Phylogenetic Investigations on Ten Genera of Tintinnid Ciliates (Ciliophora: Spirotrichea: Tintinnida), Based on Small Subunit Ribosomal RNA Gene Sequences

Tintinnida is a diverse taxon that accommodates over 1,500 morphospecies, which is an important component of marine planktonic food webs. However, evolutionary relationships of tintinnids are poorly known because molecular data of most groups within this order are lacking. In our study, the small subunit (SSU) rRNA genes representing 10 genera, 5 families of Tintinnida were sequenced, including the first SSU rRNA gene sequences for Coxliella, Dadayiella, Epiplocyloides, and Protorhabdonella, and phylogenetic trees were constructed to assess their intergeneric relationships. Phylogenies inferred from different methods showed that (1) Three newly sequenced Eutintinnus species fell into Eutintinnus clade forming a sister group to the clade containing Amphorides, Steenstrupiella, Amphorellopsis, and Salpingella; (2) Surprisingly, the genetic distances between Amphorides amphora and Amphorellopsis acuta population 1 was even smaller than that between the two populations of Amphorellopsis acuta, casting doubt on the validity of Amphorides and Amphorellopsis as presently defined; (3) The SSU rRNA sequences of Dadayiella ganymedes and Parundella aculeata were almost identical. Therefore, Parundella ganymedes novel combination is proposed; (4) Coxliella, which is currently assigned within Metacylididae, branched instead with some Tintinnopsis species. Furthermore, the validation of Coxliella, which was considered to be a “questionable” genus, was confirmed based on evidences from morphology, ecology, and molecular data; (5) Protorhabdonella and Rhabdonella showed rather low intergeneric distance and grouped together with strong support suggesting that Rhabdonellidae is a well‐defined taxon; and (6) Epiplocyloides branched with species in Cyttarocylididae indicating their close relationship.     

A New Genus of Marine Scuticociliate (Protozoa, Ciliophora) from Northern China, with a Brief Note on Its Phylogenetic Position Inferred from Small Subunit Ribosomal DNA Sequence Data

ABSTRACT. The morphology, infraciliature, and silverline system of a new marine scuticociliate, Wilbertia typica n. g., n. sp., collected from coastal waters off northern China, were investigated. The new genus Wilbertia is characterized as follows: sculptured and dorso-ventrally flattened body; dominant buccal field that is almost completely surrounded by the paroral membrane; three apically positioned long membranelles, arranged in parallel; membranelle (M)1 and M2 prominent, M3 small; reticulate silverline system. The type species W. typica n. sp. is defined by having a conspicuous anterior beak-like protrusion; five to eight caudal cilia; M1 four-rowed, M2 two-rowed; M3, single-rowed, bipartite; 15 or 16 somatic kineties; contractile vacuole positioned just posterior to the buccal field; globular macronucleus. The small subunit ribosomal DNA sequence of W. typica is 98.5% similar to the similar morphotype, Eurystomatella sinica . Phylogenetic analyses indicate that Wilbertia groups with Eurystomatella sinica forming a branch that diverges at a deep level from all other pleuronematid scuticociliates. The molecular and morphological data indicate that Wilbertia should be placed within the family Eurystomatidae, which is closely related to the well-known Cyclidiidae.     

Three New Loxophyllum Species (Ciliophora: Pleurostomatida) from China with a Brief Review of the Marine and Brackish Loxophyllum Species

Recent studies indicate that there is a high diversity of pleurostomatid ciliates in the coastal waters of China. Here, three new congeners of Loxophyllum, L. caudatum sp. n., L. rugosum sp. n., and L. chinense sp. n., are described following observations of live cells and protargol‐impregnated specimens. All three species usually have two macronuclear nodules and prominent warts along the dorsal margin formed by clustered extrusomes. In addition, L. caudatum sp. n. is characterized by its long conspicuous tail, dot‐like cortical granules, 4 or 5 left and 9 or 10 right kineties, and a single subterminal contractile vacuole. Loxophyllum rugosum sp. n. is distinguished by possessing three prominent ridges on the left side, 7–11 right and 5–7 left kineties. Loxophyllum chinense sp. n. is characterized by having several contractile vacuoles distributed along the ventral margin, 13–18 right and 6–8 left kineties. The small subunit ribosomal DNA (SSU rDNA) sequence similarities among six congeners range from 96.46% to 99.94%. Phylogenetic trees based on the SSU rDNA sequences indicate that all Loxophyllum spp. form a well‐supported monophyletic group. A brief review of the marine and brackish Loxophyllum species is supplied and one new combination, Litonotus multiplicatus (Kahl 1931) comb. n. (basionym Loxophyllum multiplicatum Kahl 1931), and one new name, Litonotus dragescoi nom. n. (basionym L. fasciolatus Dragesco 1966), are suggested.     

Morphology and Small Subunit rDNA Phylogeny of Two New Marine Urostylid Ciliates,Caudiholosticha marina sp. nov. and Nothoholosticha flava sp. nov. (Ciliophora,Hypotrichia)

Two marine urostylid ciliates, Caudiholosticha marina sp. nov. and Nothoholosticha flava sp. nov., isolated from intertidal sediment in the Yellow Sea, are investigated using morphological and small subunit rDNA phylogenetic analyses. Caudiholosticha marina is 210?310 μm × 40?55 μm in vivo, and has 10?20 macronuclear nodules, 23?37 midventral cirral pairs extending to 5?8 transverse cirri, and two caudal cirri. It differs from congeners by its marine habitat, larger size, macronuclear arrangement pattern and high number of midventral pairs. Molecular phylogenetic analyses indicate a polyphyly of Caudiholosticha. Nothoholosticha flava is yellow to brownish and 240?320 μm × 40?60 μm sized, and has a bipartite adoral zone, six frontal cirri in atypical bicorona, usually four frontoterminal, one buccal and 5?7 transverse cirri and 28?54 midventral pairs. Phylogenetic analyses allocate N. flava as sister of N. fasciola, type of the genus. The two Nothoholosticha species differ distinctly by the presence/absence of frontoterminal cirri, a feature often used to define genera in the Hypotrichia. However, the SSU rDNA sequence similarity between these two species is 99.3%, which weakens the justification for separating the new isolate at genus level. The taxonomic significance of frontoterminal cirri is discussed based on morphological and molecular data.     

Two New Brackish Ciliates,Amphileptus spiculatus sp. n. and A. bellus sp. n. from Mangrove Wetlands in Southern China,with Notes on the Molecular Phylogeny of the Family Amphileptidae (Protozoa,Ciliophora, Pleurostomatida)

Two new brackish pleurostomatid ciliates, Amphileptus spiculatus sp. n. and A. bellus sp. n. were collected from mangrove wetlands of southern China and their morphology and molecular phylogeny were studied. Amphileptus spiculatus sp. n. can be distinguished from congeners by the presence of 11–14 right and 6–8 left kineties, two macronuclear nodules and a conspicuous beak‐like anterior body end. Amphileptus bellus sp. n. is characterized by the presence of 2–4 macronuclear nodules, 31–35 right and 6 or 7 left kineties and two types of extrusomes. Phylogenetic analyses based on SSU rDNA sequences data indicate that the family Amphileptidae is paraphyletic.     

Redescription of Favella ehrenbergii (Claparède and Lachmann, 1858) Jörgensen, 1924 (Ciliophora: Choreotrichia), with Phylogenetic Analyses Based on Small Subunit rRNA Gene Sequences

ABSTRACT. The identification of Favella ehrenbergii, a marine planktonic ciliate, has largely been based on its lorica features. This approach is potentially problematic given the polymorphic lorica during this organism's life cycle. We isolated a population of F. ehrenbergii from the coastal waters of Incheon, Korea, and revealed its infraciliature using the protargol staining method. Phylogenetic analysis based on small subunit rRNA gene sequences was also performed. Results showed that this population possessed 16 collar membranelles (CM) and about 100 somatic kineties. These features are highly conserved, even in later dividers. As such, the number of CM and somatic kineties can be used as key characteristics for identification of Favella species.     

Phylogenetic Analyses Based on Small Subunit rRNA and Glycosomal Glyceraldehyde-3-Phosphate Dehydrogenase Genes and Ultrastructural Characterization of Two Snake Trypanosomes: Trypanosoma serpentis n. sp. from Pseudoboa nigra and Trypanosoma cascavelli from Crotalus durissus terrificus

ABSTRACT. We sequenced the small subunit (SSU) rRNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes of two trypanosomes isolated from the Brazilian snakes Pseudoboa nigra and Crotalus durissus terrificus . Trypanosomes were cultured and their morphometrical and ultrastructural features were characterized by light microscopy and scanning and transmission electron microscopy. Phylogenetic trees inferred using independent or combined SSU rRNA and gGAPDH data sets always clustered the snake trypanosomes together in a clade closest to lizard trypanosomes, forming a strongly supported monophyletic assemblage (i.e. lizard–snake clade). The positioning in the phylogenetic trees and the barcoding based on the variable V7–V8 region of the SSU rRNA, which showed high sequence divergences, allowed us to classify the isolates from distinct snake species as separate species. The isolate from P. nigra is described as a new species, Trypanosoma serpentis n. sp., whereas the isolate from C. d. terrificus is redescribed here as Trypanosoma cascavelli .     

Morphology of Three Species of Amphileptus (Protozoa,Ciliophora, Pleurostomatida) from the South China Sea,with Note on Phylogeny of A. dragescoi sp. n.

Two new and one problematic species of pleurostomatids, Amphileptus dragescoi sp. n., A. wilberti sp. n., and A. marinus from coastal areas of the South China Sea, are described based on observations of live and protargol‐impregnated specimens. Amphileptus dragescoi is different from its congeners by the presence of an apical group of extrusomes and the possession of 12–15 right and five left somatic kineties, two macronuclear nodules, and a single terminally positioned contractile vacuole. Amphileptus wilberti is diagnosed by oval or pyriform body, 15–19 right and seven or eight left somatic kineties, extrusomes arranged only in anterior portion of oral slit, usually three ventrally located contractile vacuoles, and two macronuclear nodules. Amphileptus marinus (Kahl, 1931) Song et al., 2004 is redescribed and its diagnosis is improved. One isolate which was misidentified as A. marinus by Song et al. (2004) is believed to represent an unknown form, named here as Amphileptus songi sp. n. Phylogenetic analyses of the SSU rDNA sequences indicate that the genus Amphileptus is paraphyletic, but its monophyly is not rejected by statistical tree topology tests.     

Kiitricha minuta n. sp., a peculiar hypotrichous ciliate (Ciliophora, Spirotrichea) from the Yellow Sea

The morphology and infraciliature of a new ciliate, Kiitricha minuta n. sp., isolated from the Yellow Sea, were investigated using live observation and protargol impregnation. Kiitricha minuta represents a third member of the rarely known order Kiitrichida. It is unique in the subclass Hypotrichia in having many rows of small uniform cirri along the right side of the body and the dorsal kineties composed of dikinetids, most of which bear two cilia. Kiitricha minuta n. sp. is ovoid and measures about 60 x 45 microm in vivo. It has a huge buccal cavity occupying about 80% of the body length, numerous body extrusomes, one macronucleus and two micronuclei, 27-27 adoral membranelles, 9-12 frontoventral cirral rows, a submarginal row of 7-9 cirri, 6 or 7 transverse cirri, and roughly 7-9 dorsal kineties. This new species differs distinctly from its only congener Kiitricha marina by its smaller size (60 microm vs. 80-150 microm), the presence of body extrusomes (vs. absent), the different macronuclear pattern (one vs. two nodules), and the lower number of frontoventral cirral rows (9-12 vs. 21-26), which terminate at the anterior two-thirds of body (vs. extend to the posterior). The new term "submarginal cirral row" is introduced to distinguish from the marginal cirral row in typical hypotrichs sensu lato. Based on our new observations and the literature, an improved diagnosis for the genus Kiitricha is provided and its phylogenetic importance is discussed.     

Phylogeny of the order Tintinnida (Ciliophora, Spirotrichea) inferred from small- and large-subunit rRNA genes

Concatenated sequences of small- and large-subunit rRNA genes were used to infer the phylogeny of 29 species in six genera of Tintinnida. We confirmed previous results on the positions of major clusters and the grouping of various genera, including Stenosemella, the paraphyletic Tintinnopsis, the newly investigated Helicostomella, and some species of the polyphyletic Favella. Tintinnidium and Eutintinnus were found to be monophyletic. This study contributes to tintinnid phylogenetic reconstruction by increasing both the number of species and the range of genetic markers analyzed.     

Morphology and Small Subunit rDNA Gene Sequence of Pseudoamphisiella quadrinucleata n. sp. (Ciliophora,Urostylida) from the South China Sea

ABSTRACT. The urosylid genus Pseudoamphisiella was established by Song (1996) with hitherto only two known congeners. In the present work, the morphology and infraciliature of a new member, Pseudoamphisiella quadrinucleata n. sp., a form with conspicuous alveolar layer and four macronuclear nodules isolated from the coastal waters both near Hong Kong and near Guangzhou, South China were investigated using living observation and protargol silver impregnation methods. Pseudoamphisiella quadrinucleata differs from other two known forms mainly by the number of macronuclear nodules: constantly four vs. two in Pseudoamphisiella alveolata and 24–57 in Pseudoamphisiella lacazei. To support this, the sequence of the small subunit rDNA of P. quadrinucleata n. sp. showed 14 and 74 nucleotides in comparison with that of the two known congeners, respectively, which hence firmly supports the validity of the new species.     

Reconsideration of the Phylogenetic Positions of Five Peritrich Genera,Vorticella, Pseudovorticella,Zoothamnopsis, Zoothamnium,and Epicarchesium (Ciliophora,Peritrichia, Sessilida), Based on Small Subunit rRNA Gene Sequences

ABSTRACT. In order to re‐evaluate the systematics of sessilid peritrich ciliates, small subunit (SSU) rRNA gene sequences were determined for 12 species belonging to five genera: Vorticella, Pseudovorticella, Epicarchesium, Zoothamnium, and Zoothamnopsis. Phylogenetic trees were deduced using Bayesian inference, maximum parsimony, and maximum likelihood methods. The phylogenetic analyses suggest that (1) sessilids which have stalks with continuous myonemes that contract in a zig‐zag fashion form a separate clade from those which have stalks that contract independently and in a spiral fashion, supporting the separation of the family Zoothamniidae from the family Vorticellidae and (2) Epicarchesium and Pseudovorticella, both of which have reticulate silverline systems, are more closely related to each other than to other vorticellids, suggesting that differences in the silverline system (i.e. transverse vs. reticulate) may be the result of genuine evolutionary divergence among sessilid peritrichs. However, the newly sequenced Zoothamnopsis sinica, which has a reticulate silverline pattern, nests within the unresolved Zoothamnium species that have transverse silverline patterns. Thus, there were at least two evolutions of the reticulate silverline pattern character state from a plesiomorphic transverse state in the peritrichid ciliates. The molecular work demonstrates the genus Zoothamnium to be paraphyletic in relation to morphological studies, and suggests that Astylozoon, Opisthonecta, and Vorticella microstoma possibly share a SSU rRNA secondary structure in the helix E10‐1 region.     

从rRNA基因序列探讨云斑蛛属Cyrtophora的系统发生地位

由于云斑蛛属Cyrtophora蜘蛛所织的网形态上较为特殊,所以该属分类地位长期以来有异议.为从分子水平探讨该属系统发生地位,本文对相关的11种蜘蛛线粒体12S rRNA基因和核18S rRNA基因部分序列进行了测定.基于12S rRNA和18S rRNA基因序列联合数据进行的分子系统发生分析结果表明云斑蛛属属于园蛛科,该结果提示在园蛛总科分类实践中以蛛网形态特征为分类依据时要慎重考虑其可靠性.     

Phylogenetic Relationships of Orders Within the Class Colpodea (Phylum Ciliophora) Inferred from Small Subunit rRNA Gene Sequences

Molecular analyses have been used recently to refine our knowledge of phylogenetic relationships within the ciliated protozoa (phylum Ciliophora). A current Hennigian phylogeny of the orders in the class Colpodea, based on light and electron microscopic analyses, makes three important assumptions with regard to apomorphic character states, namely, (1) that the kreyellid silver line evolved early in colpodean phylogeny, separating bryometopids, such as Bryometopus, from all other colpodeans; (2) that the macro–micronuclear complex is an autapomorphy of the cyrtolophosidids, such as Platyophrya; and (3) that merotelokinetal stomatogenesis is an apomorphic character of colpodids, such as Colpoda, Bresslaua, and Pseudoplatyophrya. These predictions of relationships within the class Colpodea were investigated by determining the complete small subunit rRNA gene sequences for the colpodid Bresslaua vorax, the grossglockneriid Pseudoplatyophrya nana, and the cyrtolophosidid Platyophrya vorax and a partial sequence for the bryometopid Bryometopus sphagni. These sequences were combined with the previously published complete SSrRNA sequences for the colpodid Colpoda inflata and the bursariomorphid Bursaria truncatella. The affiliations were assessed using both distance matrix and maximum-parsimony analyses. The tree topologies for the class Colpodea were identical in all analyses, with bootstrap support for bifurcations always exceeding 60%. The results suggest the following. (1) Since the clade including Bryometopus and its sister taxon, Bursaria, is never basal, the kreyellid silver-line system evolved later in colpodean phylogeny and does not separate bryometopids from all other colpodeans. (2) Since Platyophrya is always the sister taxon to the other five genera, there is a fundamental phylogenetic significance for its macro–micronuclear complex. (3) Since the colpodids, Colpoda, Bresslaua, and Pseudoplatyophrya, always group in one clade, merotelokinetal stomatogenesis appears to be a derived character state. Received: 30 June 1998 / Accepted: 3 December 1998     

Morphology and Phylogeny of Two New Pleurostomatid Ciliates,Epiphyllum shenzhenense n. sp. and Loxophyllum spirellum n. sp. (Protozoa,Ciliophora) from A Mangrove Wetland,South China

ABSTRACT. The morphology, infraciliature, and small subunit (SSU) rRNA gene sequences of two new pleurostomatid ciliates, Epiphyllum shenzhenense n. sp. and Loxophyllum spirellum n. sp., isolated from a mangrove wetland near Shenzhen, South China, were investigated. Epiphyllum shenzhenense n. sp. is morphologically characterized by leaf‐shaped cell about 150 × 35 μm in vivo, usually with four contractile vacuoles, 20–29 right kineties and 10–26 left kineties, ca. four macronuclear nodules, and two types of extrusomes (i.e. short spindle‐shaped and long bar‐shaped). As a new species, L. spirellum n. sp. is distinguished from its congeners by its posterior dorsal margin twisted onto the left side, the distribution of extrusomes (evenly arranged along the oral slit, the posterior end, and clustered to 7–13 warts on dorsal margin), the subterminally positioned contractile vacuole, the number of kineties (8–10 on right side, 4–5 on left side), and its genetic distance from congeners. Phylogenetic trees based on the SSU rRNA gene sequence for both organisms were constructed, which indicate that Epiphyllum is a distinct genus and occupies a basal position in the Pleurostomatida clade; L. spirellum n. sp. falls well into the Loxophyllum clade, which has a close relationship with Litonotus and Spiroloxophyllum.     

Pseudonotohymena antarctica n. g., n. sp. (Ciliophora,Hypotricha), a New Species from Antarctic Soil

A new soil ciliate, Pseudonotohymena antarctica n. g., n. sp., from King George Island, Antarctica, is described based on live observation, protargol impregnation, and its 18S rRNA gene. The new genus Pseudonotohymena is morphologically similar to the genus Notohymena Blatterer and Foissner 1988 in the following characteristics: 18 fronto‐ventral‐transverse cirri, a flexible body, undulating membranes, dorsomarginal kineties, and the number of cirri in the marginal rows. However, Pseudonotohymena differs from Notohymena particularly in the dorsal ciliature, that is, in possessing a nonfragmented dorsal kinety (vs. fragmented). In addition, the molecular phylogenetic relationship of the new species differs from that of Notohymena species. On the basis of the morphological features, the genetic data, and morphogenesis, we establish P. antarctica n. g., n. sp. In addition, the cyst morphology of this species is described.     

Description and Phylogenetic Relationships of Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. (Amoebozoa, Arcellinida)

ABSTRACT. Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. were isolated and described from dry epiphytic moss. The morphology and ultrastructure of both species clearly demonstrate that they belong to the genus Spumochlamys (family Microchlamyiidae). They differ from its only described member, Spumochlamys iliensis (as well as from species of Microchlamys ), in the relief of the dorsal surface of the test, revealed by scanning electron microscopy, which can represent a good characteristic for species identification. They also differ in the structure of the dorsal part of the test wall (especially S. perforata ). Small subunit ribosomal DNA-based molecular phylogenetic analyses show that Spumochlamys is a deeply branching lineage of the Arcellinida, without any close affinities. Actin gene sequence analysis places this genus within the Tubulinea, close to two other arcellinid lineages but without forming a monophyletic group with them. These data together strongly suggest that the lack of resolution in the arcellinid molecular phylogenies is due to serious undersampling of taxa, a limited number of sequence data, and high divergence rates in most of the species.     

A New Freshwater Amoeba: Cochliopodium pentatrifurcatum n. sp. (Amoebozoa,Amorphea)

Cochliopodium pentatrifurcatum n. sp. (ATCC^© 30935^TM) is described based on light microscopic morphology, fine structure, and molecular genetic evidence. Cochliopodium pentatrifurcatum n. sp. (length ~ 25 μm) is characterized by surface microscales (0.3 μm tall) containing a circular porous base (~ 0.6 μm diam.) with a thin peripheral rim. Five radially arranged feet, emanating from the base, support a short central column terminating apically as a funnel‐shaped collar (~ 0.5 μm diam.) composed of five radial, trifurcate rays extending from the center toward a thin peripheral rim. The central spine is 0.5–0.6 μm long. The comparative morphologies and combined molecular genetic evidence, SSU‐rDNA and COI, indicate that the new species falls in a clade sufficiently different from other species to suggest that it is a valid new species.     

Morphology and small subunit rRNA gene sequence of Uronemita parabinucleata n. sp. (Ciliophora,Uronematidae), with an improved generic diagnosis

The morphology and infraciliature of a new species, Uronemita parabinucleata n. sp., isolated from intertidal sediments in a coastal region in northern China, were investigated using live observation and silver impregnation methods. The new species is characterized by an in vivo body size of about 20–50 × 10–25 μm, 22 or 23 somatic kineties, two macronuclear nodules, and one caudal cilium. Its small subunit ribosomal RNA gene (SSU rDNA) was sequenced and compared with those of other Uronemita species to reveal nucleotide differences. Phylogenetic analyses indicated that Uronemita is monophyletic and that the new species clusters with its congener Uronemita filificum, with full support provided by both Bayesian inference and maximum likelihood algorithms. Based on previous studies and the present study, an improved diagnosis of the genus Uronemita is supplied, which has been absent since the establishment of this genus. A key to the Uronemita species is also provided.     

Morphology and Molecular Phylogeny of Two New Marine Euplotids,Pseudodiophrys nigricans n. g., n. sp., and Paradiophrys zhangi n. sp. (Ciliophora: Euplotida)

ABSTRACT. The morphology, infraciliature, and molecular phylogeny of Pseudodiophrys nigricans n. g., n. sp., and Paradiophrys zhangi n. sp., isolated from a sandy beach near Qingdao, China, were investigated. Pseudodiophrys is characterized by the Diophrys‐like ciliature pattern, but having only a single, reduced undulating membrane. Pseudodiophrys nigricans, the type species by monotypy, is described from live and silver‐impregnated specimens. Paradiophrys zhangi is similar to the type species Paradiophrys irmgard but can be recognized by its border body and the number (7 vs. 8–10) and arrangement (in rows vs. sparsely distributed) of the frontoventral cirri. Small subunit (SSU) rRNA gene sequence data support the validity of both species. Phylogenetic analyses based on the SSU rRNA gene sequence data currently available for uronychiids and other related taxa indicate that P. nigricans is most closely related to Diophrys scutum and Diophrys apoligothrix, while P. zhangi clusters most closely with Apodiophrys ovalis within a clade that also includes two other Paradiophrys species.