Beyond the "Code": A Guide to the Description and Documentation of Biodiversity in Ciliated Protists (Alveolata, Ciliophora).

Recent advances in molecular technology have revolutionized research on all aspects of the biology of organisms, including ciliates, and created unprecedented opportunities for pursuing a more integrative approach to investigations of biodiversity. However, this goal is complicated by large gaps and inconsistencies that still exist in the foundation of basic information about biodiversity of ciliates. The present paper reviews issues relating to the taxonomy of ciliates and presents specific recommendations for best practice in the observation and documentation of their biodiversity. This effort stems from a workshop that explored ways to implement six Grand Challenges proposed by the International Research Coordination Network for Biodiversity of Ciliates (IRCN-BC). As part of its commitment to strengthening the knowledge base that supports research on biodiversity of ciliates, the IRCN-BC proposes to populate The Ciliate Guide, an online database, with biodiversity-related data and metadata to create a resource that will facilitate accurate taxonomic identifications and promote sharing of data.

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.

Phylogenetic framework of the systematically confused Anteholosticha-Holosticha complex (Ciliophora, Hypotrichia) based on multigene analysis.

The Anteholosticha-Holosticha complex is an extremely divergent group within the urostylids, especially because the genus characterization lacks suitable synapomorphies. Previous studies have shown that morphological classification of species within this group often conflicts with SSU-rDNA data, that is this complex is not recovered as a monophyletic group and Anteholosticha spp. are widely dispersed throughout the urostylid assemblage in SSU-rDNA trees. In this study, we provided 38 new sequences (including the type species of Anteholosticha) of SSU-rDNA, ITS1-5.8S-ITS2 and LSU-rDNA genes to infer molecular phylogenies of all available taxa in the Anteholosticha-Holosticha complex. The results show that: (1) Holosticha is monophyletic in all trees, suggesting it is a well-defined genus; (2) Anteholosticha is polyphyletic and distinctly separated from Holosticha in all single-gene based and concatenated phylogenies; (3) the monophyly of Arcuseries, a recently established genus split from Anteholosticha, is strongly supported by all molecular data; (4) Anteholosticha multicirrata, Anteholosticha manca, Anteholosticha paramanca and Bakuella subtropica may share a most recent common ancestor; (5) multi-gene analyses receive higher support values than the single-gene analyses.

New record of Apoholosticha sinica (Ciliophora, Urostylida) from the UK: morphology, 18S rRNA gene phylogeny and notes on morphogenesis.

The benthic urostylid ciliate Apoholosticha sinicaFan et al., 2014 was isolated from a salt marsh at Blakeney, UK, and reinvestigated using light microscopy and small-subunit rRNA gene sequencing. Morphologically, it corresponds well with the original description. Several stages of divisional morphogenesis and physiological reorganization were also observed from which the following could be deduced: (i) the oral apparatus is completely newly built in the proter; (ii) frontal-ventral-transverse cirral anlage II does not produce a buccal cirrus; (iii) each of the posteriormost three or four anlagen contributes one transverse cirrus at its posterior end; (iv) a row of frontoterminal cirri originates from the rearmost frontal-ventral-transverse cirral anlage; (v) the last midventral row is formed from the penultimate frontal-ventral-transverse cirral anlage. Based on new data, two diagnostic features were added to the genus definition: (i) the midventral complex is composed of midventral pairs and midventral row and (ii) pretransverse ventral cirri are absent. Based on a combination of morphological and morphogenetic data, the genus Apoholosticha is assigned to the recently erected subfamily Nothoholostichinae Paiva et al., 2014, which is consistent with sequence comparison and phylogenetic analyses based on SSU rRNA gene data. It is also concluded that this benthic species, previously reported only from China, is not an endemic form.

Morphology, ontogenetic features and SSU rRNA gene-based phylogeny of a soil ciliate, Bistichella cystiformans spec. nov. (Protista, Ciliophora, Stichotrichia).

The morphology, ontogeny and SSU rRNA gene-based phylogeny of Bistichella cystiformans spec. nov., isolated from the slightly saline soil of a mangrove wetland in Zhanjiang, southern China, were investigated. The novel species was characterized by having five to eight buccal cirri arranged in a row, three to five transverse cirri, four macronuclear nodules aligned, and 17-32 and 20-34 cirri in frontoventral rows V and VI, respectively, both extending to the transverse cirri. The main ontogenetic features of the novel species were as follows: (1) the parental adoral zone of the membranelles is completely inherited by the proter; (2) the frontoventral and transverse cirri are formed in a six-anlagen mode; (3) basically, the frontal-ventral-transverse cirral anlagen II-V generate one transverse cirrus each at their posterior ends, while anlage VI provides no transverse cirrus; (4) both marginal rows and dorsal kineties develop intrakinetally, no dorsal kinety fragment is formed; and (5) the macronuclear nodules fuse into a single mass at the middle stage. Phylogenetic analyses based on the SSU rRNA gene showed that the novel species groups with the clade containing Bistichella variabilis, Parabistichella variabilis, Uroleptoides magnigranulosus and two species of the genus Orthoamphisiella. Given present knowledge, it was considered to be still too early to come to a final conclusion regarding the familial classification of the genus Bistichella; further investigations of key taxa with additional molecular markers are required.

Molecular phylogeny and taxonomy of two novel brackish water hypotrich ciliates, with the establishment of a new genus, Antiokeronopsis gen. n. (Ciliophora, Hypotrichia).

Two novel brackish water urostyloid ciliates, Anteholosticha paramanca sp. n. and Antiokeronopsis flava gen. n., sp. n., isolated from the Shenzhen Mangrove Nature Protection Area on the coast of the South China Sea, were investigated using live observation and protargol impregnation techniques. Anteholosticha paramanca sp. n. is characterized by its spherical yellowish cortical granules arranged in lines, shortened midventral complex and three transverse cirri. Morphogenesis is similar to that in Anteholosticha manca. The new genus Antiokeronopsis is diagnosed by having a continuous adoral zone of membranelles, frontal cirri arranged in a bicorona, midventral complex composed of midventral pairs only, one marginal cirral row on each side, the presence of frontoterminal and transverse cirri, and the lack of buccal and caudal cirri. The type species A. flava sp. n. is characterized by its elongated body shape, brown to yellowish body color and two types of cortical granules. Small subunit ribosomal RNA gene sequence data justify the classification of both species. Phylogenetic analyses indicate that A. paramanca clusters with Bakuella subtropica within a clade that includes two other Anteholosticha species, while Antiokeronopsis groups within the core urostylids and is most closely related to the well-known genera Pseudokeronopsis and Uroleptopsis.

Morphology, morphogenesis and molecular phylogeny of a freshwater ciliate, Monomicrocaryon euglenivorum euglenivorum (Ciliophora, Oxytrichidae).

The morphology, morphogenesis and molecular phylogeny of the oxytrichid ciliate, Monomicrocaryon euglenivorum euglenivorum (Kahl, 1932) Foissner, 2016, isolated from freshwater in a seaside park, Guangzhou, China, were investigated. Monomicrocaryon euglenivorum euglenivorum can be recognized as follows: caudal cirri in midline of body; dorsal kinety 1 without a one-kinetid-wide gap; transverse cirri acicular or rod-shaped with a fringed distal end; right marginal row commences at level of buccal vertex or anterior to buccal vertex. The main events during binary fission are as follows: (1) the proter retains the parental adoral zone of membranelles entirely; (2) frontoventral-transverse cirral anlagen I-VI are segmented in the ordinary pattern 1:3:3:3:4:4 from left to right, which form three frontal, four frontoventral, one buccal, three postoral ventral, two pretransverse ventral and five transverse cirri, respectively; (3) dorsal morphogenesis is in the typical Oxytricha-pattern, but fragmentation of dorsal kinety 3 is indistinct; and (4) three caudal cirri are formed, one at the posterior end of each of dorsal kineties 1, 2 and 4. Phylogenetic analyses based on SSU rDNA sequences showed that M. euglenivorum euglenivorum clustered with Kleinstyla dorsicirrata and Heterourosomoida lanceolata rather than with its congener M. elegans. The genus Monomicrocaryon is not monophyletic in this study; however, its monophyly is not rejected by the AU test.

New contribution to the species-rich genus Euplotes: Morphology, ontogeny and systematic position of two species (Ciliophora; Euplotia).

The morphology, ontogeny and phylogeny of two Euplotes species, E. estuarinus sp. nov. and a population of E. platystoma Dragesco and Dragesco-Kernéis, 1986, both collected from tropical brackish waters in south China, were investigated based on living morphology, ciliary pattern and molecular data. Euplotes estuarinus sp. nov. is small (about 60 × 40 µm in vivo), has a dargyrome of the double-eurystomus type, and the transverse cirri are arranged in two groups, with two left and three right ones. The original description of the poorly known species, E. platystoma, is brief, and the species was never investigated using live observation and molecular methods Hence, we provided a detailed redescription. Some stages of their morphogenesis were observed which proceed in the same pattern as in their congeners. The new species E. estuarinus sp. nov. clusters with E. curdsi, differing only by 1 bp in their SSU rRNA gene sequences, which is likely due to the recent speciation event and the limited resolution of the SSU rRNA gene at species level in this group as the two species are clearly morphologically distinct.

Redescription of two little-known urostyloid ciliates, Anteholosticharandani (Grolière, 1975) Berger, 2003 and A. antecirrata Berger, 2006 (Ciliophora, Urostylida).

The morphology of two little-known urostyloid ciliates, Anteholosticha randani (Grolière, 1975) Berger, 2003 and A. antecirrataBerger, 2006, collected from freshwater biotopes in southern China, was studied based on live observations and protargol staining. Anteholosticha randani is characterized by its bipartite adoral zone and short, longitudinally aligned undulating membranes. One early stage of reorganization/morphogenesis, one early-middle stage of reorganization and one middle stage of morphogenesis are also reported. Anteholosticha antecirrata is characterized by its large body size in vivo (200-400 × 40-80 µm), a row of buccal cirri and conspicuous, yellow-green cortical granules. Phylogenetic analyses based on SSU rDNA sequence data reveal that A. antecirrata may share a most recent common ancestor with Urostyla grandis and Bakuella granulifera, whereas A. randani branches independently and is sister to a large clade that includes Pseudourostyla, Pseudokeronopsis, Caudiholosticha and several species of Anteholosticha.

Morphology and systematics of two freshwater urostylid ciliates, with description of a new species (Protista, Ciliophora, Hypotrichia).

The morphology of two freshwater urostylid species, Neourostylopsis flava spec. nov. and Pseudourostyla subtropicaChen et al., 2014, isolated from freshwater ponds in northern and southern China, respectively, was investigated following examination of specimens in vivo and following protargol staining. Neourostylopsis flava spec. nov. is distinguished from its congeners by the following characteristics: body size 150-220 × 50-75 µm in vivo; yellow in colour; bright yellow to yellow-brownish spherical cortical granules densely arranged along marginal cirral rows and in irregular short rows on dorsal side; adoral zone with 40-55 membranelles; six to eight frontal, three or four buccal, two pretransverse ventral and seven to nine transverse cirri; 27-40 midventral pairs extending to about anterior 55% of cell; four or five left and four right marginal rows; freshwater habitat. A redescription of a freshwater population of P. subtropica is also provided. Phylogenetic analyses based on small subunit ribosomal DNA sequences shows that P. subtropica and N. flava spec. nov. group with their congeners and both Neourostylopsis and Pseudourostyla are monophyletic.

Taxonomy and molecular phylogeny of two novel ciliates, with establishment of a new genus, Pseudogastrostyla n. g. (Ciliophora, Hypotrichia, Oxytrichidae).

Two novel hypotrichous ciliates, Pseudogastrostyla flava n. g., n. sp. and Urosomoida subtropica n. sp., isolated from Daya Bay Mangrove Park, Huizhou, southern China, were observed in vivo and after protargol staining. Pseudogastrostyla n. g. is diagnosed by having more than 18 frontal-ventral-transverse cirri, an indistinct frontoventral row, undulating membranes in Oxytricha-pattern and dorsomarginal kinety, but lacking dorsal kinety fragmentation during morphogenesis. The monotypic species, P. flava n. sp., is characterized by its brownish body colour, brown-yellowish cortical granules mostly arranged in groups, frontoventral row terminating near mid-body, and one caudal cirrus. Urosomoida subtropica n. sp. can be recognized by having one micronucleus located between two macronuclear nodules, four transverse cirri, pretransverse ventral cirrus (V/2) anteriorly located, four dorsal kineties and three caudal cirri. Phylogenetic analyses based on SSU rRNA gene sequence data indicate that Pseudogastrostyla n. g. is nested within the oxytrichids and is most closely related to Rubrioxytricha ferruginea, and that Urosomoida subtropica n. sp. clusters with Oxytricha elegans rather than the type species, U. agilis. Nodal support values are, however, too low to draw any conclusions about the molecular systematics of the genus Urosomoida.

Morphology and morphogenesis of Apoholosticha sinica n. g., n. sp. (Ciliophora, Hypotrichia), with consideration of its systematic position among urostylids.

This paper investigates the morphology, morphogenesis and SSU rRNA gene-based phylogeny of Apoholosticha sinica n. g., n. sp., isolated from mangrove wetland in Shenzhen, southern China. The new genus Apoholosticha is characterized by its bipartite adoral zone, clearly differentiated frontal cirri arranged in a bicorona, midventral complex composed of midventral pairs only, one marginal cirral row on each side, presence of frontoterminal and transverse cirri, and the lack of a buccal cirrus and caudal cirri. The type species, Apoholosticha sinica n. sp. is diagnosed by the elongated body shape and two kinds of cortical granules. Its main morphogenetic features are similar to that of Pseudokeronopsis except for (1) no buccal cirrus is formed and (2) its macronuclear nodules fuse into a single mass during cell division. Phylogenetic analyses for the new taxon indicate that Apoholosticha n. g. is most closely related to Nothoholosticha and Heterokeronopsis, and falls into the family Pseudokeronopsidae within the core Urostylida clade. In addition, a species that had been misidentified in previous literature is here recognized and assigned to the new genus as Apoholosticha sepetibensis (Wanick and Silva-Neto, 2004) n. comb. (basionym: Pseudokeronopsis sepetibensis Wanick and Silva-Neto, 2004).

Morphology and SSU rRNA gene-based phylogeny of two Diophrys-like ciliates from northern China, with notes on morphogenesis of Pseudodiophrys nigricans (Protozoa, Ciliophora).

Two free-living marine euplotid ciliates, Pseudodiophrys nigricans and Diophrys japonica, collected from the coastal waters off Qingdao, northern China, were investigated using live observations and protargol impregnation methods. The cortical development of P. nigricans was observed during binary division. Although its general pattern of morphogenesis is similar to that of other Diophrys-like species, three unusual features are noteworthy: 1) the frontoventral transverse cirral anlagen develop in the secondary mode, similar to that of Euplotes; 2) the undulating membrane anlage migrates far from the cytostome and does not split into two membranes; and 3) the parental adoral zone of membranelles remains nearly intact throughout the entire morphogenetic process. Diophrys japonica is redescribed based on a Chinese population and can be recognized by having one left marginal cirrus, densely arranged cortical granules, and a fragment kinety with three dikinetids. Phylogenetic analyses based on the small subunit rRNA (SSU rRNA) gene sequence data indicate that D. japonica is placed within the Diophrys clade and is most closely related to the well-known D. apoligothrix.