18S ribosomal RNA gene sequences of Cochliopodium (Himatismenida) and the phylogeny of Amoebozoa

Cochliopodium is a very distinctive genus of discoid amoebae covered by a dorsal tectum of carbohydrate microscales. Its phylogenetic position is unclear, since although sharing many features with naked "gymnamoebae", the tectum sets it apart. We sequenced 18S ribosomal RNA genes from three Cochliopodium species (minus, spiniferum and Cochliopodium sp., a new species resembling C. minutum). Phylogenetic analysis shows Cochliopodium as robustly holophyletic and within Amoebozoa, in full accord with morphological data. Cochliopodium is always one of the basal branches within Amoebozoa but its precise position is unstable. In Bayesian analysis it is sister to holophyletic Glycostylida, but distance trees mostly place it between Dermamoeba and a possibly artifactual long-branch cluster including Thecamoeba. These positions are poorly supported and basal amoebozoan branching ill-resolved, making it unclear whether Discosea (Glycostylida, Himatismenida, Dermamoebida) is holophyletic; however, Thecamoeba seems not specifically related to Dermamoeba. We also sequenced the small-subunit rRNA gene of Vannella persistens, which constantly grouped with other Vannella species, and two Hartmannella strains. Our trees suggest that Vexilliferidae, Variosea and Hartmannella are polyphyletic, confirming the existence of two very distinct Hartmannella clades: that comprising H. cantabrigiensis and another divergent species is sister to Glaeseria, whilst Hartmannella vermiformis branches more deeply.     

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.     

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.     

Cochliopodium gallicum n. sp. (Himatismenida), an amoeba bearing unique scales, from cyanobacterial mats in the Camargue (France)

Cochliopodium gallicum n. sp., isolated from cyanobacterial mats in the Camargue (France) is the smallest marine species of Cochliopodium to date. Its unusual tectum consists of flat plate-shaped scales with honeycomb-like centres, underlain by a layer of filamentous structures connected to each other in the basal and apical parts. The tectum is very fine and can be easily lost under inappropriate EM fixation. In its light-microscopical features, this species resembles Ovalopodium carrikeri Sawyer, 1980, a himatismenid that is believed to possess a scaleless, fuzzy or hairy "glycocalyx". We suggest that O. carrikeri might have been a similar species that lost scales under fixation. Our finding makes desirable a re-investigation of the genus Ovalopodium.     

The novel marine gliding zooflagellate genus Mantamonas (Mantamonadida ord. n.: Apusozoa)

Mantamonasis a novel genus of marine gliding zooflagellates probably related to apusomonad and planomonad Apusozoa. Using phase and differential interference contrast microscopy we describe the type species Mantamonas plasticasp. n. from coastal sediment in Cumbria, England. Cells are ～5μm long, ～5μm wide, asymmetric, flattened, biciliate, and somewhat plastic. The posterior cilium, on which they glide smoothly over the substratum, is long and highly acronematic. The much thinner, shorter, and almost immobile anterior cilium points forward to the cell's left. These morphological and behavioural traits suggest thatMantamonasis a member of the protozoan phylum Apusozoa. Analyses of 18S and 28S rRNA gene sequences of Mantamonas plasticaand a second genetically very different marine species from coastal sediment in Tanzania show Mantamonasas a robustly monophyletic clade, that is very divergent from all other eukaryotes. 18S rRNA trees mostly placeMantamonaswithin unikonts (opisthokonts, Apusozoa, and Amoebozoa) but its precise position varies with phylogenetic algorithm and/or taxon and nucleotide position sampling; it may group equally weakly as sister to Planomonadida, Apusomonadida or Breviata. On 28S rRNA and joint 18/28S rRNA phylogenies (including 11 other newly obtained apusozoan/amoebozoan 28S rRNA sequences) it consistently strongly groups with Apusomonadida (Apusozoa).     

Phylogeny of the Centrohelida Inferred from SSU rRNA, Tubulins, and Actin Genes

Amoeboid protists are major targets of recent molecular phylogeny in connection with reconstruction of global phylogeny of eukaryotes as well as the search for the root of eukaryotes. The Centrohelida are one of the major groups of Heliozoa, classified in the Actinopodida, whose evolutionary position is not well understood. To clarify the relationships between the Centrohelida and other eukaryotes, we sequenced SSU rRNA, α-tubulin, and β-tubulin genes from a centroheliozoan protist, Raphidiophrys contractilis. The SSU rRNA phylogeny showed that the Centrohelida are not closely related to other heliozoan groups, Actinophryida, Desmothoracida, or Taxopodida. Maximum likelihood analyses of the combined phylogeny using a concatenate model for an α- + β-tubulin + actin data set, and a separate model for SSU rRNA, α- and β-tubulin, and actin gene data sets revealed the best tree, in which the Centrohelida have a closer relationship to Rhodophyta than to other major eukaryotic groups. However, both weighted Shimodaira–Hasegawa and approximately unbiased tests for the concatenate protein phylogeny did not reject alternative trees in which Centrohelida were constrained to be sisters to the Amoebozoa. Moreover, alternative trees in which Centrohelida were placed at the node branching before and after Amoebozoa or Viridiplantae were not rejected by the WSH tests. These results narrowed the possibilities for the position of Centrohelida to a sister to the Rhodophyta, to the Amoebozoa, or to an independent branch between the branchings of Amoebozoa and Rhodophyta (or possibly Plantae) at the basal position within the bikonts clade in the eukaryotic tree. [Reviewing Editor: Dr. Martin Kreitman]     

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).     

18S rDNA phylogeny of lamproderma and allied genera (Stemonitales, Myxomycetes, Amoebozoa)

The phylogenetic position of the slime-mould genus Lamproderma (Myxomycetes, Amoebozoa) challenges traditional taxonomy: although it displays the typical characters of the order Stemonitales, it appears to be sister to Physarales. This study provides a small subunit (18S or SSU) ribosomal RNA gene-based phylogeny of Lamproderma and its allies, with new sequences from 49 specimens in 12 genera. We found that the order Stemonitales and Lamproderma were both ancestral to Physarales and that Lamproderma constitutes several clades intermingled with species of Diacheopsis, Colloderma and Elaeomyxa. We suggest that these genera may have evolved from Lamproderma by multiple losses of fruiting body stalks and that many taxonomic revisions are needed. We found such high genetic diversity within three Lamproderma species that they probably consist of clusters of sibling species. We discuss the contrasts between genetic and morphological divergence and implications for the morphospecies concept, highlighting the phylogenetically most reliable morphological characters and pointing to others that have been overestimated. In addition, we showed that the first part (~600 bases) of the SSU rDNA gene is a valuable tool for phylogeny in Myxomycetes, since it displayed sufficient variability to distinguish closely related taxa and never failed to cluster together specimens considered of the same species.     

Vannella epipetala n. sp. isolated from the leaf surface of Spondias mombin (Anacardiaceae) growing in the dry forest of Costa Rica

As part of a Microbial Observatory of Caterpillars located in the Area de Conservacíon Guanacaste (ACG) in northwestern Costa Rica, we isolated a novel species of the genus Vannella associated with the food of the caterpillars of the saturniid moth Rothschildia lebeau, namely the leaves of the dry forest deciduous tree Spondias mombin (Anacardiaceae). The new species can be distinguished from other described species of the genus by the presence of a plasmalemma coated with a thickened, osmiophilic lamina containing glycostyles, and by its unusual habitat, the leaf surfaces or phylosphere of S. mombin. We further established the novelty of our isolate by sequencing its nuclear small-subunit (SSU) rRNA gene and inferring its phylogenetic position among all other currently sequenced members of the genera Vannella and Platyamoeba. Our results reveal that our isolate shares most recent common ancestry with three strains of Platyamoeba placida, the type species of the genus Platyamoeba. Despite this placement, the isolate clearly possesses glycostyles that are the hallmark of the genus Vannella. In addition to the cultured isolate, we also present a closely related sequence from a SSU rRNA gene clone library constructed from a DNA extract of leaf-wash of S. mombin with sterile water.     

Molecular characterization of the myxosporean associated with parasitic encephalitis of farmed Atlantic salmon Salmo salar in Ireland

During seasonal epizootics of neurologic disease and mass mortality in the summers of 1992, 1993 and 1994 on a sea-farm in Ireland, Atlantic salmon Salmo salar smolts suffered from encephalitis associated with infection by a neurotropic parasite. Based on ultrastructural studies, this neurotropic parasite was identified as an intercellular presporogonic multicellular developmental stage of a histozoic myxosporean, possibly a Myxobolus species. In order to generate sequence data for phylogenetic comparisons to substantiate the present morphological identification of this myxosporean in the absence of detectable sporogony, polymerase chain reaction (PCR), Southern blot hybridization, dideoxynucleotide chain-termination DNA sequencing, and in situ hybridization (ISH) were used in concert to characterize segments of the small subunit ribosomal RNA (SSU rRNA) gene. Oligonucleotide primers were created from sequences of the SSU rRNA gene of M. cerebralis and were employed in PCR experiments using DNA extracted from formalin-fixed paraffin-embedded tissue sections of brains from Atlantic salmon smolts in which the myxosporean had been detected by light microscopy. Five segments of the SSU rRNA gene of the myxosporean, ranging in length from 187 to 287 base pairs, were amplified, detected by hybridization with sequence-specific probes, and sequenced. Consensus sequences from these segments were aligned to create a partial sequence of the SSU rRNA gene of the myxosporean. Assessments of sequence identity were made between this partial sequence and sequences of SSU rRNA genes from 7 myxosporeans, including Ceratomyxa shasta, Henneguya doori, M. arcticus, M. cerebralis, M. insidiosus, M. neurobius, and M. squamalis. The partial SSU rRNA gene sequence from the myxosporean had more sequence identity with SSU rRNA gene sequences from neurotropic and myotropic species of Myxobolus than to those from epitheliotropic species of Myxobolus or Henneguya, or the enterotropic species of Ceratomyxa, and was identical to regions of the SSU rRNA gene of M. cerebralis. Digoxigenin-labeled oligonucleotide DNA probes complementary to multiple segments of the SSU rRNA gene of M. cerebralis hybridized with DNA of the parasite in histologic sections of brain in ISH experiments, demonstrating definitively that the segments of genome amplified were from the organisms identified by histology and ultrastructural analysis. Based on sequence data derived entirely from genetic material of extrasporogonic stages, the SSU rDNA sequence identity discovered in this study supports the hypothesis that the myxosporean associated with encephalitis of farmed Atlantic salmon smolts is a neurotropic species of the genus Myxobolus, with sequences identical to those of M. cerebralis.     

基于核糖体小亚基的波豆类鞭毛虫分子系统发育研究

波豆类鞭毛虫(动基体目)是一类非常重要的原生动物,结构独特,分布广泛.但是,这一类群的系统发育关系尚存有很多争议.为了更好的了解该类群的系统发育关系,作者分离、纯化并培养了Bodo designis DH,测定了它的SSU rRNA序列.根据该序列和GenBank中的相关序列,用最大简约法和邻接法分别构建了基于全序列和保守区序列的系统树.结果如下:1)波豆属是多系发育的;2)波豆类鞭毛虫和锥虫类鞭毛虫的系统发育关系仍有待于深入研究;3)虽然所得的系统树都显示出一种基本的双歧式树型结构,但采用不同方法所构建的系统树之间有明显的差异.这些似乎表明,就波豆类鞭毛虫而言,SSU rRNA不是一个令人满意的系统发育标记.     

Variability of the Mitochondrial SSU rDNA of <Emphasis Type="Italic">Nomuraea</Emphasis> Species and Other Entomopathogenic Fungi from Hypocreales

Hypocrealean arthropod pathogenic fungi have profound impact on the regulation of agricultural and medical pests. However, until now the genetic and phylogenetic relationships among species have not been clarified, such studies could clarify host specificity relationships and define species boundaries. Our purpose was to compare the sequences of the mitochondrial SSU rDNA fragments from several mitosporic entomopathogenic Hypocreales to infer relationships among them and to evaluate the possibility to use these sequences as species diagnostic tool in addition to the more commonly studied sequences of nuclear SSU rDNA. The SSU mt-rDNA proved to be useful to help in differentiation of species inside several genera. Clusters obtained with Parsimony, Bayesian, and Maximum Likelihood analyses were congruent with a new classification of the Clavicipitaceae (Sung et al. Stud Mycol. 2007;57:5-59) in which the anamorphic genera Nomuraea and Metarhizium species remain in the Clavicipitaceae and Isaria species sequenced here are assigned to the family Cordycipitaceae. Mitochondrial genomic information indicates the same general pattern of relationships demonstrated by nuclear gene sequences.     

The phylogenetic position of the oxymonad Saccinobaculus based on SSU rRNA

The oxymonads are a group of structurally complex anaerobic flagellates about which we know very little. They are found in association with complex microbial communities in the guts of animals. There are five recognized families of oxymonads; molecular data have been acquired for four of these. Here, we describe the first molecular data from the last remaining group, represented by Saccinobaculus, an organism that is found exclusively in the hindgut of the wood-eating cockroach Cryptocercus. We sequenced small subunit ribosomal RNA (SSU rRNA) from total gut DNA to describe Saccinobaculus SSU rRNA diversity. We also sequenced SSU rRNA from manually isolated cells of the two most abundant and readily identifiable species: the type species Saccinobaculus ambloaxostylus and the taxonomically contentious Saccinobaculus doroaxostylus. We inferred phylogenetic trees including all five known oxymonad subgroups in order to elucidate the internal phylogeny of this poorly studied group, to resolve some outstanding issues of the taxonomy and identification of certain Saccinobaculus species, and to investigate the evolution of character states within it. Our analysis recovered strong support for the existence of the five subgroups of oxymonads, and consistently united the subgroups containing Monocercomonoides and Streblomastix, but was unable to resolve any further higher-order branching patterns.     

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.     

Testing the new animal phylogeny: first use of combined large-subunit and small-subunit rRNA gene sequences to classify the protostomes

Although the small-subunit ribosomal RNA (SSU rRNA) gene is widely used in the molecular systematics, few large-subunit (LSU) rRNA gene sequences are known from protostome animals, and the value of the LSU gene for invertebrate systematics has not been explored. The goal of this study is to test whether combined LSU and SSU rRNA gene sequences support the division of protostomes into Ecdysozoa (molting forms) and Lophotrochozoa, as was proposed by Aguinaldo et al. (1997) (Nature 387:489) based on SSU rRNA sequences alone. Nearly complete LSU gene sequences were obtained, and combined LSU + SSU sequences were assembled, for 15 distantly related protostome taxa plus five deuterostome outgroups. When the aligned LSU + SSU sequences were analyzed by tree-building methods (minimum evolution analysis of LogDet-transformed distances, maximum likelihood, and maximum parsimony) and by spectral analysis of LogDet distances, both Ecdysozoa and Lophotrochozoa were indeed strongly supported (e.g., bootstrap values >90%), with higher support than from the SSU sequences alone. Furthermore, with the LogDet-based methods, the LSU + SSU sequences resolved some accepted subgroups within Ecdysozoa and Lophotrochozoa (e.g., the polychaete sequence grouped with the echiuran, and the annelid sequences grouped with the mollusc and lophophorates)-subgroups that SSU-based studies do not reveal. Also, the mollusc sequence grouped with the sequences from lophophorates (brachiopod and phoronid). Like SSU sequences, our LSU + SSU sequences contradict older hypotheses that grouped annelids with arthropods as Articulata, that said flatworms and nematodes were basal bilateralians, and considered lophophorates, nemerteans, and chaetognaths to be deuterostomes. The position of chaetognaths within protostomes remains uncertain: our chaetognath sequence associated with that of an onychophoran, but this was unstable and probably artifactual. Finally, the benefits of combining LSU with SSU sequences for phylogenetic analyses are discussed: LSU adds signal, it can be used at lower taxonomic levels, and its core region is easy to align across distant taxa-but its base frequencies tend to be nonstationary across such taxa. We conclude that molecular systematists should use combined LSU + SSU rRNA genes rather than SSU alone.     

A Description of a New "Amoebozoan" Isolated from the American Lobster, Homarus americanus

ABSTRACT. Our knowledge of the diversity of amoeboid protists is rapidly expanding as new and old habitats are more fully explored. In 2003, while investigating the cause of an amoeboid disease afflicting lobsters on the East Coast, samples were examined for the presence of amoebae from the carapace washings of the American lobster, Homarus americanus . During this survey a unique community of gymnamoebae was discovered. Among the new taxa discovered was a small Thecamoeba -like organism with a single posteriorly directed pseudopodium. Although resembling Parvamoeba rugata , this amoeba displayed distinctive morphology from that isolate or any other amoebozoan. Phylogenetic analysis shows this amoeba is distantly related to the Thecamoebidae. In this paper we describe the unique morphology of a second species of Parvamoeba and discuss its phylogenetic position with respect to the "Amoebozoa."     

New considerations on the phylogeny of cyrtophorian ciliates (Protozoa,Ciliophora): expanded sampling to understand their evolutionary relationships

To rationalize the confusing relationships among the cyrtophorian ciliates, we expanded the taxon sampling by sequencing the small subunit ribosomal RNA (SSU rRNA) gene of representatives of 12 genera (20 species, 23 new sequences). The SSU rRNA sequences of Spirodysteria, Agnathodysteria, Brooklynella and Odontochlamys are reported for the first time. Phylogenetic trees were constructed, and secondary structures of variable region 4 (V4) of all genera for which SSU rRNA gene sequence data are available were predicted. The results indicate that (i) Brooklynella is likely an intermediate taxon between Dysteriidae and Hartmannulidae; (ii) the genus Dysteria is paraphyletic with Spirodysteria and Mirodysteria nested within it; (iii) the genus Agnathodysteria is well separated from Dysteria based on both molecular and morphological data; and (iv) Trithigmostoma is a basal genus of Chilodonellidae, based on both the morphological and molecular data.     

Phylogenetic analysis of Nosema antheraeae (Microsporidia) isolated from Chinese oak silkworm, Antheraea pernyi

The microsporidian Nosema antheraeae is a pathogen that infects the Chinese oak silkworm, Antheraea pernyi. We sequenced the complete small subunit (SSU) rRNA gene and the internal transcribed spacer (ITS) of N. antheraeae, and compared the SSU rRNA sequences in other microsporidia. The results indicated that Nosema species, including N. antheraeae, formed two distinct clades, consistent with previous observations. Furthermore, N. antheraeae is clustered with N. bombycis with high bootstrap support. The organization of the rRNA gene of N. antheraeae is LSU-ITS1-SSU-ITS2-5S, also following a pattern similar to the Nosema type species, N. bombycis. Thus, N. antheraeae is a Nosema species and has a close relationship to N. bombycis.