Ribosomal RNA genes challenge the monophyly of the Hyalospheniidae (Amoebozoa: Arcellinida)

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.     

SSU rRNA reveals a sequential increase in shell complexity among the euglyphid testate amoebae (Rhizaria: Euglyphida)

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).     

Reinvestigation of Phryganella paradoxa (Arcellinida,Amoebozoa) Penard 1902

A major drawback in testate amoeba research is a general lack of scientific studies combining molecular approaches and classical laboratory experiments. We isolated five yet uncultured testate amoebae of the genus Phryganella Penard, 1902 from three different rivers and one pond in Germany. Based on established cultures we show their morphology, which we studied by light and electron microscopy, and present their unique feeding mode on abundant and common pennate diatoms like Nitzschia spp. and Synedra spp., whose frustules are bent and frequently, but not always, broken during the feeding process. We further obtained the first SSU rDNA sequences of strains of the family Phryganellidae, all of which contain introns. We used the sequences to confirm the taxonomic placement of the Phryganellidae in the Arcellinida (Amoebozoa), branching as a sister group to the Cryptodifflugiidae.     

Description and Phylogenetic Relationships of Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. (Amoebozoa, Arcellinida)

ABSTRACT. Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. were isolated and described from dry epiphytic moss. The morphology and ultrastructure of both species clearly demonstrate that they belong to the genus Spumochlamys (family Microchlamyiidae). They differ from its only described member, Spumochlamys iliensis (as well as from species of Microchlamys ), in the relief of the dorsal surface of the test, revealed by scanning electron microscopy, which can represent a good characteristic for species identification. They also differ in the structure of the dorsal part of the test wall (especially S. perforata ). Small subunit ribosomal DNA-based molecular phylogenetic analyses show that Spumochlamys is a deeply branching lineage of the Arcellinida, without any close affinities. Actin gene sequence analysis places this genus within the Tubulinea, close to two other arcellinid lineages but without forming a monophyletic group with them. These data together strongly suggest that the lack of resolution in the arcellinid molecular phylogenies is due to serious undersampling of taxa, a limited number of sequence data, and high divergence rates in most of the species.     

Phylogenetic analyses of small subunit ribosomal RNA coding regions reveal a monophyletic lineage of euglyphid testate amoebae (Order Euglyphida)

The Testaceafilosia includes amoebae with filopodia and with a proteinaceous, agglutinated or siliceous test. To explore the deeper phylogeny of this group, we sequenced the small subunit ribosomal RNA coding region of 13 species, including the first sequence of an amoeba with an agglutinated test, Pseudodifflugia sp. Phylogenetic analyses using maximum parsimony and maximum likelihood methods as well as neighbor joining method yielded the following results: the order Euglyphida forms a monophyletic lineage with the sarcomonads as sister group. The next related taxa are the Chlorarachnea and the unidentified filose strain N-Por. In agreement with the previous studies the Phytomyxea branch off at the base of this lineage. The Monadofilosa (Testaceafilosia and Sarcomonadea) appear monophyletic. The Testaceafilosia are polyphyletic, because Pseudodifflugia sp. is positioned as the sister taxon to the sarcomonads. Within the order Euglyphida Paulinella branches off first, together with Cyphoderia followed by Tracheleuglypha. In maximum likelihood and neighbor joining analyses, the genus Euglypha is monophyletic. The branching pattern within the order Euglyphida reflects the evolution of shell morphology from simple to complex built test.     

SSU rRNA phylogeny of Arcellinida (Amoebozoa) reveals that the largest Arcellinid genus, Difflugia Leclerc 1815, is not monophyletic

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.     

Morphology and phylogeny of three trachelocercids (Protozoa,Ciliophora, Karyorelictea), with description of two new species and insight into the evolution of the family Trachelocercidae

Although trachelocercid ciliates are common in marine sandy intertidal zones, methodological difficulties mean that their biodiversity and evolutionary relationships have not been well documented. This paper investigates the morphology and infraciliature of two novel Trachelolophos and one rarely known form, Tracheloraphis similis Raikov and Kovaleva, 1968, collected from the coastal waters of southern and eastern China. The small subunit (SSU) rRNA gene sequences of two of the species are presented, allowing the phylogenetic position of the genus Trachelolophos to be revealed for the first time. Phylogenetic analyses based on SSU rRNA gene sequences indicate that Trachelolophos branches with Kovalevaia and forms a sister clade with the group including Prototrachelocerca, Trachelocerca and Tracheloraphis. The monophyly of Trachelocerca is not rejected by the approximately unbiased (AU) test (P = 0.209, > 0.05) but that of Tracheloraphis is rejected (P = 3e‐033, < 0.05). The newly sequenced genus Trachelolophos, and recent studies on the morphology and phylogeny of the family Trachelocercidae, suggest two new hypotheses about the evolution of the seven genera within Trachelocercidae, based on either infraciliature or molecular evidence. Both hypotheses suppose the compound circumoral kineties in the oral apparatus is a plesiomorphic feature while the single circumoral kinety is synapomorphic. More evidence is still needed, however, as to whether the closed circumoral kinety with no brosse feature in Trachelocerca is ancestral or secondarily reduced. © 2015 The Linnean Society of London     

Diverse Legionella‐Like Bacteria Associated with Testate Amoebae of the Genus Arcella (Arcellinida: Amoebozoa)

Diverse species of Legionella and Legionella‐like amoebal pathogens (LLAPs) have been identified as intracellular bacteria in many amoeboid protists. There are, however, other amoeboid groups such as testate amoeba for which we know little about their potential to host such bacteria. In this study, we assessed the occurrence and diversity of Legionella spp. in cultures and environmental isolates of freshwater arcellinid testate amoebae species, Arcella hemispherica, Arcella intermedia, and Arcella vulgaris, via 16S rRNA gene sequence analyses and fluorescent in situ hybridization (FISH). Analysis of the 16S rRNA gene sequences indicated that A. hemispherica, A. intermedia, and A. vulgaris host Legionella‐like bacteria with 94–98% identity to other Legionella spp. based on NCBI BLAST search. Phylogenetic analysis placed Legionella‐like Arcella‐associated bacteria (LLAB) in three different clusters within a tree containing all other members of Legionella and LLAPs. The intracellular localization of the Legionella within Arcella hosts was confirmed using FISH with a Legionella‐specific probe. This study demonstrates that the host range of Legionella and Legionella‐like bacteria in the Amoebozoa extends beyond members of “naked” amoebae species, with members of the testate amoebae potentially serving an ecological role in the dispersal, protection, and replication of Legionella spp. in natural environments.     

The amoebae of Idionectes vortex (Cutosea,Amoebozoa): Motility,cytoskeleton architecture and extracellular scales

The Cutosea represent a deep-branching lineage within the phylum Amoebozoa that is still relatively poorly explored. Currently, there are four cutosean representatives known – the monotypic genera Armaparvus, Idionectes, Sapocribrum, and Squamamoeba – with marked genetic distances. Idionectes vortex is the deepest-branching species and differs markedly from the other Cutosea in ecology, life history, and most importantly, in its ability to form a flagellated swarmer with an exceptional swimming mechanism. As far as we know, the other Cutosea lack flagella and rather represent small, marine amoebae with a characteristic cell coat. The present paper focuses on the amoeboid life history stage of the algivorous amoeboflagellate Idionectes vortex to provide data for a first in-depth comparison with other Cutosea and to document structural specialties. The amoeboid stage of Idionectes is mainly associated with the specific feeding process, that is, the interaction with algal prey cells and phagocytosis of protoplast material. Yet, the present data from time-lapse microscopy, cytochemical stainings, and electron microscopy demonstrate clear similarities with the other cutosean species concerning amoeboid locomotion and cell coat ultrastructure. Furthermore, Idionectes amoebae exhibit a well-developed microtubular cytoskeleton, and an unusual basal apparatus that seems to undergo marked changes during the life history of this exceptional amoebozoan.     

Molecular Phylogeny of the Ocelloid-Bearing Dinoflagellates Erythropsidinium and Warnowia (Warnowiaceae, Dinophyceae)

ABSTRACT. Members of the family Warnowiaceae are unarmored phagotrophic dinoflagellates that possess an ocelloid. The genus Erythropsidinium (= Erythropsis ) has also developed a unique dynamic appendage, the piston, which is able to independently retract and extend for at least 2 min after the cell lyses. We provide the first small subunit ribosomal RNA gene sequences of warnowiid dinoflagellates, those of the type Erythropsidinium agile and one species of Warnowia . Phylogenetic analyses show that warnowiid dinoflagellates branch within the Gymnodinium sensu stricto group, forming a cluster separated from the Polykrikos clade and with autotrophic Pheopolykrikos beauchampii as closest relative. This reinforces their classification as unarmored dinoflagellates based on the shape of the apical groove, despite the strong ecological and ultrastructural diversity of the Gymnodinium s.s. group. Other structures, such as the ocelloid and piston, have no systematic value above the genus level.     

Sappinia sp. (Amoebozoa: Thecamoebida) and Rosculus sp. (SAR: Cercozoa) Isolated From King Penguin Guano Collected in the Subantarctic (South Georgia,Salisbury Plain) and their Coexistence in Culture

Two amoeboid organisms of the genera Sappinia Dangeard, 1896 and Rosculus Hawes, 1963 were identified in a sample containing king penguin guano. This sample, collected in the Subantarctic, enlarges the list of fecal habitats known for the presence of coprophilic amoebae. The two organisms were co‐isolated and subcultured for over 6 mo, with continuous efforts being invested to separate each one from the mixed culture. In the mixed culture, Rosculus cells were fast growing, tolerated changes in culturing conditions, formed cysts, and evidently were attracted by Sappinia trophozoites. The separation of the Rosculus strain was accomplished, whereas the Sappinia strain remained intermixed with inseparable Rosculus cells. Sappinia cell populations were sensitive to changes in culturing conditions; they improved with reduction of Rosculus cells in the mixed culture. Thick‐walled cysts, reportedly formed by Sappinia species, were not seen. The ultrastructure of both organisms was congruent with the currently accepted generic characteristics; however, some details were remarkable at the species level. Combined with the results of phylogenetic analyses, our findings indicate that the ultrastructure of the glycocalyx and the presence/absence of the Golgi apparatus in differential diagnoses of Sappinia species require a critical re‐evaluation.     

Pyramidodinium spinulosum sp. nov. (Dinophyceae), a sand‐dwelling non‐motile dinoflagellate from the seafloor (36 m deep) off Mageshima Island,Kagoshima, Japan

A new species of benthic marine dinoflagellate, Pyramidodinium spinulosum Horiguchi, Moriya, Pinto & Terada is described from the deep (36 m) seafloor off Mageshima Island, Kagoshima Prefecture, Japan in the subtropical region of the northwest Pacific. The life cycle of the dinoflagellate consists of a dominant, attached, dome‐shaped, vegetative form and short‐lasting, motile cell. Asexual reproduction takes place by the formation of two motile cells within each non‐motile cell. The released motile cells swim only for a short period and transform directly into the dome‐shaped vegetative form. The duration of the cell cycle varies and can be extremely long, ranging 5–38 days under culture conditions. The non‐motile cell is enclosed by a cell wall and its surface is covered with many (80 – 130) spines of various length. The dinoflagellate is photosynthetic and contains many (more than 50) discoidal chloroplasts. Phylogenetic analysis reveals that the dinoflagellate is closely related to the type species of the genus Pyramidodinium, P. atrofuscum which also possesses a dominant, attached, non‐motile form. However, P. spinulosum can be clearly distinguished from P. atrofuscum by the cell shape (dome‐shaped vs. pyramid‐shaped) and surface ornamentation (spines vs. wart‐like processes) of the non‐motile form. Based on these morphological differences together with molecular evidence, it was concluded that this organism from a deep water sand sample should be described as a second species of the genus Pyramidodinium, P. spinulosum.     

Phylogenetic positions and taxonomic assignments of the systematically controversial genera,Spirotrachelostyla, Uroleptopsis and Tunicothrix (Protozoa,Ciliophora, Stichotrichia) based on small subunit rRNA gene sequences

The phylogenetic positions of three systematically controversial genera of ciliates, Spirotrachelostyla, Uroleptopsis and Tunicothrix, have never been established by molecular data. The small subunit rRNA genes of three species, S. tani, U. citrina and T. wilberti, were sequenced and added to existing sequences of stichotrichs and other ciliates to construct phylogenetic trees. Results indicate the following: (1) Uroleptopsis is most closely related to species of Pseudokeronopsis, supporting its assignment to the family Pseudokeronopsidae; (2) one sampled Tunicothrix branches sister to the two sampled Parabirojimia, and this supports the placement of Tunicothrix in the Parabirojimidae; (3) Spirotrachelostyla clusters consistently with Trachelostyla to form a distinct, divergent clade that associates with Amphisiella at the base of the entire sporadotrich-urostylid clade, confirming the hypothesis that Spirotrachelostyla should be placed in the family Trachelostylidae.