Three redescriptions in Tintinnopsis (Protista: Ciliophora: Tintinnina) from coastal waters of China, with cytology and phylogenetic analyses based on ribosomal RNA genes.

BACKGROUND: The taxonomy of tintinnine ciliates is vastly unresolved because it has traditionally been based on the lorica (a secreted shell) and it has only recently incorporated cytological and molecular information. Tintinnopsis, the most speciose tintinnine genus, is also the most problematic: it is known to be non-monophyletic, but it cannot be revised until more of its species are studied with modern methods. RESULTS: Here, T. hemispiralis Yin, 1956, T. kiaochowensis Yin, 1956, and T. uruguayensis Balech, 1948, from coastal waters of China, were studied. Lorica and cell features were morphometrically investigated in living and protargol-stained specimens, and sequences of three ribosomal RNA (rRNA) loci were phylogenetically analyzed. The three species show a complex ciliary pattern (with ventral, dorsal, and posterior kineties and right, left, and lateral ciliary fields), but differ in lorica morphology, details of the somatic ciliature and rRNA gene sequences. Tintinnopsis hemispiralis is further distinguished by a ciliary tuft (a ribbon of very long cilia originated from the middle portion of the ventral kinety and extending out of the lorica) and multiple macronuclear nodules. Both T. kiaochowensis and T. uruguayensis have two macronuclear nodules, but differ in the number of somatic kineties and the position of the posterior kinety. Two neotypes are fixed for T. hemispiralis and T. kiaochowensis to stabilize the species names objectively, mainly because of the previous unavailability of type materials. By phylogenetic analysis and comparison with closely-related species, we infer that the ciliary tuft and details such as the commencement of the rightmost kinety in the lateral ciliary field are synapomorphies that may help clarify the systematics of Tintinnopsis-like taxa. CONCLUSION: The redescriptions of three poorly known Tintinnopsis species, namely T. hemispiralis, T. kiaochowensis, and T. uruguayensis firstly revealed their ciliary patterns and rRNA sequences. This study expands knowledge and database of tintinnines and helps in identifying potential synapomorphies for future taxonomic rearrangements.

Redescription of Antetintinnidium mucicola (Claparède and Lachmann, 1858) nov. gen., nov. comb. (Alveolata, Ciliophora, Tintinnina).

Tintinnid ciliates have traditionally been described and classified exclusively based on their lorica features. Although information on the cell characters is urgently needed for a natural classification, more molecular than cytological data has been accumulated over recent years. Apparently, the tintinnids developed in the marine environment and entered freshwater several times independently. Typical freshwater tintinnids belong to the genera Tintinnidium and Membranicola. The species are comparatively well-known regarding their morphology and characterised by two unusual de novo originating ciliary rows, the ventral organelles. In contrast, the cell features in the marine/brackish Tintinnidium species, specifically their somatic ciliary patterns, are insufficiently known or not known at all. Therefore, the morphology of a common marine/brackish representative, Tintinnidium mucicola, is redescribed based on live observation and protargol-stained material. Furthermore, biogeographical and autecological data of the species are compiled from literature and own records. The phylogenetic relationships of T. mucicola are inferred and the diversity of the family Tintinnidiidae is assessed from 18S rDNA sequences. The study shows that T. mucicola is not only molecularly distinct, but also characterised by many plesiomorphic features, for instance, it does not possess a verifiable homologue to the ventral organelles. Hence, a new genus, Antetintinnidium nov. gen., is established for T. mucicola. The new insights into the diversity of Tintinnidiidae shed light on the early evolution of tintinnids and might provide clues on their adaptions to freshwater.

Pelagostrobilidium liui n. sp. (Ciliophora, Choreotrichida) from the Coastal Waters of Northeastern Taiwan and an Improved Description of Pelagostrobilidium minutum Liu et al., 2012.

The number of somatic kineties in Pelagostrobilidium ranges from 4 to 6 according to the present state of knowledge. This study investigates Pelagostrobilidium liui n. sp. using live observation, protargol stain, and small subunit rDNA data sequencing. Pelagostrobilidium liui n. sp. is characterized by having a spherical-shaped body, four somatic kineties, with kinety 2 spiraled around the left side of body, about six elongated external membranelles, and invariably no buccal membranelle. It differs from its most similar congener, Pelagostrobilidium minutum Liu et al., , in (i) cell shape; (ii) macronucleus width; (iii) oral apparatus; (iv) anterior orientation of kinety 2; (v) location where kinety 2 commences; (vi) arrangement of kinety 1; (vii) distance between the anterior cell end and the locations where kineties commence; and (viii) the presence of 12 different bases (including two deletions) in the small subunit rDNA sequences. The diagnosis of P. minutum Liu et al., is also improved to include the following new characteristics: invariably four somatic kineties; kineties 2 and 4 alone commence at the same level; kinety 2 originates from right anterior cell half on ventral side, extends sinistrally posteriorly, over kinety 1, around left posterior region, terminates near posterior cell end on dorsal side; kinety 1 commences below anterior third of kinety 2.

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 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.

Phylogenetic position of the genus Cyrtostrombidium, with a description of Cyrtostrombidium paralongisomum nov. spec. and a redescription of Cyrtostrombidium longisomum Lynn & Gilron, 1993 (Protozoa, Ciliophora) based on live observation, protargol impregnation, and 18S rDNA sequences.

We redescribe Cyrtostrombidium longisomum Lynn & Gilron, 1993, the type species of the genus Cyrtostrombidium, and describe the new species Cyrtostrombidium paralongisomum n. sp. using live observation, protargol staining and molecular data. The morphological characters of these two species are clearly distinct, i.e., dikinetid numbers in the girdle and ventral kineties; however, it is difficult to separate them by 18S rDNA sequences because they differ by only 8 bp, indicating that 18S rDNA sequences are insufficient for separating different species in the genus Cyrtostrombidium. We not only observed the position of the oral primordium in the genus Cyrtostrombidium but also observed a possibly homoplasious trait, a dorsal split in the girdle kinety, in (1) Apostrombidium, (2) Varistrombidium, and (3) Cyrtostrombidium/Williophrya. This partially supports the hypothesis of somatic ciliary pattern evolution recently put forth by Agatha and Strüder-Kypke.

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.

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.

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.

Morphological, Molecular, and Growth Characteristics of a Cryptic Species, Strombidium parasulcatum n. sp. (Alveolata: Ciliophora: Oligotrichida).

A new marine planktonic ciliate from Taiwan, Strombidium parasulcatum sp. n., is described based on live observations, protargol staining, and molecular data. Its morphological characters are similar to those of Strombidium sulcatum Claparède and Lachmann, 1859 sensu Song et al., 2000 but differ from S. sulcatum sensu Fauré-Fremiet, 1912 and sensu Fauré-Fremiet and Ganier, 1970 by several morphological characters. The 18S rRNA gene sequences of the two forms display 76 base pair differences (about 5%), indicating that they should be considered separate species. The highest observed specific growth rates of S. parasulcatum in culture were 1.79 day-1 over 1 day and 1.52 day-1 over 2 days, both starting at day 5. Bacteria appear to be an important food resource for the cultivation of this medium-sized oligotrich ciliate. This and other recent studies suggest that cryptic species may be common in the genus Strombidium, and an integrative approach including morphological, ecological, and molecular data should be used to address this question.

Overview of the Diversity, Phylogeny and Biogeography of Strombidiid Oligotrich Ciliates (Protista, Ciliophora), With a Brief Revision and a Key to the Known Genera.

Strombidiids are common free-living ciliates that have colonized coastal and open oceanic waters across the world. In recent years, numerous new taxa and gene sequences of strombidiids have been reported, revealing a large diversity of both their morphologic and genetic features. Here, we compare the taxonomic characters of all genera in the family Strombidiidae, provide a key to their identification, and investigate their molecular phylogeny. In addition, we analyze their regional distribution based on faunal data accumulated in China and attempt to infer their global distribution based on SSU rRNA gene sequence data. The current work revises the systematics of strombidiids based on morphologic, phylogenetic, and biogeographic evidence and provides a genus-level review of marine strombidiids.

Taxonomy and Phylogeny of Three Species of Dysteria (Ciliophora, Phyllopharyngea) Including the Description of Dysteria ozakii nom. nov.

In the last two decades, cyrtophorian ciliates have been revealed to demonstrate a high species diversity. But this group remains difficult to study, mainly because of their low abundance and relatively few taxonomically informative morphological characters. As a contribution to the taxonomy of cyrtophorians, here we investigate three Dysteria species based on their live morphology, ciliary pattern, and molecular phylogeny. Dysteria ozakii nom. nov. can be recognized by its elongate body shape and four right kineties, including three frontoventral kineties. A neotype has to be fixed for the species as no type materials were deposited. The other two species, D. brasiliensis Faria et al., 1922 and D. compressa (Gourret & Roeser, 1886) Kahl, 1931, are redescribed and supplementary information for each is supplied. Phylogenetic analyses based on small-subunit (SSU) rRNA gene sequences support the validity of the species. In addition, four species of bacterial epibionts were observed on the surface of the three Dysteria spp. The identities of these bacterial species are discussed based on the newly obtained 16S rRNA gene sequences.

Redescriptions of three tintinnine ciliates (Ciliophora: Tintinnina) from coastal waters in China based on lorica features, cell morphology, and rDNA sequence data.

Three species of tintinnines, namely Tintinnopsis tentaculata Nie and Cheng, 1947, Tintinnopsis orientalis Kofoid and Campbell, 1929, and Eutintinnus lususundae (Entz, 1885) Kofoid and Campbell, 1939, were isolated from coastal waters of China. The morphology of each was investigated based on observations of live and protargol-stained specimens, and their SSU rDNA- and LSU rDNA-based phylogenetic relationships were analyzed. The ciliary patterns of these species are revealed for the first time. Based on the original descriptions and data from the present study, an improved diagnosis is given for each species. Unlike its congeners, the second dorsal kinety of Eutintinnus lususundae is displaced below the left ciliary field, which may suggest that the second dorsal kinety is evolving into a posterior kinety by a migration process. The ventral kinety in Eutintinnus is redefined. A neotype is fixed for T. tentaculata to stabilize the species name objectively, mainly because of the unavailability of type material.

Morphology and phylogeny of two Phialina species (Ciliophora, Haptoria) from northern China.

The morphology and infraciliature of two haptorian ciliates, Phialina clampi nov. spec. and P. caudata (Kahl, 1933) nov. comb. (original combination: Lacrymaria caudataKahl, 1933), isolated from sandy sediments of an estuary in Yantai, northern China, were investigated using live observations and protargol preparations. Phialina clampi nov. spec. is distinguished from its congeners by the following traits: extended cells about 80-300 × 18-50 µm in vivo; a single oval-shaped macronucleus; caudally located contractile vacuole; two types of extrusomes: type I about 20-35 × 1-4 µm in size, type II 1.5-3.0 × 0.7-1.0 µm in size and attached to somatic cortex; 27-31 somatic kineties. Phialina caudata is characterized as follows: extended cells about 80-170 × 20-45 µm in vivo; body radish-shaped, with a sharp posterior end; a single globular to oval macronucleus and micronucleus; subterminal contractile vacuole; 20-24 somatic kineties. Phylogenetic analyses based on SSU rRNA gene sequences indicate that P. caudata clusters with an unidentified Phialina before grouping with Phialina clampi, which form a basal clade of the family Lacrymariidae.

Lithium-induced developmental anomalies in the spirotrich ciliate Stylonychia lemnae (Ciliophora, Hypotrichida).

Lithium is known to have profound biological effects of varying intensity in different life forms. In the present investigation, the effect of lithium was studied on the spirotrich ciliate Stylonychia lemnae. Lithium treatment brings about quantitative changes in the patterning of ciliary structures in S. lemnae. The dorsal surface of the affected cells develops supernumerary ciliary kineties due to excessive proliferation of the kinetosomes. The ventral surface on the other hand develops fewer than normal cirri formed from reduced numbers of ciliary primordia. The adoral zone of membranelles (AZM) fails to remodel properly as, in certain segments, membranelles become disarranged and misaligned. Lithium-induced changes are transitory as the normal pattern is restored during recovery after the cells are shifted to normal medium, suggesting non-genic regulation of cortical pattern. Lithium also affects the process of cell proliferation as the number of cells undergoing division is negligible as compared to reorganizing cells. The results point to the extremely complex and heterogeneous organization of the cellular cortex (plasma membrane and cytoskeleton) which is capable of exerting autonomous control over the phenotype and cortical pattern.