Molecular investigation of Phryganella acropodia Hertwig et Lesser, 1874 (Arcellinida,Amoebozoa)

Phryganella acropodia Hertwig and Lesser, 1874, is one of the most common and abundant testate amoeba species. It represents the type species of the genus Phryganella Penard, 1902, which in turn is the type genus for the suborder Phryganellina (Arcellinida) Bovee, 1985, but despite its taxonomic importance it was not yet analyzed with molecular methods. We established two cultures of putative Phryganella acropodia, designed Phryganellina-specific primers, amplified SSU rDNA data and subjected these sequences to phylogenetic analyses. Morphological and genetic differences were found between both strains. With SSU rDNA phylogenetic analyses we confirm that Phryganella acropodia branches with Phryganella paradoxa Penard, 1902 and Cryptodifflugia Penard, 1890 in the Phryganellina. We thus give further evidence that pseudopodia morphology in the Arcellinida is a character of high taxonomic value, as suggested by Bovee and Jung when erecting the suborder Phryganellina. Moreover, we provide evidence for cryptic diversity and for the first time confirm the existence of a naked life stage in Arcellinida by molecular means.     

Rediscovery of Nebela ansata (Amoebozoa: Arcellinida) in eastern North America: biogeographical implications

Aim The question whether free‐living protists are generally cosmopolitan is currently a matter of debate. In this study we investigate the geographical distribution of a distinctive testate amoeba species, Nebela ansata, and use our data to assess the potential for highly restricted distribution patterns in some protist species. Location Global. Methods We analysed (1) 3400 testate amoeba publications from North America and other continents, (2) unpublished slides of the Penard Collection of the Natural History Museum, London, UK, and (3) 104 Sphagnum samples from eastern North America. Non‐metric multidimensional scaling (NMDS) was used to visualize the similarities in testate amoeba community composition among 1012 North American samples, including two communities that contained N. ansata. Results We rediscovered N. ansata at a site in New Jersey located close to its type locality, and in Nova Scotia. We also report the existence of an apparently unpublished museum specimen originally collected from New Jersey. Our extensive literature survey confirmed the presence of this species only in the temperate part of eastern North America. The NMDS revealed that communities with N. ansata were less similar to each other than to communities from other parts of North America, suggesting that favourable habitats for N. ansata occur in other Sphagnum‐dominated peatlands, a habitat type that has been extensively sampled in North America and elsewhere. Main conclusions These data provide an unusually convincing case of a free‐living microorganism with a very limited distribution range in the temperate part of eastern North America. The remarkably restricted distribution of N. ansata highlights the extent of our ignorance about the natural history of free‐living microorganisms, and raises questions about the lack of attention to microbial diversity in conservation biology.     

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.     

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.     

New record of Arcella gandalfi (Amoebozoa: Arcellinida) from Colombia

The reports of testate amoebae species in Colombia are limited to a few studies in lentic and peat bog systems; however, the diversity in lotic systems has been poorly investigated. Zooplankton sampling was performed in lotic environments of the Piedemonte Llanero in Colombia. Ten individuals of the species Arcella gandalfi were identified to document the first record of A. gandalfi from the Piedemonte Llanero in Colombia, expanding its distribution from Brazil to Colombia. This finding corroborates the status of A. gandalfi as a flagship species from the South America continent.     

Not all free-living microorganisms have cosmopolitan distributions – the case of Nebela (Apodera) vas Certes (Protozoa: Amoebozoa: Arcellinida)

Aim  To review and critically assess the evidence that the testate amoeba species Nebela vas does not have a cosmopolitan distribution, in contradiction to the paradigm of microbial distribution that 'everything is everywhere'.
Location  Terrestrial and wetland habitats world-wide.
Methods  Relevant data were assembled on the morphology, ecology and global geographical distribution of N. vas from a comprehensive literature review of published original records. The data were collated, analysed and summarized. The roles of long-term and short-term agents of dispersal are discussed.
Results  A clear and repeated pattern has been elucidated of a microbial species, with a distinctive and unmistakeable morphology, that inhabits acid, damp terrestrial and wetland habitats in the southern cool-temperate and sub-Antarctic zones – including similar high-altitude habitats in the Tropics. It is almost entirely confined to the Gondwanaland continents and Southern Hemisphere islands. It is definitively absent from Holarctic regions. It is proposed that the continental distribution of N. vas is a consequence of continental drift following its original speciation in the Mesozoic or earlier, whereas its distribution on sub-Antarctic islands (glaciated in the Pleistocene) has been effected by dispersal from South America by wind around the Southern Ocean.
Main conclusion  Nebela vas is a proven instance of a microbial species that does not have a cosmopolitan distribution.     

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).     

Nebela jiuhuensis nov. sp. (Amoebozoa; Arcellinida; Hyalospheniidae): A New Member of the Nebela saccifera ‐ equicalceus ‐ ansata Group Described from Sphagnum Peatlands in South‐Central China

Hyalospheniids are among the most common and conspicuous testate amoebae in high‐latitude peatlands and forest humus. These testate amoebae were widely studied as bioindicators and are increasingly used as models in microbial biogeography. However, data on their diversity and ecology are still very unevenly distributed geographically: notably, data are lacking for low‐latitude peatlands. We describe here a new species, Nebela jiuhuensis, from peatlands near the Middle Yangtze River reach of south‐central China with characteristic morphology. The test (shell) has hollow horn‐like lateral extensions also found in N. saccifera, N. equicalceus (=N. hippocrepis), and N. ansata, three large species restricted mostly to Sphagnum peatlands of Eastern North America. Mitochondrial cytochrome oxidase (COI) data confirm that N. jiuhuensis is closely related to the morphologically very similar North American species N. saccifera and more distantly to N. ansata within the N. penardiana group. These species are all found in wet mosses growing in poor fens. Earlier reports of morphologically similar specimens found in South Korea peatlands suggest that N. jiuhuensis may be distributed in comparable peatlands in Eastern Asia (China and Korea). The discovery of such a conspicuous new species in Chinese peatlands suggests that many new testate amoebae species are yet to be discovered, including potential regional endemics. Furthermore, human activities (e.g., drainage, agriculture, and pollution) have reduced the known habitat of N. jiuhuensis, which can thus be considered as locally endangered. We, therefore, suggest that this very conspicuous micro‐organism with a probably limited geographical distribution and specific habitat requirement should be considered as a flagship species for microbial biogeography as well as local environmental conservation and management.     

A needle in a haystack: A new metabarcoding approach to survey diversity at the species level of Arcellinida (Amoebozoa: Tubulinea)

Environmental DNA-based diversity studies have increased in popularity with the development of high throughput sequencing technologies. This permits the potential simultaneous retrieval of vast amounts of molecular data from many different organisms and species, thus contributing to a wide range of biological disciplines. Environmental DNA protocols designed for protists often focused on the highly conserved small subunit of the ribosome gene, that does not permit species-level assignments. On the other hand, eDNA protocols aiming at species-level assignments allow a fine level ecological resolution and reproducible results. These protocols are currently applied to organisms living in marine and shallow lotic freshwater ecosystems, often in a bioindication purpose. Therefore, in this study, we present a species-level eDNA protocol designed to explore diversity of Arcellinida (Amoebozoa: Tubulinea) testate amoebae taxa that is based on mitochondrial cytochrome oxidase subunit I (COI). These organisms are widespread in lentic water bodies and soil ecosystems. We applied this protocol to 42 samples from peatlands, estuaries and soil environments, recovering all the infraorders in Glutinoconcha (with COI data), except for Hyalospheniformes. Our results revealed an unsuspected diversity in morphologically homogeneous groups such as Cylindrothecina, Excentrostoma or Sphaerothecina. With this protocol we expect to revolutionize the design of modern distributional Arcellinida surveys. Our approach involves a rapid and cost-effective analysis of testate amoeba diversity living in contrasted ecosystems. Therefore, the order Arcellinida has the potential to be established as a model group for a wide range of theoretical and applied studies.     

中国有壳肉足虫(原生动物)五新纪录描述

描述了采自云南、湖北和广东的5种有壳肉足虫,隶属原生动物亚界,肉鞭门,叶足纲,表壳目,均为国内新纪录种:非游巧装表壳虫Arcella artocrtea aplanata Grospietsch,1954;具角肋表壳虫A.costata angulosa (Perty) Playfair,1918;拟普通盘状表壳虫A.discoides pseudovulgaris Deflandre ,1928;光滑弯凸表壳虫A.gibbosa laevis Deflandre ,1928;球形叉口砂壳虫Difflugia gramen globulosa Stěpánek et Jirí,1958.     

Transfer of the Thecate Amoeba Lecythium mutabilis to a Novel Genus Omnivora (Fiscullidae,Thecofilosea, Cercozoa)

Thecofilosea is a class in Cercozoa (Rhizaria) comprising mainly freshwater‐inhabiting algivores. Recently, numerous isolates of thecofilosean amoebae have been cultured and were characterized by an integrated morphological and molecular approach. As attempts to establish a culture of Lecythium mutabilis repeatedly failed, it was not yet investigated by molecular means. We isolated single cells of L. mutabilis directly from their habitat and successfully sequenced the V4 region of their SSU rDNA. Phylogenetic analyses showed that L. mutabilis is not directly related to the genus Lecythium and instead branches within the Fiscullidae (Tectofilosida, Thecofilosea). Accordingly, we transfer the species L. mutabilis to a novel genus Omnivora gen. nov.     

Phylogeny and Redescription of the Testate Amoeba Diaphoropodon archeri (Chlamydophryidae,Thecofilosea, Cercozoa), De Saedeleer 1934, and Annotations on the Polyphyly of Testate Amoebae with Agglutinated Tests in the Cercozoa

The genus Diaphoropodon, Archer 1869, comprises filose amoebae with agglutinated tests made of quartz grains, diatom frustules and other particulate materials. The key trait of the genus is a hyaline theca covered with numerous 5‐ to 10‐μm‐long, hairlike rods. Based on SSU rDNA phylogeny, we show that Diaphoropodon groups closely to Lecythium, a testate amoeba genus with a flexible but naked theca. Electron microscopic images reveal that the rods of Diaphoropodon are not perforating the test but lie randomly distributed on the surface of the amoeba. Comparing fairly naked cells from our cultures with cells from the environment leads to the conclusion that these rods play a role in agglutinating the material on the test.     

Phylogenetic divergence within the Arcellinida (Amoebozoa) is congruent with test size and metabolism type

Arcellinida (lobose testate amoebae) are abundant and diverse in many ecosystems, especially in moist to aquatic environments. Molecular phylogeny has shown that overall test morphology (e.g., spherical or elongate) is generally conserved in Arcellinida lineages, but the taxonomic value of other traits (e.g., size, ornamentation, mixotrophy/heterotrophy metabolism type) has not been systematically evaluated. Morphological and physiological traits that correspond to genetic differences likely represent adaptive traits of ecological significance. We combined high-resolution phylogenetics (NAD9-NAD7 genes) and advanced morphometrics to assess the phylogenetic signal of morphological traits of a group of elongate Difflugia species (Arcellinida). The phylogenetic analyses revealed two clades which could be reliably separated by test size and the presence/absence of mixotrophy. Differences in test size may reflect trophic level, with smaller organisms occupying lower trophic levels. In addition to having larger tests, elongate mixotrophic Difflugia are characterised by wide, flat bases and an inflation of the lower two thirds of their test. These morphological traits may provide additional volume for endosymbionts and/or increased surface area to aid light transmission. Our results showcase greater diversity within the elongate Difflugia and highlight morphological traits of ecological and evolutionary significance.