Phylogenetic placement of diverse amoebae inferred from multigene analyses and assessment of clade stability within 'Amoebozoa' upon removal of varying rate classes of SSU-rDNA

Placing amoeboid lineages on the eukaryotic tree of life is difficult due to the paucity of comparable morphological characters and the limited molecular data available for many groups. This situation has led to the lumping of distantly related lineages into large inclusive groups, such as Sarcodina, that do not reflect evolutionary relationships. Previous analyses of molecular markers with limited taxon sampling reveal members of Sarcodina are scattered in five of the six proposed supergroups. We have used multigene analyses to place seven diverse amoeboid lineages-two Nolandella spp., Rhizamoeba sp., Pessonella sp., Arcella hemisphaerica, Arachnula sp. and Trichosphaerium sp.-on the eukaryotic tree of life. Bayesian analysis of the concatenated data of the four genes sequenced (SSU-rDNA, actin, alpha-tubulin and beta-tubulin), including diverse representatives of eukaryotes, indicates that all seven taxa group within the 'Amoebozoa' supergroup. We further performed separate analyses of the well-sampled SSU-rDNA and actin genes using Bayesian and Maximum Likelihood analyses to assess the positions of our newly characterized taxa. In the case of SSU-rDNA, we performed extensive analyses with removal of the fastest rates classes to evaluate the stability and resolution of various taxonomic hypotheses within 'Amoebozoa'. Five of our seven amoeboid lineages fall within well-supported clades that are corroborated by morphology. In contrast, the positions of Arachnula sp. and Trichosphaerium sp. in the SSU-rDNA gene trees are unstable and vary by analyses. Placement of these taxa will require additional data from slowly evolving genes combined with taxon-rich phylogenetic analyses. Finally, the analyses without the fastest rate classes demonstrate that SSU-rDNA has a limited signal for deep relationships within the 'Amoebozoa'.     

Scanning electron microscopy and molecular characterization of a new Haplosporidium species (Haplosporidia), a parasite of the marine gastropod Siphonaria pectinata (Mollusca: Gastropoda: Siphonariidae) in the Gulf of Mexico

Based on scanning electron microscopy and the small subunit ribosomal RNA (SSU rRNA), Haplosporidium tuxtlensis n. sp. (Haplosporidia), a parasite found in the visceral tissues of the false limpet Siphonaria pectinata (Linnaeus, 1758), is described. The spores are ellipsoidal (3.61 ± 0.15 μm × 2.69 ± 0.19 μm), with a circular lid (2.94 ± 0.5 μm) representing the operculum. The spore wall bears filaments occurring singly, or in clusters, of 2 to 8, fusing distally. Phylogenetic relationships of H. tuxtlensis n. sp. were assessed with other described species using the SSU rRNA sequence. Haplosporidium tuxtlensis n. sp. is sister taxon to Haplosporidium pickfordi Barrow, 1961. The morphological characteristics (spore wall structure, shape, size, and filament structure) and the unique host identity corroborate it as a new species. Additionally, this is the first record of Haplosporidia infecting striped false limpets in the Gulf of Mexico.     

Subulatomonas tetraspora nov. gen. nov. sp. is a member of a previously unrecognized major clade of eukaryotes

While a large number of aerobic free-living protists have been described within the last decade, the number of new anaerobic or microaerophilic microbial eukaryotic taxa has lagged behind. Here we describe a microaerophilic genus and species of amoeboflagellate isolated from a near-shore marine site off the coast at Plymouth, Massachusetts: Subulatomonas tetraspora nov. gen. nov. sp. This taxon is closely related to Breviata anathema based on both microscopical features and phylogenetic analyses of sequences of three genes: SSU-rDNA, actin, and alpha-tubulin. However, Subulatomonas tetraspora nov. gen. nov. sp. and B. anathema are morphologically distinctive, differ by 14.9% at their SSU-rDNA locus, and were isolated from marine and 'slightly brackish' environments, respectively. Phylogenetic analyses of these two taxa plus closely related sequences from environmental surveys provide support for a novel clade of eukaryotes that is distinct from the major clades including the Opisthokonta, Excavata, Amoebozoa and 'SAR' (Stramenopile, Alveolate, Rhizaria).     

Evolution of Rhizaria: new insights from phylogenomic analysis of uncultivated protists

Background  

Recent phylogenomic analyses have revolutionized our view of eukaryote evolution by revealing unexpected relationships between and within the eukaryotic supergroups. However, for several groups of uncultivable protists, only the ribosomal RNA genes and a handful of proteins are available, often leading to unresolved evolutionary relationships. A striking example concerns the supergroup Rhizaria, which comprises several groups of uncultivable free-living protists such as radiolarians, foraminiferans and gromiids, as well as the parasitic plasmodiophorids and haplosporids. Thus far, the relationships within this supergroup have been inferred almost exclusively from rRNA, actin, and polyubiquitin genes, and remain poorly resolved. To address this, we have generated large Expressed Sequence Tag (EST) datasets for 5 species of Rhizaria belonging to 3 important groups: Acantharea (Astrolonche sp., Phyllostaurus sp.), Phytomyxea (Spongospora subterranea, Plasmodiophora brassicae) and Gromiida (Gromia sphaerica).     

Three new species of deep‐sea Gromia (Protista,Rhizaria) from the bathyal and abyssal Weddell Sea,Antarctica

We describe three new species of the genus Gromia from bathyal and abyssal depths in the Weddell Sea. The new species are characterized by a combination of morphological and molecular criteria. All three species possess a distinct oral capsule and a layer of ‘honeycomb membranes’, which form the inner part of the organic test wall. Both these features are typical of gromiids. Their identification as gromiids is confirmed by analyses of partial small subunit ribosomal DNA (SSU rDNA) gene sequences. Gromia marmorea sp. nov. is a rounded species with a prominent oral capsule and a characteristically mottled appearance. In Gromia melinus sp. nov. , the test surface exhibits a polygonal pattern of ridges, with a layer of clay particles coating the surface between the ridges. Gromia winnetoui sp. nov. represents an elongate morphotype in which the organic test is enclosed within an agglutinated case, a feature previously unknown in gromiids. Phylogenetic analysis using the maximum‐likelihood method revealed that all three species form distinct clades, reflecting the morphological differences among Weddell Sea species, as well as between deep‐water Southern Ocean Gromia and previously described gromiids. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 157 , 451–469.     

Molecular phylogenetic evidence that the phylum Haplosporidia has an alveolate ancestry

The phylogenetic position of the phylum Haplosporidia among other protists was investigated with the complete 16S-like rRNA gene sequences from two species in the phylum: Haplosporidium nelsoni, a parasite of oysters, and Minchinia teredinis, a parasite of shipworms. Because the lack of obvious morphological homologies with other protists hampered decisions regarding taxonomic composition for sequence alignment and phylogenetic analysis, the complete sequences for these two haplosporidians were directed as search queries to the blast/ncbi.nlm.nih.gov electronic mail server. The results of this heuristic similarity search provided a basis for constructing a preliminary higher-taxonomic-level analysis comparing the haplosporidians with species from the slime molds, fungi, algae, amoebae, ciliates, dinoflagellates, and apicomplexans. Maximum parsimony yielded equivocal results, whereas transversionally weighted parsimony suggested an affinity with the alveolates (i.e., the ciliates, dinoflagellates, and apicomplexans). Multiple alignment of the two haplosporidian sequences against 17 taxa in a secondary analysis focusing on the alveolates and subsequent parsimony analysis placed the phylum Haplosporidia as a monophyletic group within the Alveolata and as a taxon of equal rank with the other three alveolate phyla. The precise placement within the Alveolata was sensitive to weighting.      

A new viviparous acoel Childia vivipara sp. nov. with observations on the developing embryos, sperm ultrastructure, body wall and stylet musculatures

A free‐living viviparous acoel, Childia vivipara sp. nov., from the Gullmar fjord of the Swedish coast is described. The new species is assigned to the taxon Childia based on histological, ultrastructural and molecular sequence similarities. All available molecular markers (18S rRNA, 28S rRNA and histone H3) and several morphological characters, obtained using transmission electron microscopy and confocal scanning laser microscopy of whole mount specimen stained with TRITC‐labelled phalloidin, support the placement of C. vivipara in the taxon Childia. Childia vivipara and other Childia species share the following morphological synapomorphies: well‐developed copulatory organs built of tightly packed stylet needles, proximal part of the stylet inserted into the seminal vesicle, reversed body‐wall musculature, absence of ventral diagonal muscles, presence of dorsal diagonal muscles, and presence of ventral straight longitudinal muscles between frontal pore and mouth, 9 + 1 sperm axoneme structure, six distal sperm cytoplasmic microtubules, and extensive overlap of axonemes and nucleus. The new species can be easily distinguished from other Childia species by its viviparous mode of reproduction and single curved stylet. Observations on late embryonic development based on the oldest developing embryos are discussed.     

Phylogenetic position of Gromia oviformis Dujardin inferred from nuclear-encoded small subunit ribosomal DNA

Gromia oviformis Dujardin is a common marine protist characterised by a large, globular test and filose pseudopodia. First considered a foraminifer, Gromia was later placed within the Filosea and recently included among amoebae of uncertain affinities. In order to clarify the phylogenetic position of this genus, we sequenced the complete small-subunit ribosomal DNA gene of G. oviformis collected at five different geographic localities. The high divergence of obtained sequences suggests that G. oviformis is a species complex composed of several genetically distinct sibling species. Sequence analyses show Gromia to be a member of the Cercozoa, a heterogeneous assemblage which includes filose amoebae, the amoeboflagellate cercomonads, the chlorarachniophytes and the plasmodiophorid plant pathogens. Contrary to traditional classification, Gromia is not closely related to other testate filose amoebae (the Euglyphida), but seems to branch early among the Cercozoa. Our analyses also show a close relationship between the Cercozoa and the Acantharea. Because the Cercozoa are related to the Foraminifera based on other molecular data, we propose that most protists possessing filopodia, reticulopodia and axopodia have a common origin.     

Phylogeny of novel naked Filose and Reticulose Cercozoa: Granofilosea cl. n. and Proteomyxidea revised

Naked filose and reticulose protozoa were long lumped as proteomyxids or left outside higher groups. We cultivated eight naked filose or reticulose strains, did light microscopy, 18S rDNA sequencing and phylogeny (showing all are Cercozoa), and sequenced 80 environmental 18S-types. Filose species belong in subphylum Filosa and reticulose ones in subphylum Endomyxa, making proteomyxids polyphyletic. We therefore transfer the classically mainly reticulose Proteomyxidea to Endomyxa, removing evident filosans as new class Granofilosea (including Desmothoracida, Acinetactis and new heliomonad family Heliomorphidae (new genus Heliomorpha (=Dimorpha)). Five new species of Limnofila gen. n. (L. mylnikovi; L. anglica; L. longa; L. oxoniensis; L. borokensis, previously misidentified as Biomyxa (=Gymnophrys) cometa) form a large freshwater clade (new order Limnofilida). Mesofila limnetica gen., sp. n. and Nanofila marina gen., sp. n. group separately in Granofilosea (Cryptofilida ord. n.). In Endomyxa, a new genus of reticulose proteomyxids (Filoreta marina, F. japonica, F. turcica spp. n., F. (=Corallomyxa) tenera comb. n.) forms a clade (Reticulosida) related to Gromiidea/Ascetosporea. Platyreta germanica gen., sp. n. and Arachnula impatiens are related vampyrellids (Aconchulinida) within a large clade beside Phytomyxea. Biomyxidae and Rhizoplasmidae fam. n. remain incertae sedis within Proteomyxidea. Gymnophrydium and Borkovia are revised. The reticulose Corallomyxa are unlike Filoreta and possibly Amoebozoa, not Cercozoa.     

伊贝母葡萄孢盘(新种)的分类学研究

在陕西省太白县采集到伊贝母(Fritillaria pallidiflora Schrenk.)的菌核病致病真菌,伊贝母葡萄孢盘 Botryotinia fritillarii-pallidiflori Q.T.Chen et J.L.Li sp.nov.新种。并报道了其形态特征及其与椭圆葡萄孢 [Botrytis elliptica(Berk.)Cooke]和驴蹄草葡萄孢盘(Botryotinia calthae Hennebert et Elliott)的区别。     

Revised small subunit rRNA analysis provides further evidence that Foraminifera are related to Cercozoa

There is accumulating evidence that the general shape of the ribosomal DNA-based phylogeny of Eukaryotes is strongly biased by the long-branch attraction phenomenon, leading to an artifactual basal clustering of groups that are probably highly derived. Among these groups, Foraminifera are of particular interest, because their deep phylogenetic position in ribosomal trees contrasts with their Cambrian appearance in the fossil record. A recent actin-based phylogeny of Eukaryotes has proposed that Foraminifera might be closely related to Cercozoa and, thus, branch among the so-called crown of Eukaryotes. Here, we reanalyze the small-subunit ribosomal RNA gene (SSU rDNA) phylogeny by removing all long-branching lineages that could artifactually attract foraminiferan sequences to the base of the tree. Our analyses reveal that Foraminifera branch together with the marine testate filosean Gromia oviformis as a sister group to Cercozoa, in agreement with actin phylogeny. Our study confirms the utility of SSU rDNA as a phylogenetic marker of megaevolutionary history, provided that the artifacts due to the heterogeneity of substitution rates in ribosomal genes are circumvented.     

New species of Clavulina from Guyana

Two new species of Clavulina are described from rainforests dominated by ectomycorrhizal Dicymbe spp. (Caesalpiniaceae) in the Pakaraima Mountains of Guyana. Clavulina nigricans, sp. nov. is unique for Clavulina in having 4-6 spores per basidium. Clavulina craterelloides, sp. nov. has an infundibuliform basidiome, previously unknown in the otherwise coralloid genus Clavulina. Macromorphological, micromorphological and habitat data are provided for each taxon, as well as justification for their placement in Clavulina based on morphological and molecular features.     

Sympatry in Mantophasmatodea,with the description of a new species and phylogenetic considerations

We describe a new genus of Mantophasmatodea, Viridiphasma gen. n. (Austrophasmatidae), represented by one new species, V. clanwilliamense sp. n. The new species differs from previously described species in features of the male and female postabdomen including the genitalia, in morphometrics and details of colouration. The new species occurs syntopically with another austrophasmatid, Karoophasma biedouwense Klass et al., 2003; this is the first well-documented case of sympatry of two mantophasmatodean species. We therefore survey the morphological differences between these two species, document the absence of any morphological evidence of hybridisation, and also report on differences in life history. While a previous molecular phylogeny using COI and 16S genes ambiguously placed V. clanwilliamense sp. n. near the base of Austrophasmatidae (but not as sister to all other Austrophasmatidae), morphological characters strongly support V. clanwilliamense sp. n. to be the sister taxon of a clade comprising all remaining Austrophasmatidae. This phylogenetic placement challenges the current hypothesis of a linear north-to-south diversification of Austrophasmatidae.     

The second species of Gromia (Protista) from the deep sea: its natural history and association with the Pakistan margin oxygen minimum zone

We describe a gromiid protist Gromia pyriformis sp. nov., from bathyal depths on the Pakistan margin (NE Arabian Sea), an area characterised by a well-developed Oxygen Minimum Zone (OMZ). The new species is smaller (length usually <1 mm) than the only other described deep-sea gromiid species (Gromia sphaerica) or the well-known coastal species Gromia oviformis. Its identification as a gromiid is based on the test-wall ultrastructure. This includes (i) an outer wall (165-300 nm thick) limited by an electron-opaque layer and perforated by pore structures which typically extend through its entire thickness, and (ii) inner "honeycomb membrane" structures which form a discontinuous sheet (18-20 nm thick) lying parallel to the outer wall. An outermost glycocalyx (approximately 75 nm thick), not observed in other gromiid species, is also present and imparts a finely granular appearance to the outer test surface, as seen by Scanning Electron Microscopy (SEM). Numerous rod-shaped prokaryotes are attached to the exterior of the glycocalyx. Gromia pyriformis sp. nov. typically occurs above the sediment-water interface, attached to the large arborescent foraminiferan Pelosina sp. It is confined to a very narrow bathymetric zone (approximately 1000 m water depth) in the lower portion of the OMZ, where bottom-water oxygen concentrations are approximately 0.2 ml l(-1).     

Identity and systematic position of Paradinium poucheti and other Paradinium-like parasites of marine copepods based on morphology and nuclear-encoded SSU rDNA

Paradinium and Paradinium-like parasites were detected in various copepod hosts collected in the NW Mediterranean Sea, the North Atlantic Ocean, and the Godth?bsfjord (Greenland). The identity and systematic position of the parasitic, plasmodial protist Paradinium was investigated on the basis of SSU rDNA and morphology. SSU rDNA sequences were obtained from 3 specimens of Paradinium poucheti isolated from their cyclopoid copepod host, Oithona similis. In addition, a comparable sequence was obtained from a hitherto undescribed species of Paradinium from the harpactacoid copepod Euterpina acutifrons. Finally, SSU rDNA sequences were acquired from 2 specimens of a red plasmodial parasite (RP parasite) isolated from Clausocalanus sp. Both morphological and SSU rDNA sequence data supported that P. poucheti and Paradinium sp. are closely related organisms. In phylogenetic analyses based on SSU rDNA sequences, Paradinium spp. clustered with sequences from an uncultured eukaryote clone from the Pacific Ocean and two sequences from haplosporidian-like parasites of shrimps, Pandalus spp. This Paradinium clade branched as a sister group to a clade comprising the Haplosporidia and the Foraminifera. The RP parasite had a superficial morphological resemblance to Paradinium and has previously been interpreted as a member of this genus. However, several morphological characters contradict this and SSU rDNA sequence data disagree with the RP parasite and Paradinium being related. The phylogenetic analyses suggested that the RP parasite is a fast-evolved alveolate and a member of the so-called marine alveolate Group I (MAGI) and emerging data now suggest that this enigmatic group may, like the syndinian dinoflagellates, consist of heterotrophic parasites.     

<Emphasis Type="Italic">Calcarisporium xylariicola</Emphasis> sp. nov. and introduction of <Emphasis Type="Italic">Calcarisporiaceae</Emphasis> fam. nov. in Hypocreales

During a survey of fungicolous fungi, a novel taxon from the surface of stroma of an unidentified Xylaria species was collected. Phylogenetic analyses showed that this taxon clustered with Calcarisporium sp. and C. arbuscula isolates, but was resolved as a distinct species. A detailed morphological examination coupled with phylogenetic analysis indicated that the taxon represented a new species. Calcarisporium xylariicola sp. nov. is thus introduced. The new taxon is characterized by short conidiophores with swollen bases and less length/width ratio of conidia that distinguish it from other Calcarisporium species. Calcarisporium is presently placed in Hypocreales genera, incertae sedis genus. Species in the genus are largely fungicolous, or occasionally caulicolous or foliicolous, and have hyaline, erect, verticillate conidiophores and sympodial, polyblastic conidiation. A phylogenetic analysis of combined SSU, ITS, LSU, TEF and RPB2 sequence data from Calcarisporium species and other taxa in Hypocreales indicate that Calcarisporium is a distinct lineage from other families. Therefore, a new family, Calcarisporiaceae, in Hypocreales is introduced.     

Reptodigitus Chapmanii (Nostocales,Hapalosiphonaceae) Gen. Nov.: A Unique Nostocalean (Cyanobacteria) Genus Based on a Polyphasic Approach1

The Nostocales is a monophyletic, heterocytous lineage of cyanobacteria capable of akinete production and division in multiple planes, depending upon family-level clade. While present in a variety of ecosystems, the diversity of the Nostocales has been poorly elucidated. Due to environmentally -induced phenotypic plasticity, morphology alone is often insufficient to determine the true phylogenetic placement of these taxa. In order to bridge this gap, taxonomists now employ the polyphasic approach, combining methods such as morphological analysis, phylogenetic analysis based on DNA sequence and genetic identity based on ribosomal genes, and secondary structure of the 16S-23S ITS and 16S rRNA gene sequences, as well as ecological characterization. Using this combined approach, a new genus and species (Reptodigitus chapmanii gen. et sp. nov.) isolated from the St. Johns River (Jacksonville, Florida, USA) within the Nostocales is herein described. Phylogenetic analyses place this taxon within the Hapalosiphonaceae, sister to the clade containing Fischerella, Hapalosiphon, and Westiellopsis. The 16S-23S ITS secondary folding structure analysis also supports the erection of this new genus.     

Evolution of elongation factor-like (EFL) protein in Rhizaria is revised by radiolarian EFL gene sequences

Elongation factor 1α (EF-1α) and elongation factor-like (EFL) proteins are considered to carry out equivalent functions in translation in eukaryotic cells. Elongation factor 1α and EFL genes are patchily distributed in the global eukaryotic tree, suggesting that the evolution of these elongation factors cannot be reconciled without multiple lateral gene transfer and/or ancestral co-occurrence followed by differential loss of either of the two factors. Our current understanding of the EF-1α/EFL evolution in the eukaryotic group Rhizaria, composed of Foraminifera, Radiolaria, Filosa, and Endomyxa, remains insufficient, as no information on EF-1α/EFL gene is available for any members of Radiolaria. In this study, EFL genes were experimentally isolated from four polycystine radiolarians (i.e. Dictyocoryne, Eucyrtidium, Collozoum, and Sphaerozoum), as well as retrieved from publicly accessible expressed sequence tag data of two acantharean radiolarians (i.e. Astrolonche and Phyllostaurus) and the endomyxan Gromia. The EFL homologs from radiolarians, foraminiferans, and Gromia formed a robust clade in both maximum-likelihood and Bayesian phylogenetic analyses, suggesting that EFL genes were vertically inherited from their common ancestor. We propose an updated model for EF-1α/EFL evolution in Rhizaria by incorporating new EFL data obtained in this study.     

华北地区莴苣属（广义）植物花粉特征的研究

本文研究了华北地区莴苣属（广义）７种植物的花粉形态与微形态特征。结果表明：莴苣属（广义）内这些种具有相同的花粉类型。该属的花粉为球形,３孔沟;极面观为三裂圆形,近六边形;赤道面观为圆形;花粉表面具网状纹饰,同胞１５个,网脊上有许多小穴和规则排列的刺。与邻近属形成明显差异,使该属成为一个自然类群。新属毛鳞菊属ＣｈａｅｔｏｓｅｒｉｓＳｈｉｈｇｅｎ．ｎｏｖ．以其花粉网状纹饰的网脊上具不规则排列的短柱而有别于莴苣属（广义）。花粉的形态特征与微形态特征可为菊科属级水平分类提供重要依据。