Claudia Wylezich

Large-scale patterns in biodiversity of microbial eukaryotes from the abyssal sea floor

Eukaryotic microbial life at abyssal depths remains “uncharted territory” in eukaryotic microbiology. No phylogenetic surveys have focused on the largest benthic environment on this planet, the abyssal plains. Moreover, knowledge of the spatial patterns of deep-sea community structure is scanty, and what little is known originates primarily from morphology-based studies of foraminiferans. Here we report on the great phylogenetic diversity of microbial eukaryotic communities of all 3 abyssal plains of the southeastern Atlantic Ocean---the Angola, Cape, and Guinea Abyssal Plains---from depths of 5,000 m. A high percentage of retrieved clones had no close representatives in genetic databases. Many clones were affiliated with parasitic species. Furthermore, differences between the communities of the Cape Abyssal Plain and the other 2 abyssal plains point to environmental gradients apparently shaping community structure at the landscape level. On a regional scale, local species diversity showed much less variation. Our study provides insight into the community composition of microbial eukaryotes on larger scales from the wide abyssal sea floor realm and marks a direction for more detailed future studies aimed at improving our understanding of deep-sea microbes at the community and ecosystem levels, as well as the ecological principles at play.

Molecular Comparisons of Freshwater and Marine Isolates of the Same Morphospecies of Heterotrophic Flagellates

ABSTRACT Heterotrophic flagellates are key components of all ecosystems. Understanding the patterns of biodiversity of these organisms is thus particularly important. Here we analyzed the intraspecific diversity of 10 morphospecies of heterotrophic flagellates comprising representatives of the Apusozoa (2 morphospecies) and Kinetoplastea (8 morphospecies), all belonging to the most common flagellates with worldwide distribution. Most morphospecies showed a mixing of lineages isolated from diverse habitats, indicating that some lineages of these morphospecies had been able to colonize different habitats several times. Furthermore, our results revealed remarkable levels of genetic divergence within most of the morphospecies studied, underlining the difficulty of correctly determining species by means of morphology alone. Many cryptic or pseudocryptic species seem to occur. Our results revealed clear divergence between marine and freshwater lineages of the morphospecies Ancyromonas sigmoides, showing that freshwater lineages have not been able to colonize marine environments and marine lineages have not been able to colonize freshwater environments for a long time.

Protist diversity in suboxic and sulfidic waters of the Black Sea

The oxic-anoxic transition zone of the Black Sea comprises a large suboxic zone as well as anoxic and sulfidic waters. While the prokaryotes and biogeochemical cycles that characterize this zone have been frequently studied, little is known about the diversity or ecology of its microbial eukaryotes. Here, we present the first broad qualitative report of the protist species composition in the Black Sea redoxcline using molecular tools. Fingerprint analysis from the whole redoxcline revealed a complex community structure of metabolically active protists with distinct shifts along the redox gradient. Additionally, 18S rRNA gene clone libraries were used to compare protist species composition of suboxic and sulfidic water layers. Among the ciliates, sequences related to Pleuronema and Strombidium were dominant in both water layers whereas sequences affiliated with anaerobic plagiopylids and Cyclidium were detected only in the sulfidic zone. Among the flagellates, mainly stramenopiles (mostly bicosoecids and chrysophytes) occurred throughout the redoxcline. In the sulfidic zone we found stramenopile sequences but also euglenozoans, jakobids and choanoflagellates that were related to clonal sequences from other anoxic marine habitats, thus indicating the existence of globally distributed groups of anoxic flagellates. Higher species diversity in the sulfidic zone and about twice as many novel sequence types of ciliates and stramenopiles compared with the suboxic layer emphasizes the importance of anoxic, sulfidic waters as habitat for high protist diversity although the function of these organisms is yet unknown.

Ovalopodium desertum n. sp. and the phylogenetic relationships of Cochliopodiidae (Amoebozoa).

An amoeba isolated from a weakly saline semi-desert pond in Kazakhstan (Central Asia) resembles a small Cochliopodium in the light microscope, but has a dorsal fibrous cell coat without scales. Thus it can be identified morphologically as a new species of Ovalopodium Sawyer, 1980, and it is herein named O. desertum. Phylogenetic analysis of the SSU rRNA gene sequences of the new species and four Cochliopodium spp. sequenced additionally shows that Ovalopodium desertum is a sister clade to a robustly monophyletic Cochliopodium. The close relationship between Ovalopodium and Cochliopodium is also confirmed by the analysis of SSU rRNA secondary structure showing the specific helices in the region V5 in all species of both genera. Analysis of actin gene sequences fails to resolve the position of Ovalopodium but demonstrates that Parvamoeba Rogerson, 1993 is probably related to Cochliopodium. The position of Cochliopodiidae within Amoebozoa remains unresolved, despite our efforts to resolve it using broader taxonomic sampling of Amoebozoa, testing alternative tree topologies and removing the fast-evolving sites. Among sequenced genera, Parvamoeba and Endostelium Olive et al., 1984 are probable relatives to Cochliopodiidae. Molecular trees weakly support an inclusion of the family in Flabellinia (Discosea), but more phylogenomic data are necessary to test this hypothesis.

Distribution and Phylogenetic Relationships of Freshwater Thaumatomonads with a Description of the New Species Thaumatomonas coloniensis n. sp.

ABSTRACT. The order Thaumatomonadida includes biflagellated heterotrophic flagellates that form filopodia and typically possess siliceous surface scales. We found thaumatomonads to contribute on average about 5%–10% to flagellate abundance in different benthic habitats. A new species of thaumatomonads, Thaumatomonas coloniensis n. sp., is described on the basis of morphological and molecular biological features. This new species was isolated both from groundwater at Appeldorn near Rees (Germany) and from the Rhine River at Cologne (Germany). We have sequenced the small subunit rRNA (ssu rRNA) gene and a fragment of the large subunit rRNA (lsu rRNA) gene (D3–D5 region) from the isolates of the new species, including the first sequence of a representative of the thaumatomonad genus Gyromitus. In agreement with previous studies, the differences in ribosomal genes of different thaumatomonad species are very small. For understanding the phylogenetic relationships of Thaumatomonadida and to explore their sister group relationships, we have created three sequence data sets (ssu rRNA, partial lsu rRNA, concatenated alignment of both) with the same composition of isolates (from Thaumatomonadida, Euglyphida, Cercomonadidae, and Heteromitidae). According to a Kishino–Hasegawa test, Thaumatomonadida evolved within the Cercozoa as a sister taxon to the Heteromitidae. A possibly close relationship to the Euglyphida, recently grouped together with the Thaumatomonadida in the class Imbricatea/Silicofilosea based on the rRNA data sets was not supported by our analyses.

Unveiling Trophic Functions of Uncultured Protist Taxa by Incubation Experiments in the Brackish Baltic Sea

Abstract Background Our knowledge of the phylogeny and diversity of aquatic protists is rapidly increasing due to molecular surveys and next-generation sequencing approaches. This has led to a considerable discrepancy between the taxa known from cultures and those known from environmental 18S rRNA gene sequences. Hence, it is generally difficult to assign ecological functions to new taxa detected by culture-independent molecular approaches. Methodology/Principal Findings A combination of unamended dark incubations and 18S rRNA sequencing was chosen to link molecular diversity data of uncultured protists with heterotrophic, presumably bacterivorous, growth. The incubations, conducted with Baltic Sea brackish water, resulted in a consistent shift from a protistan community dominated by phototrophs to one in which heterotrophs predominated. This was determined on the basis of cell abundance and 18S rRNA sequences derived from fingerprint analysis and clone libraries. The bulk of enriched phylotypes after incubation were related to hitherto uncultured marine taxa within chrysophytes, ochrophytes, choanoflagellates, cercozoans, and picobiliphytes, mostly represented in recently established or here defined environmental clades. Their growth in the dark, together with coinciding results from studies with a similar objective, provides evidence that these uncultured taxa represent heterotrophic or mixotrophic species. Conclusions/Significance These findings shed some light into the trophic role of diverse uncultured protists especially within functionally heterogeneous groups (e.g., chrysophytes, ochrophytes) and groups that appear to be puzzling with regard to their nutrition (picobiliphytes). Additionally, our results indicate that the heterotrophic flagellate community in the southwestern Baltic Sea is dominated by species of marine origin. The combination of unamended incubations with molecular diversity analysis is thus confirmed as a promising approach to explore the trophic mode of environmentally relevant protist taxa for which only sequence data are currently available.

An Evaluation of the Use of the LSU rRNA D1-D5 Domain for DNA-based Taxonomy of Eukaryotic Protists

Diagnostic signature DNA sequences are important tools for the identification of species. There is an active debate in the literature on the choice of the best markers applicable for a broad range of organisms. Protists have seldom been included in these evaluations. Mitochondrial gene sequences are inappropriate for protists since several groups do not possess mitochondria. Here we studied the application of the large subunit (LSU) rRNA gene fragments (D1-D5) regarding their usefulness to discriminate between a wide range of heterotrophic nanoflagellates. Phylogenetic analyses based on the LSU rRNA fragments showed similar results compared to phylogenetic trees based on the small subunit (SSU) rRNA. The data set indicates the power of the use of the D1-D5 region as a marker for a DNA-based taxonomy. Our results, together with the available sequences in Genbank, form a comprehensive database for unicellular eukaryotes, especially heterotrophic flagellates. It is now possible to assign new sequences to the different groups of heterotrophic flagellates which we have tested for different closely related Cercomonas and Paracercomonas strains from groundwater.

Molecular identification of Nucleophaga terricolae sp. nov. (Rozellomycota), and new insights on the origin of the Microsporidia

Microsporidia are widespread endoparasites of animals, including humans. They are characterized by highly modified morphological and genetic features that cause difficulties in elucidating their enigmatic origin and evolution. Recent advances, however, indicate that the Microsporidia have emerged from the Rozellomycota, forming together either the most basal lineage of the Fungi or its closer relative. The Rozellomycota comprise a huge diversity of uncultured environmental clones, with a very few known species endoparasitic of algae and water moulds, like the chytrid-like Rozella, and of free-living amoebae, like Nucleophaga and the microsporidia-like Paramicrosporidium. A possible ancestral microsporidium, Mitosporidium, has recently been described from the water flea Daphnia, since the phylogenomic reconstruction showed that it branches to the root of the microsporidian tree, while the genome analysis revealed a fungal-like nuclear genome and the persistence of a mitochondrial genome. Here we report the 18S rDNA molecular phylogeny of an additional microsporidium-like endoparasite of amoebae, which has a developmental cycle almost identical to that of Nucleophaga amoebae. Our results show that the endoparasite is closely related to N. amoebae, forming a distinct species, for which we propose the name Nucleophaga terricolae. Furthermore, the Nucleophaga lineage is recovered as sister to the Microsporidia while Mitosporidium turns out to be member of a well-supported group of environmental clones. These results raise the question about the actual ancestry of the Microsporidia within the Rozellomycota. A precise and robust phylogeny will require further comparative genomic studies of these various strains, and should also consider the primitive microsporidia, for which genetic data are still lacking, because all these organisms are essentially morphologically similar.

High genetic diversity of Sappinia-like strains (Amoebozoa, Thecamoebidae) revealed by SSU rRNA investigations

Sappinia diploidea is known as a free-living amoeba of worldwide distribution and has also been reported as causative agent of a brain infection in an immunocompetent young man. In the current study, we were able to isolate eight strains of S. diploidea-like amoebae identified by light microscopy from different habitats. Cultures of all strains were established successfully for molecular characterization. The small subunit ribosomal RNA genes of all strains were sequenced and compared to one another, to the neotype of S. diploidea, and to strains of Sappinia pedata, the only other Sappinia species known to date, from GenBank by multiple sequence alignment and cluster analysis. Altogether, the phylogenetic position of the genus Sappinia within the Thecamoebidae was corroborated; however, it was shown that the genus splits into several well-separated clusters making the establishment of new species within this genus inevitable. Furthermore, two of the S. diploidea-like strains were actually more closely related to S. pedata than to S. diploidea, although the diagnostically relevant standing form which seems to be characteristic for S. pedata was not observed in either of the two strains.

Ecologically relevant choanoflagellates collected from hypoxic water masses of the Baltic Sea have untypical mitochondrial cristae

BackgroundProtist communities inhabiting oxygen depleted waters have so far been characterized through both microscopical observations and sequence based techniques. However, the lack of cultures for abundant taxa severely hampers our knowledge on the morphology, ecology and energy metabolism of hypoxic protists. Cultivation of such protists has been unsuccessful in most cases, and has never yet succeeded for choanoflagellates, even though these small bacterivorous flagellates are known to be ecologically relevant components of aquatic protist communities.ResultsQuantitative data for choanoflagellates and the vertical distribution of Codosiga spp. at Gotland and Landsort Deep (Baltic Sea) indicate its preference for oxygen-depleted zones. Strains isolated and cultivated from these habitats revealed ultrastructural peculiarities such as mitochondria showing tubular cristae never seen before for choanoflagellates, and the first observation of intracellular prokaryotes in choanoflagellates. Analysis of their partial 28S rRNA gene sequence complements the description of two new species, Codosiga minima n. sp. and C. balthica n. sp. These are closely related with but well separated from C. gracilis (C. balthica and C. minima p-distance to C. gracilis 4.8% and 11.6%, respectively). In phylogenetic analyses the 18S rRNA gene sequences branch off together with environmental sequences from hypoxic habitats resulting in a wide cluster of hypoxic Codosiga relatives so far only known from environmental sequencing approaches.ConclusionsHere, we establish the morphological and ultrastructural identity of an environmental choanoflagellate lineage. Data from microscopical observations, supplemented by findings from previous culture-independent methods, indicate that C. balthica is likely an ecologically relevant player of Baltic Sea hypoxic waters. The possession of derived mitochondria could be an adaptation to life in hypoxic environments periodically influenced by small-scale mixing events and changing oxygen content allowing the reduction of oxygen consuming components. In view of the intricacy of isolating and cultivating choanoflagellates, the two new cultured species represent an important advance to the understanding of the ecology of this group, and mechanisms of adaptations to hypoxia in protists in general.