Invalidation of Hyperamoeba by Transferring its Species to Other Genera of Myxogastria

ABSTRACT. The genus Hyperamoeba Alexeieff, 1923 was established to accommodate an aerobic amoeba exhibiting three life stages—amoeba, flagellate, and cyst. As more species/strains were isolated, it became increasingly evident from small subunit (SSU) gene phylogenies and ultrastructure that Hyperamoeba is polyphyletic and its species occupy different positions within the class Myxogastria. To pinpoint Hyperamoeba strains within other myxogastrid genera we aligned numerous myxogastrid sequences: whole small subunit ribosomal (SSU or 18S rRNA) gene for 50 dark‐spored (i.e. Stemonitida and Physarida) Myxogastria (including a new “Hyperamoeba”/Didymium sequence) and a ～400‐bp SSU fragment for 147 isolates assigned to 10 genera of the order Physarida. Phylogenetic analyses show unambiguously that the type species Hyperamoeba flagellata is a Physarum (Physarum flagellatum comb. nov.) as it nests among other Physarum species as robust sister to Physarum didermoides. Our trees also allow the following allocations: five Hyperamoeba strains to the genus Stemonitis; Hyperamoeba dachnaya, Pseudodidymium cryptomastigophorum, and three other Hyperamoeba strains to the genus Didymium; and two further Hyperamoeba strains to the family Physaridae. We therefore abandon the polyphyletic and redundant genus Hyperamoeba. We discuss the implications for the ecology and evolution of Myxogastria, whose amoeboflagellates are more widespread than previous inventories supposed, being now found in freshwater and even marine environments.     

Impact of soil warming and shading on colonization and community structure of arbuscular mycorrhizal fungi in roots of a native grassland community

Arbuscular mycorrhizal (AM) fungi have a major influence on the structure, responses and below‐ground C allocation of plant communities. Our lack of understanding of the response of AM fungi to factors such as light and temperature is an obstacle to accurate prediction of the impact of global climate change on ecosystem functioning. In order to investigate this response, we divided a grassland site into 24 plots, each either unshaded or partly shaded with soil either unheated or heated by 3°C at 2 cm depth. In both short‐term studies in spring and autumn, and in a 1‐year‐long study, we measured root length colonization (L_RC) by AM and non‐AM fungi. For selected root samples, DNA sequences were amplified by PCR with fungal‐specific primers for part of the small sub‐unit (SSU) rRNA gene. In spring, the total L_RC increased over 6 weeks from 12% to 25%. Shading significantly reduced AM but increased non‐AM fungal colonization, while soil warming had no effect. In the year‐long study, colonization by AM fungi peaked in summer, whereas non‐AM colonization peaked in autumn, when there was an additive effect of shading and soil warming that reduced AM but increased non‐AM fungi. Stepwise regression revealed that light received within the 7 days prior to sampling was the most significant factor in determining AM L_RC and that mean temperature was the most important influence on non‐AM L_RC. Loglinear analysis confirmed that there were no seasonal or treatment effects on the host plant community. Ten AM fungal sequence types were identified that clustered into two families of the Glomales, Glomaceae and Gigasporaceae. Three other sequence types were of non‐AM fungi, all Ascomycotina. AM sequence types showed seasonal variation and shading impacts: loglinear regression analysis revealed changes in the AM fungal community with time, and a reduction of one Glomus sp. under shade, which corresponded to a decrease in the abundance of Trifolium repens. We suggest that further research investigating any impacts of climate change on ecosystem functioning must not only incorporate their natural AM fungal communities but should also focus on niche separation and community dynamics of AM fungi.     

Nuclear and Nucleomorph SSU rDNA Phylogeny in the Cryptophyta and the Evolution of Cryptophyte Diversity

The plastid-bearing members of the Cryptophyta contain two functional eukaryotic genomes of different phylogenetic origin, residing in the nucleus and in the nucleomorph, respectively. These widespread and diverse protists thus offer a unique opportunity to study the coevolution of two different eukaryotic genomes within one group of organisms. In this study, the SSU rRNA genes of both genomes were PCR-amplified with specific primers and phylogenetic analyses were performed on different data sets using different evolutionary models. The results show that the composition of the principal clades obtained from the phylogenetic analyses of both genes was largely congruent, but striking differences in evolutionary rates were observed. These affected the topologies of the nuclear and nucleomorph phylogenies differently, resulting in long-branch attraction artifacts when simple evolutionary models were applied. Deletion of long-branch taxa stabilized the internal branching order in both phylogenies and resulted in a completely resolved topology in the nucleomorph phylogeny. A comparison of the tree topologies derived from SSU rDNA sequences with characters previously used in cryptophyte systematics revealed that the biliprotein type was congruent, but the type of inner periplast component incongruent, with the molecular trees. The latter is indicative of a hidden cellular dimorphism (cells with two periplast types present in a single clonal strain) of presumably widespread occurrence throughout cryptophyte diversity, which, in consequence, has far-reaching implications for cryptophyte systematics as it is practiced today.     

Freshwater foraminiferans revealed by analysis of environmental DNA samples

Sediment-dwelling protists are among the most abundant meiobenthic organisms, ubiquitous in all types of aquatic ecosystems. Yet, because their isolation and identification are difficult, their diversity remains largely unknown. In the present work, we applied molecular methods to examine the diversity of freshwater Foraminifera, a group of granuloreticulosan protists largely neglected until now. By using specific PCR primers, we detected the presence of Foraminifera in all sediment samples examined. Phylogenetic analysis of amplified SSU rDNA sequences revealed two distinct groups of freshwater foraminiferans. All obtained sequences branched within monothalamous (single-chambered), marine Foraminifera, suggesting a repeated colonization of freshwater environments. The results of our study challenge the traditional view of Foraminifera as essentially marine organisms, and provide a conceptual framework for charting the molecular diversity of freshwater granuloreticulosan protists.     

Long branch attraction effects and the status of "basal eukaryotes": phylogeny and structural analysis of the ribosomal RNA gene cluster of the free-living diplomonad Trepomonas agilis

The three taxa emerging at the base of the eukaryotic ribosomal RNA phylogenetic tree (Diplomonadida, Microspora, and Parabasalia) include a wide array of parasitic species. and some free-living organisms that appear to be derived from a parasitic ancestry. The basal position of these taxa, which lack mitochondria, has recently been questioned. I sequenced most of the ribosomal RNA gene cluster of the free-living diplomonad Trepomonas agilis and a secondary structure model was reconstructed for the SSU rRNA. I conducted a RASA matrix analysis to identify, independently from tree reconstruction, putative long branch attraction effects in the data matrix. The results show that each of the basal clades and the euglenozoan clade act, indeed, as long branches and may have been engaged in a process of accelerated rate of evolution. A nucleotide signature analysis was conducted in the conserved regions for positions defining the three great domains of life (Eubacteria, Archea, and Eukaryota). For the three basal taxa, this analysis showed the presence of a significant number of different non-eukaryotic nucleotides. A precise study of the nature and location of these nucleotides led to conclusions supporting the results of the RASA analysis. Altogether, these findings suggest that the basal placement of these taxa in the SSU ribosomal RNA phylogenetic tree is artifactual, and flawed by long branch attraction effects.     

The systematic position of Paraspathidium Noland, 1937 (Ciliophora,Litostomatea?) inferred from primary SSU rRNA gene sequences and predicted secondary rRNA structure

Traditionally the unusual ciliate Paraspathidium has been regarded as a gymnostome haptorid (Litostomatea) based on its morphological features. In order to test this placement, the small-subunit (SSU) rRNA gene was sequenced for two isolates of Paraspathidium apofuscum and phylogenetic trees were constructed. Furthermore, the putative structure of the variable regions 2 and 4 of the SSU rRNA gene were predicted and compared with those of other ciliates. Our analyses of SSU rRNA gene sequences revealed (i) a clear separation of Paraspathidium from the haptorids and indeed the class Litostomatea, rejecting its systematic position based on morphological characters and (ii) an equally clear association with the assemblage comprising the classes Plagiopylea and Prostomatea. Putative secondary structures of the variable regions 2 and 4 of Paraspathidium are similar to those of the plagiopyleans and prostomateans but differ from the hapotrids in Helix 10, Helix E10-1 and Helix E23-5. Taken together, these results support the placement of Paraspathidium close to prostomateans and plagiopyleans, or even as a distinct group possibly at ordinal rank, within the class Plagiopylea.     

Molecular evidence demonstrating the basidiomycetous fungus Cryptococcus curvatus is the dominant microbial eukaryote in sediment at the Kuroshima Knoll methane seep

The Kuroshima Knoll, located in the southern Ryukyu Arc, is known to actively bubble with gas containing methane and hydrogen sulfide from numerous fissures in the large carbonate pavement. Although ecological studies regarding macrobenthos and bacteria from Kuroshima Knoll have been intensively conducted, the community structure and ecological importance of microbial eukaryotes (protists) have not yet been investigated. In the present study, we directly extracted DNA from sediment of the Kuroshima Knoll at a depth of 640 m and constructed genetic libraries of PCR-amplified eukaryotic small-subunit ribosomal DNA (SSU rDNA). Although the SSU rDNA sequences of several types of benthic foraminifers were retrieved from the surface of the sediment, all other sequences (just below the sediment surface to approximately 9 cm below sediment surface) were derived from the basidiomycetous yeast Cryptococcus curvatus. Furthermore, sequences of the internal transcribed spacer of rDNA (ITS-rDNA) retrieved from the same sediment were identical to that of C. curvatus originating from terrestrial habitats. The diversity of microbial eukaryotes in the Kuroshima Knoll sediment seems to be extremely low and significantly different from that of other marine environments previously reported.     

Redescription of Favella ehrenbergii (Claparède and Lachmann, 1858) Jörgensen, 1924 (Ciliophora: Choreotrichia), with Phylogenetic Analyses Based on Small Subunit rRNA Gene Sequences

ABSTRACT. The identification of Favella ehrenbergii, a marine planktonic ciliate, has largely been based on its lorica features. This approach is potentially problematic given the polymorphic lorica during this organism's life cycle. We isolated a population of F. ehrenbergii from the coastal waters of Incheon, Korea, and revealed its infraciliature using the protargol staining method. Phylogenetic analysis based on small subunit rRNA gene sequences was also performed. Results showed that this population possessed 16 collar membranelles (CM) and about 100 somatic kineties. These features are highly conserved, even in later dividers. As such, the number of CM and somatic kineties can be used as key characteristics for identification of Favella species.