus, hinders progress in lineage- and taxon-specific ecological surveys. The smallest representatives of colourless chrysophytes have been addressed in very few taxonomic studies although they are among the dominant flagellates in field communities. To overcome the blurred picture and set the field for further investigation in biogeography and ecology of the organisms in question, we studied a set of strains of specifically small, colourless, nonscaled chrysomonad flagellates by means of electron microscopy and molecular analyses. They were isolated by a filtration-acclimatisation approach focusing on flagellates of around 5 µm. We present the phylogenetic position of eight different lineages on both the ordinal and the generic level. Accordingly, we describe the new genera Apoikiospumella, Chromulinospumella, Segregatospumella, Cornospumella and Acrispumella Boenigk et Grossmann n. g. and different species within them.

Scale evolution in Paraphysomonadida (Chrysophyceae): Sequence phylogeny and revised taxonomy of Paraphysomonas, new genus Clathromonas, and 25 new species.

Heterotrophic chrysomonads of the genus Paraphysomonas are ubiquitous phagotrophs with diverse silica scale morphology. Over 50 named species have been described by electron microscopy from uncultured environmental samples. Sequence data exist for very few, but the literature reveals misidentification or lumping of most previously sequenced. For critically integrating scale and sequence data, 59 clonal cultures were studied light microscopically, by sequencing 18S ribosomal DNA, and recording scale morphology by transmission electron microscopy. We found strong congruence between variations in scale morphology and rDNA sequences, and unexpectedly deep genetic diversity. We now restrict Paraphysomonas to species with nail-like spine scales, establishing 23 new species and eight subspecies (Paraphysomonadidae). Species having base-plates with dense margins form three distinct subclades; those with a simple margin only two. We move 29 former Paraphysomonas species with basket scales into a new genus, Clathromonas, and describe two new species. Clathromonas belongs to a very distinct rDNA clade (Clathromonadidae fam. n.), possibly distantly sister to Paraphysomonas. Molecular and morphological data are mutually reinforcing; both are needed for evaluating paraphysomonad diversity and confirm excessive past lumping. Former Paraphysomonas species with neither nail-like nor basket scales are here excluded from Paraphysomonas and will be assigned to new genera elsewhere.

Structure of loricae and stalks of several bacterivorous chrysomonads (chrysophyceae): taxonomical importance and possible ecological significance.

Stalks and loricae of chrysomonads were studied by fluorescence microscopy employing Calcoflor White-Evans blue (CW-Eb) staining, and by uranyl acetate staining of dried, whole mount preparations for electron microscopy. These structures were composed of microfibrils approximately 4 nm in diameter embedded in a matrix. The organization of the loricae of Poterioochromonas malhamenesis, "Amimonas minuta", Poterioochromonas stipitata and Ochromonas gloeopara showed a similar structural plan, consisting of a foot, stalk and cup region that together resemble a wine glass. CW-Eb-stained, microfibrillar stalks were identified also in Paraphysomonas vestita, Anthophysa vegetans and "Felimonas flocculans". These results suggest that CW-Eb-stained structures composed of microfibrils approximately 4 nm in diameter may be more common in chrysomonads than previously recognized. In cultures, these structures participate in the formation of cell aggregates and attachment of cells to substrates, and thus may be of ecological importance. Additionally, non-siliceous, CW-Eb-stained cysts were identified for the first time in P. malhamensis.

Multigene evidence for the placement of a heterotrophic amoeboid lineage Leukarachnion sp. among photosynthetic stramenopiles.

The colorless amoeboid eukaryote genus Leukarachnion represents one of a long list of microbial lineages for which there have been few taxonomic studies. In this study, we analyze molecular data to assess the placement of a species of Leukarachnion on the eukaryotic tree of life and we report fine structural data to provide additional information on the identity of this taxon. Our multigene analyses indicate that Leukarachnion sp. (ATCC PRA-24) is a member of the stramenopiles, sister to the Chrysophyceae/Synurophyceae clade. It also forms a sister group relationship to the clade containing Chlamydomyxa labyrinthuloides and Synchroma grande, both of which are characterized by net-like amoeboid phases. Leukarachnion sp. and Chlamydomyxa labyrinthuloides also share fine structural cyst morphology such as bilayered structure of the cyst wall. The amoeboid form and heterotrophic habit of Leukarachnion sp. highlight the multiple origins of diverse body forms and multiple plastid losses within the stramenopiles.