Morphology,Morphogenesis, and Molecular Phylogeny of Anteholosticha multicirrata n. sp. (Ciliophora,Spirotrichea) with a Note on Morphogenesis of A. pulchra (Kahl, 1932) Berger, 2003

Two marine urostylid ciliates, Anteholosticha multicirrata n. sp. and Anteholosticha pulchra (Kahl, 1932) Berger, 2003, were collected from South Korea. These species were identified based on morphology, morphogenesis, and SSU rRNA gene sequence comparison. Anteholosticha multicirrata n. sp. is characterized by the following features: body size 90–125 × 30–45 μm in vivo, shape slender to ellipsoidal in outline, with yellow‐greenish cortical granules distributed along and between dorsal kineties and cirri; single contractile vacuole positioned on left at mid‐body; three frontal, five to seven frontoterminal, one buccal, one to two pretransverse and four to six transverse cirri; three complete dorsal kineties; one left and one right marginal cirral row; about 117 macronuclear nodules; and three to four micronuclei observed during morphogenesis. In addition, based on the observations of morphogenesis, we found that A. pulchra has pretransverse cirri, which were not described in detail in previous studies. Nuclear small subunit ribosomal RNA (SSU rRNA) gene was used to analyse their phylogenetic relationship, and the gene tree supports that the genus Anteholosticha is a highly polyphyletic group.     

Morphological Description and Molecular Phylogeny of Two Species of Levicoleps (Ciliophora,Prostomatida), L. taehwae nov. spec. and L. biwae jejuensis nov. subspec., collected in Korea

Two colepid ciliates, Levicoleps taehwae nov. spec. and L. biwae jejuensis nov. subspec., were collected from the brackish water of the Taehwa River and a small freshwater pond in Jeju Island, South Korea, respectively. Their living morphology, infraciliature, and small subunit (SSU) rRNA gene sequences were determined using standard methods. Barrel‐shaped L. taehwae nov. spec. is a small ciliate with an average size of 45 × 25 μm in vivo, about 15 ciliary rows each composed of 12 monokinetids and two perioral dikinetids, and two 20 μm‐long caudal cilia. The sequence length and GC content of the SSU rRNA gene are 1,669 bp, 44.5%. This novel species is similar in body size to Coleps hirtus, and has six armor tiers and hirtus‐type tier plates, and the same number of ciliary rows as C. hirtus; however, it can be distinguished from the latter by the absence of armor spines and its sequence similarity of SSU rRNA gene is about 92.8% which indicates that it is a distinct form. Levicoleps biwae jejuensis nov. subspec., is a medium colepid ciliate which has a barrel‐shaped body, about 22 somatic kineties and 16 transverse ciliary rows, three mini adoral organelles, and four 15 μm‐long caudal cilia. The sequence length and GC content of the SSU rRNA gene are 1,666 bp and 44.4%.     

Morphological Characterizations of Four Species of Parallelostrombidium (Ciliophora,Oligotrichia), with a Note on the Phylogeny of the Genus

The morphology and phylogeny of four oligotrichid ciliates, Parallelostrombidium paraellipticum sp. n., P. dragescoi sp. n., P. jankowskii (Xu et al. 2009) comb. n., and P. kahli (Xu et al. 2009) comb. n., are described or redescribed based on live observation, protargol stained material, and SSU rRNA gene sequences. The new species P. paraellipticum sp. n. is characterized by its obovoidal cell shape, adoral zone composed of 17–21 collar, 9–11 buccal, and two thigmotactic membranelles, and extrusomes attached in one row along the girdle kinety. The new species P. dragescoi sp. n. is distinguished from its congeners by its obovoidal cell shape and a lack of thigmotactic membranelles. Based on ciliary patterns recognizable in the original slides, Omegastrombidium jankowskii Xu et al. 2009 and O. kahli Xu et al. 2009 should be transferred to the genus Parallelostrombidium Agatha 2004. Phylogenetic analyses based on SSU rRNA gene sequence data demonstrate that all four new sequences cluster with previously described congeners. The genus Parallelostrombidium is separated into two clusters, suggesting its non‐monophyly and probably corresponding to the two subgenera proposed by Agatha and Strüder‐Kypke (2014), as well as their morphological difference (cell dorsoventrally flattened vs. unflattened).     

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.     

Ultrastructural and Phylogenetic Description of Kudoa orbicularis n. sp. (Myxosporea: Multivalvulida): A Parasite Infecting the Muscle of the Fish Chaetobranchopsis orbicularis (Teleostei: Cichlidae) in the Amazon Region

A new myxosporean species is described from the muscle of the Amazonian freshwater fish Chaetobranchopsis orbicularis (Teleostei, Cichlidae), with basis on morphometric, ultrastructural and molecular data. Numerous myxospores were observed within pseudocysts located on the hosts' dorsal and ventral muscles, near the neural spines and neural canal (spinal cord). Mature myxospores quadrangular with rounded ends in apical view, measuring 4.3 (3.6–5.0) μm in length and 5.1 (4.2–5.8) μm in width. The myxospores wall is formed by four symmetric valves. Within, four pyriform polar capsules, 2.1 (1.7–2.6) μm long and 1.3 (0.9–1.7) μm wide, located two by two in opposite sides of the myxospores longitudinal axis, each containing a polar filament forming 2–3 coils. Molecular analysis of the SSU rRNA gene by maximum likelihood, neighbor‐joining and maximum parsimony confirms the parasite as a new member of the genus Kudoa, herein named Kudoa orbicularis n. sp., the second species of its genus reported from the South American freshwater fauna, and the fourth species worldwide known to occur in the freshwater environment. Furthermore, its sequence of the SSU rRNA gene constitutes the first entry of a freshwater Kudoa species in GenBank.     

Description of a New Freshwater Ciliate Epistylis wuhanensis n. sp. (Ciliophora,Peritrichia) from China,with a Focus on Phylogenetic Relationships within Family Epistylididae

Two populations of Epistylis wuhanensis n. sp., a new freshwater peritrich ciliate, were isolated from different freshwater ponds located in Hubei, China. Their morphological characteristics were investigated using live observation, protargol impregnation, and scanning electron microscopy (SEM). Specimens from the two populations showed identical arrangement of the infraciliature and identical small subunit ribosomal RNA (SSU rRNA) gene and ITS1‐5.8S‐ITS2 sequences. The zooids present bell‐shaped and 90–175 × 27–54 μm in vivo. Macronucleus is variable in shape and located in the middle of cell. Pellicle is usually smooth with 139–154 and 97–105 striations above and below the trochal band, respectively. SSU rRNA gene and ITS1‐5.8S‐ITS2 sequences of E. wuhanensis n. sp. did not match any available sequences in GenBank. Phylogenetically, E. wuhanensis n. sp. clusters with the other Epistylis within the family Epistylididae, but is distinct from the major clades of Epistylis. Above all, the morphological characteristics and molecular analyses support that the present Epistylis is a new species. Expanded phylogenetic analyses of sessilids based on both SSU rRNA gene sequences and ITS1‐5.8S‐ITS2 sequences reveal that the genus Epistylis consists of Epistylis morphospecies and taxonomic revision of the genus is needed.     

Morphological and molecular characterization of Aspidisca hongkongensis spec. nov. (Ciliophora,Euplotida) from the South China Sea

The morphology and infraciliature of a new ciliate, Aspidisca hongkongensis spec. nov., a large marine form isolated from the coastal waters near Hong Kong, were investigated. It differs from the congeners by the combination of cell size (about 90×60 μm in vivo), 9–11 conspicuous dorsal ridges, one fine and seven strong frontoventral cirri, six membranelles in anterior portion of adoral zone, and 17–23 membranelles in posterior portion of adoral zone. The comparison with similar congeners clearly supports the distinction of this new species based on morphological and small subunit (SSU) rRNA gene sequence studies. The SSU rRNA gene sequence similarity within the genus is about 75.3–92.1% which indicates that this is a distinct form. Phylogenetic trees based on SSU rRNA gene information show that A. hongkongensis groups with A. leptaspis.     

Fish Parasite Dinoflagellates Haidadinium ichthyophilum and Piscinoodinium Share a Recent Common Ancestor

The dinoflagellate Haidadinium ichthyophilum Buckland‐Nicks, Reimchen and Garbary 1997 is an ectoparasite of the spine‐deficient, three‐spine stickleback Gasterosteus aculeatus L. Reimchen 1984, a fish endemic to Rouge Lake, Haida Gwaii. Haidadinium ichthyophilum proved difficult to assign taxonomically because its morphology and complex life cycle exhibited defining characteristics of both autotrophic and heterotrophic dinoflagellates, and was tentatively assigned to the Phytodiniales. Here, we characterized a 492 bp fragment of the small subunit ribosomal RNA (SSU rRNA) from preserved H. ichthyophilum cysts. In SSU phylogeny, H. ichthyophilum branches with the fish parasites, Piscinoodinium sp., strongly supporting the inclusion of H. ichthyophilum within the Suessiales.     

The parthenogenetic Marmorkrebs (Malacostraca: Decapoda: Cambaridae) is a triploid organism

There is a close association between parthenogenesis and polyploidy. For this reason, we undertook a karyological analysis to test whether the parthenogenetic Marmorkrebs, Procambarus fallax forma virginalis, possesses an enlarged set of chromosomes. For this purpose, we karyotyped the Marmorkrebs, the sexual form of P. fallax (together called P. fallax complex), and the closely related species P. alleni. The latter shows 94 chromosomes in the haploid condition. In contrast to this, we found a haploid set of 92 chromosomes in individuals of the P. fallax complex. However, in mitotic metaphases the sexual form shows 184 chromosomes, whereas the Marmorkrebs possesses 276 chromosomes. Hence, the parthenogenetic Marmorkrebs reveals a triple amount of the haploid chromosome number. In addition, we detected a strikingly large subtelocentric chromosome which appears once in haploid and twice in diploid cells of sexual individuals of the P. fallax complex. In the parthenogenetic Marmorkrebs, this prominent chromosome occurs thrice. All this clearly reveals that the Marmorkrebs is a triploid organism. The applicability of the used methods, the significance of polyploidy in evolution of Decapoda, putative pathways to parthenogenetic triploidy, a possible hybrid origin and the scientific and ecological consequences of an increased chromosome set in Marmorkrebs are discussed.     

Experimental warming alters the community composition,diversity, and N2 fixation activity of peat moss (Sphagnum fallax) microbiomes

Sphagnum‐dominated peatlands comprise a globally important pool of soil carbon (C) and are vulnerable to climate change. While peat mosses of the genus Sphagnum are known to harbor diverse microbial communities that mediate C and nitrogen (N) cycling in peatlands, the effects of climate change on Sphagnum microbiome composition and functioning are largely unknown. We investigated the impacts of experimental whole‐ecosystem warming on the Sphagnum moss microbiome, focusing on N₂ fixing microorganisms (diazotrophs). To characterize the microbiome response to warming, we performed next‐generation sequencing of small subunit (SSU) rRNA and nitrogenase (nifH) gene amplicons and quantified rates of N₂ fixation activity in Sphagnum fallax individuals sampled from experimental enclosures over 2 years in a northern Minnesota, USA bog. The taxonomic diversity of overall microbial communities and diazotroph communities, as well as N₂ fixation rates, decreased with warming (p < 0.05). Following warming, diazotrophs shifted from a mixed community of Nostocales (Cyanobacteria) and Rhizobiales (Alphaproteobacteria) to predominance of Nostocales. Microbiome community composition differed between years, with some diazotroph populations persisting while others declined in relative abundance in warmed plots in the second year. Our results demonstrate that warming substantially alters the community composition, diversity, and N₂ fixation activity of peat moss microbiomes, which may ultimately impact host fitness, ecosystem productivity, and C storage potential in peatlands.     

Morphology and Phylogeny of Two Species of Loxodes (Ciliophora,Karyorelictea), with Description of a New Subspecies,Loxodes striatus orientalis subsp. n.

The morphology and phylogeny of Loxodes vorax and L. striatus orientalis subsp. n. were investigated based on infraciliature and small subunit (SSU) rRNA gene sequence data. Loxodes striatus orientalis subsp. n. was separated from L. striatus striatus stat. n. by having fewer dikinetids in the intrabuccal kinety (35–55 vs. 50–70) and a variable number of macronuclei (2–4 vs. 2). In addition, the SSU rRNA gene sequence of the new subspecies differs in 13 and 11 nucleotides from that of two populations of the nominotypic subspecies. We also summarized the morphological differences between Loxodes and Remanella based on the data available. Phylogenetic analyses revealed that the genus Loxodes was monophyletic and nested within Remanella species. This study might, therefore, support the hypothesis that the freshwater genus Loxodes evolved from the marine genus Remanella.     

Morphology,Ontogeny, and Molecular Phylogeny of Two Freshwater Species of Deviata (Ciliophora,Hypotrichia) from Southern China

The morphology and partial morphogenesis of two freshwater hypotrichous ciliates, Deviata brasiliensis Siqueira‐Castro et al., 2009 and Deviata rositae Küppers et al., 2007, isolated from southern China, were investigated using live observation and protargol staining. Our populations resemble the original ones in terms of their live characters and ciliary patterns. The main determinable morphogenetic features of D. brasiliensis basically correspond with those of the type population. However, the origin of anlage V for either proter or opisthe is ambiguous: whether anlage V for the proter originates from parental frontoventral row 2 (the same as in the original population) or parental frontoventral row 3 (the same as in Deviata abbrevescens) or even de novo is not clear; the anlage V for the opisthe is possibly derived from frontoventral row 3 and further migrates to frontoventral row 2, like that in D. abbrevescens. In addition, the SSU rRNA gene was first sequenced for both species. Molecular phylogenetic analyses suggest that the genus Deviata is non‐monophyletic and has a close relationship with Perisincirra paucicirrata.     

Polyphasic approach using multilocus analyses supports the establishment of the new aerophytic cyanobacterial genus Pycnacronema (Coleofasciculaceae,Oscillatoriales)

A new Phormidium‐like genus was found during an investigation of Oscillatoriales diversity in Brazil. Eight aerophytic populations from south and southeastern regions were isolated in monospecific cultures and submitted to polyphasic evaluation. The populations presented homogeneous morphology with straight trichomes, not attenuated, and apical cell with thickened cell wall. Phylogenetic analyses based on 16S rRNA gene sequences showed that these populations, plus the Brazilian strain Phomidium sp. B‐Tom from GenBank, formed a highly supported and distinctive clade, which corresponds to the new genus Pycnacronema, comprising six new species: P. brasiliensis (type species), P. arboriculum, P. conicum, P. marmoreum, P. rubrum, and P. savannensis. These results were confirmed and supported by rpoC1 and rbcL genes evaluated independently and by the concatenated analysis of 16S rRNA, rpoC1 and rbcL genes (for all species but P. savannensis). Secondary structures of the D1‐D1′, box‐B, and V3 regions of the internal transcribed spacer were informative at specific level, being conserved in P. brasiliensis and variable among the other strains, also confirming the phylogenetic analyses. The generic name and specific epithets of the new taxa are proposed under the provisions of the International Code of Nomenclature of algae, fungi, and plants.     

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.     

A New Heterotrophic Dinoflagellate from the North‐eastern Pacific,Protoperidinium fukuyoi: Cyst–Theca Relationship,Phylogeny, Distribution and Ecology

The cyst–theca relationship of Protoperidinium fukuyoi n. sp. (Dinoflagellata, Protoperidiniaceae) is established by incubating resting cysts from estuarine sediments off southern Vancouver Island, British Columbia, Canada, and San Pedro Harbor, California, USA. The cysts have a brown‐coloured wall, and are characterized by a saphopylic archeopyle comprising three apical plates, the apical pore plate and canal plate; and acuminate processes typically arranged in linear clusters. We elucidate the phylogenetic relationship of P. fukuyoi through large and small subunit (LSU and SSU) rDNA sequences, and also report the SSU of the cyst‐defined species Islandinium minutum (Harland & Reid) Head et al. 2001. Molecular phylogenetic analysis by SSU rDNA shows that both species are closely related to Protoperidinium americanum (Gran & Braarud 1935) Balech 1974. Large subunit rDNA phylogeny also supports a close relationship between P. fukuyoi and P. americanum. Three subgroups in total are further characterized within the Monovela group. The cyst of P. fukuyoi shows a wide geographical range along the coastal tropical to temperate areas of the North‐east Pacific, its distribution reflecting optimal summer sea‐surface temperatures of ~14–18 °C and salinities of 22–34 psu.     

Morphogenesis and Molecular Phylogeny of a Soil Ciliate Uroleptoides longiseries (Foissner,Agatha and Berger, 2002) Berger 2008 (Ciliophora,Hypotrichia)

Morphogenesis of the hypotrich ciliate Uroleptoides longiseries, isolated from sandy soil beside the Yellow River, Suide, Yulin, Shaanxi Province, China, was investigated using protargol staining. The main events during binary fission are as follows: (1) the long frontoventral row is formed by a single anlage; (2) five frontoventral‐transverse cirral anlagen are formed in primary mode; (3) only the posterior part of the parental adoral zone of the membranelles is renewed; and (4) the oral primordium of the opisthe is formed intrakinetally. This is the first detailed record of all stages of morphogenesis for Uroleptoides. We also provide the first record of the small subunit ribosomal DNA (SSU rDNA) sequences for U. longiseries. Phylogenetic analyses based on the SSU rDNA sequences data show that Uroleptoides longiseries clusters with U. magnigranulosa with moderate to high support which together form a clade with Orthoamphisiella breviseries. These three species share the morphogenetic feature of the long frontoventral row being formed by a single anlage.     

New considerations on the phylogeny of cyrtophorian ciliates (Protozoa,Ciliophora): expanded sampling to understand their evolutionary relationships

To rationalize the confusing relationships among the cyrtophorian ciliates, we expanded the taxon sampling by sequencing the small subunit ribosomal RNA (SSU rRNA) gene of representatives of 12 genera (20 species, 23 new sequences). The SSU rRNA sequences of Spirodysteria, Agnathodysteria, Brooklynella and Odontochlamys are reported for the first time. Phylogenetic trees were constructed, and secondary structures of variable region 4 (V4) of all genera for which SSU rRNA gene sequence data are available were predicted. The results indicate that (i) Brooklynella is likely an intermediate taxon between Dysteriidae and Hartmannulidae; (ii) the genus Dysteria is paraphyletic with Spirodysteria and Mirodysteria nested within it; (iii) the genus Agnathodysteria is well separated from Dysteria based on both molecular and morphological data; and (iv) Trithigmostoma is a basal genus of Chilodonellidae, based on both the morphological and molecular data.     

Taxonomic and phylogenetic evaluation of Limnothrix strains (Oscillatoriales, Cyanobacteria) by adding Limnothrix planktonica strains isolated from central China

Six Limnothrix strains, isolated for the first time from a shallow eutrophic lake in central China, were taxonomically and phylogenetically evaluated by investigating their polyphasic characteristics, including morphological features, cellular ultrastructures, and 16S rRNA gene sequences. All the six strains were morphologically similar, and their trichomes were in average 1.7 μm wide and cells 4.0 μm long, and having small gas vesicles within cells, and therefore identified as Limnothrix planctonica (Woloszynska) Meffert. Cellular ultrastructures of them showed that peripheral thylakoids with 3–5 parallel layers were parietally distributed in the cells. The phylogenetic results based on the 16S rRNA gene sequences showed that all the Limnothrix strains, including the six in this study and those from the Genbank, formed two distinct clusters. The similarity in 16S rDNA sequences between these two clusters was lower than 90%, indicating that these Limnothrix strains belong to different genera. This is the first report on the morphology and phylogeny of L. planctonica strains, providing the new information on taxonomy of the genus Limnothrix.     

Molecular and Morphological Characterization of Anncaliia azovica sp. n. (Microsporidia) Infecting Niphargogammarus intermedius (Crustacea,Amphipoda) from the Azov Sea

Five out of one hundred adults of Niphargogammarus intermedius caught at the Azov sea shore were found to be infected with microsporidia. The infection was found in the subcuticular fat body and myocytes. Parasites developed in direct contact with host cells, displayed a disporoblastic sporogony and diplokaryotic arrangement of nuclei at all stages. Spores were oval, 4.6–5.8 × 2.6–3.0 μm. Exospore appendages, vesicular–tubular secretions, and the anisofilar polar filament indicated a similarity to Anncaliia species. Sporont surfaces displayed ridges of amorphous material. Meronts and sporonts formed protoplastic extensions, similar to A. vesicularum and A. meligheti. Mature spores possessed a bipartite polaroplast. The polar tube was arranged in one row of 13–18 coils including 0–3 distal coils of lesser diameter. Partial sequencing of SSU, ITS, and LSU regions of rRNA gene (GenBank accessions: KY288064 – KY288065 ) confirmed this new species to be congeneric with A. algerae (# AF069063 ) and A. meligheti (# AY894423 ). The SSU gene of this novel microsporidium shared 99.4% sequence similarity to A. algerae and 98.9% to A. meligheti. Genes for HSP70 and RPB1 amplified with primers designed for A. algerae orthologs displayed 99.7% and 97.4% similarity, respectively, between A. algerae and the novel microsporidium. A new species, Anncaliia azovica, is described based on morphological and molecular characterization.