Molecular phylogeny of Chonotrichia (Ciliophora, Phyllopharyngea) inferred from SSU rDNA sequences.

The ciliophoran taxon Chonotrichia Wallengren, 1895 are epibionts typically on Crustacea. There are about 112 species reported from all around the world. These species are known essentially from microscopical observations, and molecular data are far from sufficient, which largely hinders the study on their phylogenetic relationships. Here, we present the complete small-subunit (SSU) rDNA sequences of three species from Roscoff, Brittany, France, and one from Switzerland. These sequences are used to analyze the phylogentic relationships of the Chonotrichia, covering all three controversial orders, Exogemmida Jankowski, 1972, Cryptogemmida Jankowski, 1975, and Chilodochonida Batisse, 1994, for the first time. The phylogenetic tree confirms that all chonotrich sequences group together, nested in the dysteriid sequences. The tree topology also shows the separation of the chilodochonids from Exogemmida, supporting the establishment of the order Chilodochonida.

Phylogeny and Systematics of Leptomyxid Amoebae (Amoebozoa, Tubulinea, Leptomyxida).

We describe four new species of Flabellula, Leptomyxa and Rhizamoeba and publish new SSU rRNA gene and actin gene sequences of leptomyxids. Using these data we provide the most comprehensive SSU phylogeny of leptomyxids to date. Based on the analyses of morphological data and results of the SSU rRNA gene phylogeny we suggest changes in the systematics of the order Leptomyxida (Amoebozoa: Lobosa: Tubulinea). We propose to merge the genera Flabellula and Paraflabellula (the genus Flabellula remains valid by priority rule). The genus Rhizamoeba is evidently polyphyletic in all phylogenetic trees; we suggest retaining the generic name Rhizamoeba for the group unifying R. saxonica, R.matisi n. sp. and R. polyura, the latter remains the type species of the genus Rhizamoeba. Based on molecular and morphological evidence we move all remaining Rhizamoeba species to the genus Leptomyxa. New family Rhizamoebidae is established here in order to avoid paraphyly of the family Leptomyxidae. With the suggested changes both molecular and morphological systems of the order Leptomyxida are now fully congruent to each other.

Barcoding amoebae: comparison of SSU, ITS and COI genes as tools for molecular identification of naked lobose amoebae.

Morphological identification of naked lobose amoebae has always been a problem, hence the development of reliable molecular tools for species distinction is a priority for amoebae systematics. Previous studies based on SSU rDNA sequences provided a backbone for the phylogeny of Amoebozoa but were of little help for the species distinctions in this group. On one hand, the SSU rDNA sequences were rather conserved between closely related species; on the other hand, the intra-strain polymorphism of the SSU gene obscured species identification. In the present study, a 3' fragment of the SSU, a complete ITS1-5.8S-ITS2 block and a 5' fragment of COI gene were cloned and sequenced for six Vannella morphospecies, of which V. simplex was represented by six different isolates. SSU rDNA and ITS were found to be inappropriate for species differentiation, while distinctive and homogenous COI sequences were obtained for each well-defined morphospecies. Moreover, a number of distinct COI genotypes have been identified among V. simplex isolates. This suggests that COI may be a good candidate for DNA barcoding of amoebae, but further studies are necessary to confirm the accurateness of the COI gene as a barcode in other gymnamoebae, and to understand the taxonomic meaning of COI variations.

Rhizamoeba neglecta n. sp. (Amoebozoa, Tubulinea) from the bottom sediments of freshwater Lake Leshevoe (Valamo Island, North-Western Russia), with notes on the phylogeny of the order Leptomyxida.

A new species of Leptomyxida, named Rhizamoeba neglecta was found during studies of the amoeba fauna of the inner Lake Leshevoe located at Valamo archipelago (The Lake Ladoga, North-Western Russia). Light-microscopical and ultrastructural studies indicated that it represents a new species of Leptomyxida. The partial 18S rDNA sequence of this amoeba is very similar to that of Leptomyxa reticulata.. These organisms, however, are very different in LM morphology and biology. Organisms assigned to the genus Rhizamoeba do not form a single clade in the 18S rDNA tree. This may indicate that the genus is an artificial grouping or that a number of studied strains were misidentified. The phylogeny and the systematics of leptomyxids require further investigation.

Tempo and mode of spliceosomal intron evolution in actin of foraminifera.

Spliceosomal introns are present in almost all eukaryotic genes, yet little is known about their origin and turnover in the majority of eukaryotic phyla. There is no agreement whether most introns are ancestral and have been lost in some lineage or have been gained recently. We addressed this question by analyzing the spatial and temporal distribution of introns in actins of foraminifera, a group of testate protists whose exceptionally rich fossil record permits the calibration of molecular phylogenies to date intron origins. We identified 24 introns dispersed along the sequence of two foraminiferan actin paralogues and actin deviating proteins, an unconventional type of fast-evolving actin found in some foraminifera. Comparison of intron positions indicates that 20 of 24 introns are specific to foraminifera. Four introns shared between foraminifera and other eukaryotes were interpreted as parallel gains because they have been found only in single species belonging to phylogenetically distinctive lineages. Moreover, additional recent intron gain due to the transfer between the actin paralogues was observed in two cultured species. Based on a relaxed molecular clock timescale, we conclude that intron gains in actin took place throughout the evolution of foraminifera, with the oldest introns inserted between 550 and 500 million years ago and the youngest ones acquired less than 100 million years ago.

Phylogenetic position of Multicilia marina and the evolution of Amoebozoa.

Recent molecular phylogenetic studies have led to the erection of the phylum Amoebozoa, uniting naked and testate lobose amoebae, the mycetozoan slime moulds and amitochondriate amoeboid protists (Archamoebae). Molecular data together with ultrastructural evidence have suggested a close relationship between Mycetozoa and Archamoebae, classified together in the Conosea, which was named after the cone of microtubules that, when present, is characteristic of their kinetids. However, the relationships of conoseans to other amoebozoans remain unclear. Here, we obtained the complete small-subunit (SSU) rRNA gene sequence (2746 bp) of the enigmatic, multiflagellated protist Multicilia marina, which has formerly been classified either in a distinct phylum, Multiflagellata, or among lobose amoebae. Our study clearly shows that Multicilia marina belongs to the Amoebozoa. Phylogenetic analyses including 60 amoebozoan SSU rRNA gene sequences revealed that Multicilia marina branches at the base of the Conosea, together with another flagellated amoebozoan, Phalansterium solitarium, as well as with Gephyramoeba sp., Filamoeba nolandi and two unidentified amoebae. This is the first report showing strong support for a clade containing all flagellated amoebozoans and we discuss the position of the root of the phylum Amoebozoa in the light of this result.

The testate lobose amoebae (order Arcellinida Kent, 1880) finally find their home within Amoebozoa.

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.

Molecular phylogenetic analysis places Percolomonas cosmopolitus within Heterolobosea: evolutionary implications.

Percolomonas cosmopolitus is a common free-living flagellate of uncertain phylogenetic position that was placed within the Heterolobosea on the basis of ultrastructure studies. To test the relationship between Percolomonas and Heterolobosea, we analysed the primary structure of the actin and small-subunit ribosomal RNA (SSU rRNA) genes of P. cosmopolitus as well as the predicted secondary structure of the SSU rRNA. Percolomonas shares common secondary structure patterns of the SSU rRNA with heterolobosean taxa, which, together with the results of actin gene analysis, confirms that it is closely related to Heterolobosea. Phylogenetic reconstructions based on the sequences of the SSU rRNA gene suggest Percolomonas belongs to the family Vahlkampfiidae. The first Bayesian analysis of a large taxon sampling of heterolobosean SSU rRNA genes clarifies the phylogenetic relationships within this group.

How many novel eukaryotic 'kingdoms'? Pitfalls and limitations of environmental DNA surveys.

BACKGROUND: Over the past few years, the use of molecular techniques to detect cultivation-independent, eukaryotic diversity has proven to be a powerful approach. Based on small-subunit ribosomal RNA (SSU rRNA) gene analyses, these studies have revealed the existence of an unexpected variety of new phylotypes. Some of them represent novel diversity in known eukaryotic groups, mainly stramenopiles and alveolates. Others do not seem to be related to any molecularly described lineage, and have been proposed to represent novel eukaryotic kingdoms. In order to review the evolutionary importance of this novel high-level eukaryotic diversity critically, and to test the potential technical and analytical pitfalls and limitations of eukaryotic environmental DNA surveys (EES), we analysed 484 environmental SSU rRNA gene sequences, including 81 new sequences from sediments of the small river, the Seymaz (Geneva, Switzerland). RESULTS: Based on a detailed screening of an exhaustive alignment of eukaryotic SSU rRNA gene sequences and the phylogenetic re-analysis of previously published environmental sequences using Bayesian methods, our results suggest that the number of novel higher-level taxa revealed by previously published EES was overestimated. Three main sources of errors are responsible for this situation: (1) the presence of undetected chimeric sequences; (2) the misplacement of several fast-evolving sequences; and (3) the incomplete sampling of described, but yet unsequenced eukaryotes. Additionally, EES give a biased view of the diversity present in a given biotope because of the difficult amplification of SSU rRNA genes in some taxonomic groups. CONCLUSIONS: Environmental DNA surveys undoubtedly contribute to reveal many novel eukaryotic lineages, but there is no clear evidence for a spectacular increase of the diversity at the kingdom level. After re-analysis of previously published data, we found only five candidate lineages of possible novel high-level eukaryotic taxa, two of which comprise several phylotypes that were found independently in different studies. To ascertain their taxonomic status, however, the organisms themselves have now to be identified.

Small-subunit ribosomal RNA gene sequences of Phaeodarea challenge the monophyly of Haeckel's Radiolaria.

In his grand monograph of Radiolaria, Ernst Haeckel originally included Phaeodarea together with Acantharea and Polycystinea, all three taxa characterized by the presence of a central capsule and the possession of axopodia. Cytological and ultrastructural studies, however, questioned the monophyly of Radiolaria, suggesting an independent evolutionary origin of the three taxa, and the first molecular data on Acantharea and Polycystinea brought controversial results. To test further the monophyly of Radiolaria, we sequenced the complete small subunit ribosomal RNA gene of three phaeodarians and three polycystines. Our analyses reveal that phaeodarians clearly branch among the recently described phylum Cercozoa, separately from Acantharea and Polycystinea. This result enhances the morphological variability within the phylum Cercozoa, which already contains very heterogeneous groups of protists. Our study also confirms the common origin of Acantharea and Polycystinea, which form a sister-group to the Cercozoa, and allows a phylogenetic reinterpretation of the morphological features of the three radiolarian groups.

The twilight of Heliozoa and rise of Rhizaria, an emerging supergroup of amoeboid eukaryotes.

Recent molecular phylogenetic studies revealed the extraordinary diversity of single-celled eukaryotes. However, the proper assessment of this diversity and accurate reconstruction of the eukaryote phylogeny are still impeded by the lack of molecular data for some major groups of easily identifiable and cultivable protists. Among them, amoeboid eukaryotes have been notably absent from molecular phylogenies, despite their diversity, complexity, and abundance. To partly fill this phylogenetic gap, we present here combined small-subunit ribosomal RNA and actin sequence data for the three main groups of "Heliozoa" (Actinophryida, Centrohelida, and Desmothoracida), the heliozoan-like Sticholonche, and the radiolarian group Polycystinea. Phylogenetic analyses of our sequences demonstrate the polyphyly of heliozoans, which branch either as an independent eukaryotic lineage (Centrohelida), within stramenopiles (Actinophryida), or among cercozoans (Desmothoracida), in broad agreement with previous ultrastructure-based studies. Our data also provide solid evidence for the existence of the Rhizaria, an emerging supergroup of mainly amoeboid eukaryotes that includes desmothoracid heliozoans, all radiolarians, Sticholonche, and foraminiferans, as well as various filose and reticulose amoebae and some flagellates.

Phylogeny of lobose amoebae based on actin and small-subunit ribosomal RNA genes.

Lobose amoebae are abundant free-living protists and important pathogenic agents, yet their evolutionary history and position in the universal tree of life are poorly known. Molecular data for lobose amoebae are limited to a few species, and all phylogenetic studies published so far lacked representatives of many of their taxonomic groups. Here we analyze actin and small-subunit ribosomal RNA (SSU rRNA) gene sequences of a broad taxon sampling of naked, lobose amoebae. Our results support the existence of a monophyletic Amoebozoa clade, which comprises all lobose amoebae examined so far, the amitochondriate pelobionts and entamoebids, and the slime molds. Both actin and SSU rRNA phylogenies distinguish two well-defined clades of amoebae, the "Gymnamoebia sensu stricto" and the Archamoebae (pelobionts + entamoebids), and one weakly supported and ill-resolved group comprising some naked, lobose amoebae and the Mycetozoa.

The evolution of early Foraminifera.

Fossil Foraminifera appear in the Early Cambrian, at about the same time as the first skeletonized metazoans. However, due to the inadequate preservation of early unilocular (single-chambered) foraminiferal tests and difficulties in their identification, the evolution of early foraminifers is poorly understood. By using molecular data from a wide range of extant naked and testate unilocular species, we demonstrate that a large radiation of nonfossilized unilocular Foraminifera preceded the diversification of multilocular lineages during the Carboniferous. Within this radiation, similar test morphologies and wall types developed several times independently. Our findings indicate that the early Foraminifera were an important component of Neoproterozoic protistan community, whose ecological complexity was probably much higher than has been generally accepted.

Small subunit ribosomal DNA suggests that the xenophyophorean Syringammina corbicula is a foraminiferan.

Xenophyophorea are giant deep-sea rhizopodial protists of enigmatic origins. Although species were described as Foraminifera or sponges in the early literature, the xenophyophoreans are currently classified either as a class of Rhizopoda or an independent phylum. To establish the phylogenetic position of Xenophyophorea, we analysed the small subunit (SSU) rRNA gene sequence of Syringammina corbicula Richardson, a newly described xenophyophorean species from the Cape Verde Plateau. The SSUrDNA analyses showed that S. corbicula is closely related to Rhizammina algaeformis, a tubular deep-sea foraminiferan. Both species branch within a group of monothalamous (single-chambered) Foraminifera, which include also such agglutinated genera as Toxisarcon, Rhabdammina, and Saccammina, and the organic-walled genera Gloiogullmia and Cylindrogullmia. Our results are congruent with observations of similar cytoplasmic organisation in Rhizammina and Syringammina. Thus, the Xenophyophorea appear to be a highly specialised group of deep-sea Foraminifera.