Phylogeny and Establishment of a New Ciliate Family,Wilbertomorphidae fam. nov. (Ciliophora,Karyorelictea), a Highly Specialized Taxon Represented by Wilbertomorpha colpoda gen. nov., spec. nov.

The morphology and molecular phylogeny of a new marine interstitial karyorelictean ciliate, Wilbertomorpha colpoda gen. nov., spec. nov., isolated from a sandy beach at Qingdao, China, are investigated. The ciliature is mainly on the right surface with the left surface barren except for a single marginal kinety, which is typical of the order Loxodida. Nevertheless, other morphological features, such as the highly reduced buccal structure, the absence of a buccal cavity and the presence of extremely specialized cortical granules, prevent its assignation to any known family. Furthermore, the secondary structure of the variable region 2 of the SSU rRNA gene is unique among loxodids. Thus, we establish a new family, Wilbertomorphidae fam. nov. for the genus Wilbertomorpha. Phylogenetic analyses based on SSU rRNA gene sequence data suggest that this taxon is a sister group to the family Geleiidae which belongs to the order Protoheterotrichida. However, morphologically Wilbertomorpha bears a much stronger resemblance to Loxodidae which belongs to the order Loxodida. Based on these findings, we tentatively place Wilbertomorphidae nov. fam. incertae sedis within Karyorelictea.     

Molecular Phylogeny and Taxonomy of a New Freshwater Hymenostomatid from Northeastern China,with the Establishment of a New Genus Anteglaucoma gen. n. (Protista,Ciliophora, Oligohymenophorea)

The morphology, infraciliature and SSU rDNA sequence of a new freshwater hymenostomatid ciliate, Anteglaucoma harbinensis gen. nov., spec. nov., collected from a farmland pond in Harbin, China, were investigated. The new genus Anteglaucoma is characterized as follows: small to medium‐sized Glaucomidae with oral apparatus in anterior one‐third of cell; paroral membrane composed of almost longitudinally arranged dikinetids; three adoral membranelles nearly equal in length and arranged almost longitudinally in parallel; silverline pattern tetrahymenid. The improved diagnosis of family Glaucomidae Corliss 1971 is provided based on the previous and present work. The type species Anteglaucoma harbinensis spec. nov. is defined by having 32–35 somatic kineties; four or five postoral kineties; membranelle 1 and membranelle 2 having five or six kinetosomal rows, membranelle 3 having three kinetosomal rows; single macronuclear nodule; contractile vacuole on average 15% from posterior body end; locomotion characterized by crawling with a rather hectic jerking motion; freshwater habitat. Phylogenetic analyses show that Anteglaucoma clusters in the family Glaucomidae and groups with the genera Glaucoma. The molecular and morphological data indicate that Glaucomidae is related to the family Bromeliophryidae in the phylogenetic trees.     

Morphology and molecular phylogeny of the anaerobic freshwater ciliate Urostomides spinosus nov. spec. (Ciliophora,Armophorea, Metopida) from China

The morphology and molecular phylogeny of a new metopid ciliate, Urostomides spinosus nov. spec., discovered in a freshwater ditch in Qingdao, China, were investigated using live observation, morphometry and protargol staining as well as molecular phylogenetic methods. Diagnostic features of the new species include a broadly obpyriform body carrying three posterior spines, eight somatic kineties, five preoral dome kineties with specialized row 3, adoral zone composed of about 28 membranelles, making a 270° turn around body axis. Phylogenetic analyses of the SSU rDNA sequence revealed that the genus Urostomides is monophyletic, but its interspecific relationships remained unresolved. Moreover, a closer relationship of the new species with the morphologically similar Urostomides campanula was not supported by the molecular data.     

A further phylogenetic study of the heterotrophic dinoflagellate genus, Protoperidinium (Dinophyceae) based on small and large subunit ribosomal RNA gene sequences

The heterotrophic marine dinoflagellate genus Protoperidinium is the largest genus in the Dinophyceae. Previously, we reported on the intrageneric and intergeneric phylogenetic relationships of 10 species of Protoperidinium, from four sections, based on small subunit (SSU) rDNA sequences. The present paper reports on the impact of data from an additional 5 species and, therefore, an additional two sections, using the SSU rDNA data, but now also incorporating sequence data from the large subunit (LSU) rDNA. These sequences, in isolation and in combination, were used to reconstruct the evolutionary history of the genus. The LSU rDNA trees support a monophyletic genus, but the phylogenetic position within the Dinophyceae remains ambiguous. The SSU, LSU and SSU + LSU rDNA phylogenies support monophyly in the sections Avellana, Divergentia, Oceanica and Protoperidinium, but the section Conica is paraphyletic. Therefore, the concept of discrete taxonomic sections based on the shape of 1′ plate and 2a plate is upheld by molecular phylogeny. Furthermore, the section Oceanica is indicated as having an early divergence from other groups within the genus. The sections Avellana and Excentrica and a clade combining the sections Divergentia/Protoperidinium derived from Conica‐type dinoflagellates independently. Analysis of the LSU rDNA data resulted in the same phylogeny as that obtained using SSU rDNA data and, with increased taxon sampling, including members of new sections, a clearer idea of the evolution of morphological features within the genus Protoperidinium was obtained. Intraspecific variation was found in Protoperidinium conicum (Gran) Balech, Protoperidinium excentricum (Paulsen) Balech and Protoperidinium pellucidum Bergh based on SSU rDNA data and also in Protoperidinium claudicans (Paulsen) Balech, P. conicum and Protoperidinium denticulatum (Gran et Braarud) Balech based on LSU rDNA sequences. The common occurrence of base pair substitutions in P. conicum is indicative of the presence of cryptic species.     

GRACILARIOPSIS MCLACHLANII SP. NOV. AND GRACILARIOPSIS PERSICA SP. NOV. OF THE GRACILARIACEAE (GRACILARIALES,RHODOPHYCEAE) FROM THE INDIAN OCEAN1

Two new species of Gracilariopsis from the Indian Ocean are proposed—Gracilariopsis (Gp.) mclachlanii Buriyo, Bellorin et M. C. Oliveira sp. nov. from Tanzania and Gracilariopsis persica Bellorin, Sohrabipour et E. C. Oliveira sp. nov. from Iran—based on morphology and DNA sequence data (rbcL gene and SSU rDNA). Both species fit the typical features of Gracilariopsis: axes cylindrical throughout, freely and loosely ramified up to four orders, with an abrupt transition in cell size from medulla to cortex, cystocarps lacking tubular nutritive cells and superficial spermatangia. Nucleotide sequence comparisons of rbcL and SSU rDNA placed both species into the Gracilariopsis clade as distinct species from all the accepted species for this genus, forming a deeply divergent lineage together with some species from the Pacific. The new species are very difficult to distinguish on morphological grounds from other species of Gracilariopsis, stressing the importance of homologous molecular marker comparisons for the species recognition in this character‐poor genus.     

Morphology and phylogeny of four marine or brackish water spirotrich ciliates (Protozoa,Ciliophora) from China,with descriptions of two new species

In this study, we investigated the morphology and molecular phylogeny of four marine or brackish spirotrichean ciliates found in China, namely: Caryotricha sinica sp. nov., Prodiscocephalus orientalis sp. nov., P. cf. borrori, and Certesia quadrinucleata. Caryotricha sinica is characterized by its small size, seven cirral rows extending posteriorly to about 65% of the cell length, and four transverse cirri. Prodiscocephalus orientalis differs from its congeners mainly by the number of cirri in the “head” region and on the ventral side. The SSU rDNA sequence of P. cf. borrori differs from that of other population of P. borrori by ca. 40 bp. Consequently, the nominal species P. borrori is considered to be a species-complex. New data are provided for Certesia quadrinucleata. The Chinese population of C. quadrinucleata, for example, has fewer left marginal cirri than the other populations for which such data are available. Phylogenetic analyses based on SSU rDNA sequence data show that the genus Caryotricha is monophyletic. All typical “discocephalids” with a discoid “head” form a strongly supported clade that is sister to the unstable uronychiids + pseudoamphisiellids clade within the Euplotia. The genus Certesia forms a sister group to the Euplotes clade, also within the Euplotia assemblage.     

New taxa refresh the phylogeny and classification of pleurostomatid ciliates (Ciliophora,Litostomatea)

A high diversity of pleurostomatid ciliates has been discovered in the last decade, and their systematics needs to be improved in the light of new findings concerning their morphology and molecular phylogeny. In this work, a new genus, Protolitonotus gen. n., and two new species, Protolitonotus magnus sp. n. and Protolitonotus longus sp. n., were studied. Furthermore, 19 novel nucleotide sequences of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2 were collected to determine the phylogenetic relationships and systematic positions of the pleurostomatid ciliates in this study. Based on both molecular and morphological data, the results demonstrated that: (i) as disclosed by the sequence analysis of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2, Protolitonotus gen. n. is sister to all other pleurostomatids and thus represents an independent lineage and a separate family, Protolitonotidae fam. n., which is defined by the presence of a semi‐suture formed by the right somatic kineties near the dorsal margin of the body; (ii) the families Litonotidae and Kentrophyllidae are both monophyletic based on both SSU rDNA and LSU rDNA sequences, whereas Amphileptidae are non‐monophyletic in trees inferred from SSU rDNA sequences; and (iii) the genera Loxophyllum and Kentrophyllum are both monophyletic, whereas Litonotus is non‐monophyletic based on SSU rDNA analyses. ITS1‐5.8S‐ITS2 sequence data were used for the phylogenetic analyses of pleurostomatids for the first time; however, species relationships were less well resolved than in the SSU rDNA and LSU rDNA trees. In addition, a major revision to the classification of the order Pleurostomatida is suggested and a key to its families and genera is provided.     

Morphology and Morphogenesis of a Novel Saline Soil Hypotrichous Ciliate,Gonostomum sinicum nov. spec. (Ciliophora,Hypotrichia, Gonostomatidae), Including a Report on the Small Subunit rDNA Sequence

Morphology, cirral pattern, and morphogenesis of the new saline soil hypotrich, Gonostomum sinicum nov. spec. collected from Longfeng Wetland in Daqing, north China, were studied, using detailed live observations and protargol‐stained specimens. The new species is characterized as follows: (i) a size in vivo of 100–125 × 30–40 μm, (ii) colorless cortical granules, 0.5 μm across, arranged in short rows, (iii) an adoral zone composed of 28–33 membranelles, (iv) three or four frontoventral rows, one of which extends onto the postoral area, (v) left and right marginal rows composed of 18–27 and 21–35, cirri, respectively, and (vi) usually two transverse cirri. Morphogenesis is as usual for the genus Gonostomum, i.e. the cirral primordia II–VI are primary primordia which split into two sets for proter and opisthe in division middle stages, except for anlage I which develops independently. However, the number of frontoventral transverse anlagen is either five or six not only in different individuals but even in proter and opisthe of the same divider. The phylogenetic analyses based on SSU rDNA sequences showed that the genus Gonostomum is nonmonophyletic, indicating that the patterns of cirri and dorsal kineties are homoplasious characters. The new species G. sinicum nov. spec. is perhaps closely related to Cotterillia bromelicola and two congeners.     

Phylogeny and taxonomy of the Raphidophyceae (Heterokontophyta) and Chlorinimonas sublosa gen. et sp. nov., a new marine sand-dwelling raphidophyte

A new flagellate of the Raphidophyceae, Chlorinimonas sublosa gen. et sp. nov., collected from Wakayama Prefecture, Japan is described based on morphological observations, microspectrophotometry of chloroplasts, and phylogenetic analysis of SSU rDNA sequences. The cell was usually elliptical, sometimes spherical, oval or slender, and possessed two subequal heterodynamic flagella emerging from a subapical pit. Greenish yellow discoidal chloroplasts, 15–25 per cell, were situated at the periphery of the cell. The alga is very similar to the genus Heterosigma, but distinct in that there is no invagination of thylakoids into the pyrenoids and no typical girdle lamella in the chloroplast, and the chloroplasts are greenish yellow. Phylogenetic analysis of SSU rDNA revealed that this alga forms a sister clade with the clade of Chattonella and Heterosigma. Based on these results, we propose a new genus Chlorinimonas with Chlorinimonas sublosa as the type species. In addition, this paper is the first report of molecular data covering all genera of the Raphidophyceae. The phylogenetic analysis suggests that the intrusion to freshwater habitat has occurred only once in the Raphidophyceae.     

Description of the Halophile Euplotes qatarensis nov. spec. (Ciliophora,Spirotrichea, Euplotida) Isolated from the Hypersaline Khor Al‐Adaid Lagoon in Qatar

The morphology, ontogenesis, and phylogenetic relationships of a halophile euplotid ciliates, Euplotes qatarensis nov. spec., isolated from the Khor Al‐Adaid Lagoon in Qatar were investigated based on live observation as well as protargol‐ and silver nitrate‐impregnated methods. The new species is characterised by a combination of features: the halophile habitat, a cell size of 50–65 × 33–40 μm, seven dorsal ridges, 10 commonly sized frontoventral cirri, two widely spaced marginal cirri, 10 dorsolateral kineties, and a double silverline pattern. The morphogenesis is similar to that of its congeners: (i) the oral primordium develops hypoapokinetally and the parental oral apparatus is retained; (ii) the frontoventral‐transverse field of five streaks gives rise to the frontal, ventral, and transverse cirri, but not to the cirri I/1 and the marginal cirri; (iii) the dorsal somatic ciliature develops by intrakinetal proliferation of basal bodies in two anlagen per kinety that are just anterior and posterior to the future division furrow; (iv) the caudal cirri are formed by the two rightmost dorsolateral kineties. The SSU rDNA sequence of E. qatarensis branches with full support in the Euplotopsis elegans–Euplotes nobilii–Euplotopsis raikovi clade. The closest related publicly available SSU rDNA sequence is the one of E. nobilii, with which E. qatarensis has 93.4% sequence similarity. Euplotes parawoodruffi Song & Bradbury, 1997 is transferred to the genus Euplotoides based on the absence of frontoventral cirrus VI/3.     

Morphology,ontogenesis and molecular phylogeny of a new saline soil ciliate,Uroleptoides salina nov. spec. (Ciliophora,Hypotrichia)

The morphology, morphogenesis and molecular phylogeny of a new saline soil hypotrich ciliate, Uroleptoides salina nov. spec., discovered from China, was investigated. The new species is characterized as follows: body 150–215 × 40–50 μm in vivo, slender and highly flexible; usually four ellipsoidal macronuclear nodules; contractile vacuole absent; cortical granules absent; endosymbiotic algae present; amphisiellid median cirral row consists of 14–25 cirri and terminates about 47% down length of body; usually three buccal cirri and 3–13 cirri left of anterior portion of amphisiellid median cirral row; 3–5 transverse cirri. Morphogenesis during binary fission is characterized by: (1) the parental adoral zone of membranelles is retained completely, parental paroral contributes to the formation of the undulating membranes anlage for the proter; (2) the oral primordium of the opisthe is formed apokinetally; and (3) the amphisiellid median cirral row is formed from two anlagen. Phylogenetic analyses based on SSU rDNA sequence data show that Uroleptoides salina nov. spec. has a close relationship with its morphologically similar species, U. longiseries, U. magnigranulosus, Orthamphisiella breviseries, and Parabistichella variabilis.     

Multiple Independent Losses of Photosynthesis and Differing EvolutionaryRates in the Genus Cryptomonas (Cryptophyceae): Combined Phylogenetic Analyses of DNA Sequences of the Nuclear and the Nucleomorph Ribosomal Operons

In this study, evidence for at least three independent losses of photosynthesis in the freshwater cryptophyte genus Cryptomonas is presented. The phylogeny of the genus was inferred by molecular phylogenetic analyses of the nuclear internal transcribed spacer 2 (nuclear ITS2), partial nuclear large subunit ribosomal DNA (LSU rDNA), and nucleomorph small subunit ribosomal DNA (SSU rDNA, NM). Both concatenated and single data sets were used. In all data sets, the colorless Cryptomonas strains formed three different lineages, always supported by high bootstrap values (maximum parsimony, neighbor joining and maximum likelihood) and posterior probabilities (Bayesian analyses). The three leukoplast-bearing lineages displayed differing degrees of accelerated evolutionary rates in nuclear and nucleomorph rDNA. Also an increase in A+T-content in highly variable regions of the nucleomorph SSU rDNA was observed in one of the leukoplast-bearing lineages.This article contains three online-only supplementary tables.Reviewing Editor: Dr. Yves Van de Peer     

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.     

Morphology and Molecular Phylogeny of a New Hypotrich Ciliate,Pseudourostyla guizhouensis sp. nov. from Southern China,with Notes on a Chinese Population of Hemicycliostyla franzi (Foissner, 1987) Paiva et al., 2012 (Ciliophora,Hypotricha)

The morphology and molecular phylogeny of a soil hypotrich ciliate, Pseudourostyla guizhouensis sp. nov., collected from southern China, were investigated. Pseudourostyla guizhouensis sp. nov. has an elongate elliptical body measuring 180–310 × 65–85 μm in vivo; invariably two right and three or four left marginal rows; six or seven dorsal kineties; adoral zone consisting of 57–70 membranelles; 12–16 frontal cirri, one buccal cirrus, 13–20 midventral pairs, two frontoterminal cirri, two pretransverse cirri, and five to seven transverse cirri. Morphogenesis during physiological regeneration indicates that the marginal rows of each side originate from a common anlage that differentiates into several rows. Molecular phylogenetic analysis based on SSU rDNA sequence data reveals that P. guizhouensis sp. nov. clusters with the type species P. cristata (Jerka‐Dziadosz, 1964) Borror, 1972 and that the genus Pseudourostyla is monophyletic. The morphological characters of another soil hypotrich ciliate, Hemicycliostyla franzi (Foissner, 1987) Paiva et al., 2012, are also described based on a Chinese (Guizhou) population.     

Lineage-specific variations of congruent evolution among DNA sequences from three genomes,and relaxed selective constraints on <Emphasis Type="Italic">rbc</Emphasis>L in <Emphasis Type="Italic">Cryptomonas</Emphasis> (Cryptophyceae)

Background  

Plastid-bearing cryptophytes like Cryptomonas contain four genomes in a cell, the nucleus, the nucleomorph, the plastid genome and the mitochondrial genome. Comparative phylogenetic analyses encompassing DNA sequences from three different genomes were performed on nineteen photosynthetic and four colorless Cryptomonas strains. Twenty-three rbc L genes and fourteen nuclear SSU rDNA sequences were newly sequenced to examine the impact of photosynthesis loss on codon usage in the rbc L genes, and to compare the rbc L gene phylogeny in terms of tree topology and evolutionary rates with phylogenies inferred from nuclear ribosomal DNA (concatenated SSU rDNA, ITS2 and partial LSU rDNA), and nucleomorph SSU rDNA.     

Molecular Phylogeny of Conjugating Green Algae (Zygnemophyceae, Streptophyta) Inferred from SSU rDNA Sequence Comparisons

Abstract Nuclear-encoded SSU rDNA sequences have been obtained from 64 strains of conjugating green algae (Zygnemophyceae, Streptophyta, Viridiplantae). Molecular phylogenetic analyses of 90 SSU rDNA sequences of Viridiplantae (inciuding 78 from the Zygnemophyceae) were performed using complex evolutionary models and maximum likelihood, distance, and maximum parsimony methods. The significance of the results was tested by bootstrap analyses, deletion of long-branch taxa, relative rate tests, and Kishino–Hasegawa tests with user-defined trees. All results support the monophyly of the class Zygnemophyceae and of the order Desmidiales. The second order, Zygnematales, forms a series of early-branching clades in paraphyletic succession, with the two traditional families Mesotaeniaceae and Zygnemataceae not recovered as lineages. Instead, a long-branch Spirogyra/Sirogonium clade and the later-diverging Netrium and Roya clades represent independent clades. Within the order Desmidiales, the families Gonatozygaceae and Closteriaceae are monophyletic, whereas the Peniaceae (represented only by Penium margaritaceum) and the Desmidiaceae represent a single weakly supported lineage. Within the Desmidiaceae short internal branches and varying rates of sequence evolution among taxa reduce the phylogenetic resolution significantly. The SSU rDNA-based phylogeny is largely congruent with a published analysis of the rbcL phylogeny of the Zygnemophyceae (McCourt et al. 2000) and is also in general agreement with classification schemes based on cell wall ultrastructure. The extended taxon sampling at the subgenus level provides solid evidence that many genera in the Zygnemophyceae are not monophyletic and that the genus concept in the group needs to be revised.     

Redescription of Rigidohymena inquieta (Stokes, 1887) Berger, 2011 as Metahymena inquieta gen. nov., comb. nov. (Ciliophora,Hypotricha) Based on Morphology,Morphogenesis, and Molecular Phylogeny

The morphology and morphogenesis of Rigidohymena inquieta (Stokes, 1887) Berger, 2011, isolated from a lawn soil in the campus of the University of Ulsan, Korea, was studied, using live observation and protargol impregnation. The molecular phylogeny was studied based on the SSU rRNA gene sequences. The morphology of the Korean population of R. inquieta matches the previously known populations; however, the morphogenetic pattern shows differences to the species R. candens in the involvement of cirrus V/3 in the anlagen formation. A novel genus namely Metahymena gen. nov. has been erected for the present species based on the ontogenetic difference, and the new combination Metahymena inquieta gen. nov., comb. nov. is proposed. The morphology, morphogenesis, distribution, and phylogeny of M. inquieta are presented. The morphologic and morphogenetic data corroborate the phylogenetic analyses as M. inquieta clusters among the stylonychid ciliates in a clade distant from Rigidohymena candens.     

Multigene‐based phylogeny of Urostylida (Ciliophora,Hypotrichia), with establishment of a novel family

The Urostylida is a major taxon of hypotrichs with many unresolved evolutionary relationships. Due to incomplete or inaccurate character states and a paucity of morphogenetic data, the phylogeny of several taxa within urostylids is unresolved. Molecular phylogeny studies based on single gene (SSU rDNA) data may lead to conflict between morphological classification and SSU rDNA tree. In this work, 20 new sequences (SSU rDNA, ITS1‐5.8S‐ITS2 and LSU rDNA) of five genera of urostylids are provided to further investigate the phylogenetic relationships of this group. The main findings are as follows: (i) the establishment of Hemicycliostylidae, a novel family presently including Hemicycliostyla and Australothrix, is supported by both single gene and concatenated phylogenies; (ii) all molecular data support the exclusion of Eschaneustyla from the family Epiclintidae; (iii) Australothrix, Bergeriella and Thigmokeronopsis are distinctly separated in all gene trees although they share the character that each posterior streak generates the ventral row together with the midventral pair; (iv) compared with closely related genera in all trees, that is Metaurostylopsis and Apourostylopsis, Neourostylopsis is characterized by having more than three frontal cirri arranged in distinct or indistinct corona rather than the length of the midventral complex; (v) Hemicycliostyla and Pseudourostyla, two morphologically similar genera, do not form a monophyletic group in all molecular trees, suggesting that the bicorona, multiple marginal cirral rows and high numbers of dorsal kineties may result from convergent evolution; (vi) species of Bakuella fall into three separate clades in all trees suggesting that this genus needs to be split.