A detailed description of a Brazilian Holophrya teres (Ehrenberg, 1834) and nomenclatural revision of the genus Holophrya (Ciliophora, Prostomatida).

I studied a Brazilian population of Holophrya teres (Ehrenberg, 1834) Foissner, Berger and Kohmann, 1994, using live observation, morphometry, silver impregnation, and scanning electron microscopy. This showed a fair similarity with European populations, especially in having a large (about 13 × 5 µm in vivo) micronucleus pyriform in broad-side view and cuneate in narrow-side view. Several new structures were discovered, viz., an internal oral basket, teeth on anterior end of the oral basket rods, and a buccal seal closing the oral basket when not feeding. Based on this knowledge, I provide an improved diagnosis for H. teres, which can be used as template in further species descriptions. Depending on brosse implantation in the somatic ciliature, the genus was split in Holophrya, Hillerophrya nov. gen., Vdacnyophrya nov. gen., and Bardeleophrya nov. gen. Twenty-one Prorodon species were combined with the holophryid genera, using brosse and thick oral basket rods as main markers. This revealed two new species, viz., Holophrya longiarmata nov. spec. and H. agamalievi nov. spec.

Morphology and ontogenesis of two new Hemiholosticha species (Ciliophora, Hypotrichia, Hemiholostichidae nov. fam.).

The morphology and ontogenesis of two new hypotrich ciliates, Hemiholosticha solitaria and Hemiholosticha germanica, were studied using live observation, protargol impregnation, and scanning electron microscopy. Both species share a medium-sized, almost globular body with a short anterior projection; two macronuclear nodules with a single micronucleus in between; a central contractile vacuole; three or four ventral, one postoral, one right and one left marginal cirral row; and three dorsal kineties extending along ribs. However, H. germanica is distinguished from congeners by a higher number of cirri in ventral rows R1 and R2 (3-6 vs. 2 cirri in each row). Hemiholosticha solitaria differs from congeners by having four (vs. three) ventral cirral rows and by the lack (vs. presence) of intracellular green algae. The ontogenesis of H. solitaria follows the H. pantanalensis mode in that (i) the oral primordium develops in a deep pouch and generates the first two cirral streaks in addition to adoral membranelles and undulating membranes, (ii) the undulating membrane anlage does not produce any cirri, and (iii) the longitudinal ventral cirral row R3 originates from two anlagen. The ontogenetic peculiarities along with the 18S rRNA gene phylogenies suggest classification of Hemiholosticha, Psilotrichides, and Urospinula into a new family, Hemiholostichidae.

A huge diversity of metopids (Ciliophora, Armophorea) in soil from the Murray River floodplain, Australia. III. Morphology, ontogenesis and conjugation of Metopus boletus nov. spec., with implications for the phylogeny of the SAL supercluster.

The morphology, ontogenesis, conjugation, and phylogenetic position of Metopus boletus nov. spec. were studied using live observation, various silver impregnation methods, scanning electron microscopy, morphometry, and the 18S rRNA gene sequence. The new species is outstanding in having a mushroom-like appearance; a globular to broadly ellipsoid macronucleus in anterior body half; 5-10 elongated caudal cilia; 4-6 dikinetids curved rightwards in the anterior portion of the first postoral kinety; and an adoral zone composed of an average of 28 small polykinetids. Ontogenesis of M. boletus follows the metopid mode and the species-specific vegetative morphology is obtained after division. Its conjugation is temporary, isogamic and the partners unite ventral-to-dorsal, forming strongly arched to almost rod-like pairs, which indicates a heteropolar arrangement. There are only two maturation divisions and a single synkaryon division in exconjugants. The conjugation data corroborate a sister group relationship of the classes Armophorea and Litostomatea within the SAL (Spirotrichea + Armophorea + Litostomatea) supercluster in that the partners unite ventral-to-dorsal and the main body axes are antiparallel. On the other hand, the last common ancestor of the spirotricheans very likely had a ventral-to-ventral and homopolar conjugation mode with the main body axes oriented in parallel.

Re-analysis of the 18S rRNA gene phylogeny of the ciliate class Colpodea.

We critically re-analyzed the 18S rRNA gene phylogeny of the ciliate class Colpodea where four main lineages have been recognized: (1) Bursariomorphida including bryometopids, (2) Platyophryida including sorogenids, (3) Cyrtolophosidida, and (4) Colpodida including bryophryids and grossglockneriids. The Platyophryida branched off first and the Cyrtolophosidida and Colpodida were classified as sister groups. On basis of multiple statistical tests, we unraveled three problematic issues in colpodean phylogenies: the positions of the Bursariomorphida and Platyophryida are unstable and depend on alignment masking; a sister relationship of the Platyophryida and Cyrtolophosidida cannot be excluded by any statistical tree topology test; and clustering of bryophryids and grossglockneriids outside the Colpodida are also statistically valid possibilities. Natural classification of the highly diverse order Colpodida remains puzzling, possibly due to the lack of a phylogenetic signal and morphostasis of the oral ciliature in several Colpoda-like lineages. According to the "Ur-Colpoda" hypothesis, Colpoda represents the stem lineage from which both Colpoda-like and morphologically more derived taxa might have branched off. This evolutionary concept preserves not only information on morphology, ecology, and evolutionary processes of colpodid ciliates, but also aids practicability because the connection to the traditional literature is optimally maintained.

A Proposed Timescale for the Evolution of Armophorean Ciliates: Clevelandellids Diversify More Rapidly Than Metopids.

Members of the class Armophorea occur in microaerophilic and anaerobic habitats, including the digestive tract of invertebrates and vertebrates. Phylogenetic kinships of metopid and clevelandellid armophoreans conflict with traditional morphology-based classifications. To reconcile their relationships and understand their morphological evolution and diversification, we utilized the molecular clock theory as well as information contained in the estimated time trees and morphology of extant taxa. The radiation of the last common ancestor of metopids and clevelandellids very likely occurred during the Paleozoic and crown diversification of the endosymbiotic clevelandellids dates back to the Mesozoic. According to diversification analyses, endosymbiotic clevelandellids have higher net diversification rates than predominantly free-living metopids. Their cladogenic success was very likely associated with sharply isolated ecological niches constituted by their hosts. Conflicts between traditional classifications and molecular phylogenies of metopids and clevelandellids very likely come from processes, leading to further diversification without extinction of ancestral lineages as well as from morphological plesiomorphies incorrectly classified as apomorphies. Our study thus suggests that diversification processes and reconstruction of ancestral morphologies improve the understanding of paraphyly which occurs in groups of organisms with an apparently long evolutionary history and when speciation prevails over extinction.

A huge diversity of metopids (Ciliophora, Armophorea) in soil from the Murray River floodplain, Australia. I. Description of five new species and redescription of Metopus setosus Kahl, 1927.

Six metopid ciliates from soil of the Murray River floodplain in Australia were studied using live observation, various silver impregnation methods, scanning electron microscopy, and multivariate statistics. One of the species is affiliated with M. setosus while the others represent new taxa. Metopus filum nov. spec. is distinguished from most congeners by the slender body, the absence of cortical granules, and the low number of ciliary rows and adoral polykinetids. Metopus palaeformides nov. spec. most resembles Heterometopus palaeformis (Kahl, 1927) Foissner, 2016b but they can be distinguished by body size, the number of adoral polykinetids, and the oral area pattern. Metopus murrayensis nov. spec. is outstanding in having a globular macronucleus surrounded by innumerable refractive granules and a conspicuously thick preoral dome. Metopus rex nov. spec. and M. magnus nov. spec. are easily distinguished from most congeners by their large body size and the shape of the macronucleus. Moreover, M. rex displays up to 30µm long endosymbiotic bacteria while the micronucleus of M. magnus is uniquely situated in a small macronuclear concavity. Multivariate statistics corroborates the distinctness of these six metopid populations.

Morphology and ontogenesis of Stylonychia (Metastylonychia) nodulinucleata nov. subgen. (Ciliophora, Hypotricha) from Australia.

Using standard methods, we describe the morphology and ontogenesis of a possibly very rare ciliate, Stylonychia (Metastylonychia) nodulinucleata nov. subgen (basionym: Stylonychia nodulinucleataShi and Li, 1993), isolated from soil of the Murray River floodplain in Australia. Further, we provide an English translation of the Chinese original description. Whether the Australian population is conspecific with the Chinese Stylonychia nodulinucleata requires a careful redescription of the latter. The new subgenus is characterized by a moniliform macronuclear strand and an ontogenetic feature, viz., that all cirri remain intact during anlagen formation. The moniliform macronuclear strand and the curved dorsal kineties resemble the stylonychid genus Coniculostomum. The species is easily recognized by the large size of the body and oral apparatus, the moniliform macronucleus, and the enormous caudal cirri reaching half of body length. Very likely, Stylonychia (Metastylonychia) nodulinucleata has a restricted distribution.

Protists as bioindicators in activated sludge: Identification, ecology and future needs.

When the activated sludge process was developed, operators and scientists soon recognized protists as valuable indicators. However, only when Curds et al. (1968) showed with a few photographs the need of ciliates for a clear plant effluent, sewage protistology began to bloom but was limited by the need of species identification. Still, this is a major problem although several good guides are available. Thus, molecular kits should be developed for identification. Protists are indicators in two stages of wastewater treatment, viz., in the activated sludge and in the environmental water receiving the plant effluent. Continuous control of the protist and bacterial communities can prevent biological sludge foaming and bulking and may greatly save money for sludge oxygenation because several protist species are excellent indicators for the amount of oxygen present. The investigation of the effluent-receiving rivers gives a solid indication about the long term function of sewage works. The literature on protist bioindication in activated sludge is widely distributed. Thus, I compiled the data in a simple Table, showing which communities and species indicate good, mediocre, or poor plant performance. Further, many details on indication are provided, such as sludge loading and nitrifying conditions. Such specific features should be improved by appropriate statistics and more reliable identification of species. Then, protistologists have a fair chance to become important in wastewater works. Activated sludge is a unique habitat for particular species, often poorly or even undescribed. As an example, I present two new species. The first is a minute (∼30µm) Metacystis that makes an up to 300µm-sized mucous envelope mimicking a sludge floc. The second is a Phialina that is unique in having the contractile vacuole slightly posterior to mid-body. Finally, I provide a list of species which have the type locality in sewage plants.

High cryptic soil ciliate (Ciliophora, Hypotrichida) diversity in Australia.

The diversity and distribution of soil ciliates from Australia is poorly known. Thus, we studied eight taxa, using the non-flooded Petri dish culture method, live observation, silver impregnation, detailed morphometrics, ontogenesis, and reinvestigation of type slides. At first glance, the Australian taxa looked very similar to described species, however, detailed investigations resulted in the identification of six cryptic species: Afroamphisiella multinucleata minima nov. subspec., Cladotricha similis nov. spec., Erimophrya similis nov. spec., Heterogonostomum salinarum nov. gen., nov. spec., Pseudohemisincirra arabica australiensis nov. subspec., and Pattersoniella (Pattersoniellides) australiensis nov. subgen., nov. spec. This new subgenus is unique among all described hypotrichs in having reduced some anterior paroral dikinetids the fibrillar associates of which are, however, still present. Only two of the eight taxa are possibly cosmopolitans: Apourosomoida halophilaFoissner et al., 2002 and Urosoma karinaeFoissner, 1987. This supports the moderate endemicity model, i.e., that a third of protists have a restricted distribution (Foissner, Chao and Katz 2008).

Heterometopus meisterfeldi nov. gen., nov. spec. (Protozoa, Ciliophora), a new metopid from Australia.

Using standard methods, I describe Heterometopus meisterfeldi nov. gen., nov. spec., a new armophorean ciliate from dry soil of the bed of the Fogg Dam in northern Australia. It appeared in the non-flooded Petri dish culture three weeks after soil re-wetting, i.e., when the bottom zone of the culture became anaerobic. The new genus is characterized by a cylindroid body and a short, slightly oblique, J-shaped adoral zone extending only ventrally and about one third of body length. The species has an average size of 140×30µm and has 23 ciliary rows, 30 adoral polykinetids, and 25 false kineties in the perizonal stripe. Metopus palaeformis, as redescribed by Foissner et al. (2002) and in the present paper, shows very similar characteristics and is thus transferred to the new genus: Heterometopus palaeformis (Kahl, 1927) nov. comb. Heterometopus meisterfeldi and H. palaeformis differ by body size (∼140×30 vs. 100×20 in vivo), the shape of the macronucleus (ellipsoid, ∼2:1 vs. elongate ellipsoid, ∼3:1), the width of the side-stripe (12µm vs. 4µm), the number of false kineties in the perizonal stripe (25 vs. 14), and the number of adoral polykinetids (∼30 vs. ∼20). Very likely, several species from Kahl's Metopus groups II and III belong to Heterometopus but a transfer should await detailed redescriptions. Heterometopus meisterfeldi belongs very likely to the family Metopidae. The short, J-shaped adoral zone of polykinetids highly resembles the blepharismid genera Pseudoblepharisma and Blepharisma.

Biogeographic specializations of two large hypotrich ciliates: Australocirrus shii and A. australis and proposed synonymy of Australocirrus and Cyrtohymenides.

Using standard methods, we studied the morphology and distribution of an Australian population of Australocirrus shii (Shi et al., 1997) nov. comb. and a Jamaican population of A. australis (Foissner, 1995) nov. comb. We identified three features, not or rarely used earlier, that distinguish Australocirrus shii from A. australis: the distance between the anterior pretransverse cirrus and the anteriormost transverse cirrus (5-8% vs. 1.5-1.7% of body length), the arrangement of the transverse cirri (3+2 vs. an oblique row), and the resting cyst macronuclear nodules (separate vs. fused). Australocirrus shii has been reported from Asia and Australia while A. australis is possibly restricted to the Neotropic. Because of problems in getting voucher materials of A. shii, we emphasise that permanent slides should be deposited in international repositories. Based on previous studies and new data, especially a refined interpretation of the shape of the paroral membrane, we suggest synonymy of Cyrtohymenides and Australocirrus. Thus, Cyrtohymena (Cyrtohymenides) shii, C. (Cyrtohymenides) aspoecki, and C. (Cyrtohymenides) australis are transferred to Australocirrus which is, inter alia, defined by a moderately to distinctly curved, but not recurved, paroral membrane, multiple fragmentation of dorsal kinety 3 and three or more dorsomarginal kineties.

Free-living ciliates from epiphytic tank bromeliads in Mexico.

The ciliate diversity of Mexican bromeliads is poorly known. We studied the ciliate community of two species of epiphytic tank bromeliads from 48 individuals of Tillandsia heterophylla and four of T. prodigiosa. The bromeliads occurred on over 22 tree host species. Samples were collected during 2009 and 2010 in a mountain cloud forest and in two coffee plantations and in a pine-oak forest. The ciliates were identified in live and protargol preparations. We recorded 61 ciliate species distributed in 39 genera grouped in eight classes. Ten species were frequent in the 52 samples (20 ± 3.2) and Leptopharynx bromeliophilus was the most frequent recorded in 25 samples. Thirty-three species are new for the fauna of Mexico, 24 species have been recorded for the first time in tank bromeliads. The classes Spirotrichea, Oligohymenophorea and Colpodea presented the highest number of species, 16, 14, and 12, respectively. Colpoda was the most species-rich genus being present with six species. A low similarity between areas and seasons was obtained with Jaccard's index. We conclude that the two bromeliads species host a rich ciliate diversity whose knowledge contributes to the question of ciliate distribution and specifically, in tank bromeliads.

Multivariate morphometric analyses of the predatory ciliate genus Semispathidium (Ciliophora: Litostomatea), with description of S. longiarmatum nov. spec.

We studied morphometrical variation, species boundaries, and importance of morphometric features for a reliable separation of five African Semispathidium taxa. Altogether, 20 features traditionally used in alpha-taxonomy of the predatory genus Semispathidium were measured or scored on 85 protargol-impregnated interphase specimens, and were analyzed using hierarchical clustering as well as principal component and canonical discriminant analyses. This multivariate approach confirmed that a population found in Botswanan floodplain soil represents a distinct taxon. The new species is described here as S. longiarmatum, using live observation, protargol impregnation, and scanning electron microscopy. Semispathidium longiarmatum strongly resembles S. armatum and S. breviarmatum but it is clearly distinguished from these species by the extrusome pattern. The reliability of S. longiarmatum is also strengthened, according to the canonical discriminant analysis, by several quantitative features, viz., the number of ciliary rows, the length:width ratio of the macronucleus, and the number of dikinetids in brush row 1. Moreover, the present study documents the distinctness of all African Semispathidium species which can be separated by a combination of both qualitative and quantitative (morphometric) features. Consequently, Semispathidium species do not form a continuous complex but fairly discrete clusters in the phenotypic space.

The chaos prevails: molecular phylogeny of the Haptoria (Ciliophora, Litostomatea).

The Haptoria are free-living predatory ciliates living in terrestrial and aquatic habitats all around the world. They belong to a highly diverse class, Litostomatea, whose morphological and molecular classifications harmonize poorly since both approaches produce rather different frameworks. In the present study, we analyzed the genealogy of the litostomateans, including eight new haptorian 18S rRNA gene sequences. Apart from traditional tree-building methods, we also applied phylogenetic networks, split spectrum analysis and quartet likelihood mapping to assess the information content of alignments. These analyses show that: (1) there are several strongly supported monophyletic litostomatean lineages--Rhynchostomatia, Trichostomatia, Haptorida, Lacrymariida, Pleurostomatida, and Didiniida; (2) the Rhynchostomatia are the best candidates for a basal litostomatean group; (3) sister relationship of the Trichostomatia and Haptoria is very likely, which well corroborates the traditional morphology-based classifications; (4) molecular phylogeny of the order Spathidiida is only poorly resolved very likely due to one or several rapid radiation events or due to the incomplete lineage sorting at the rRNA locus; and (5) the basal position of the genera Chaenea and Trachelotractus in molecular trees and phylogenetic networks is very likely a result of class III long-branch effects.

Morphology and ontogenesis of Psilotrichides hawaiiensis nov. gen., nov. spec. and molecular phylogeny of the Psilotrichidae (Ciliophora, Hypotrichia).

The Psilotrichidae are a family of middle-sized hypotrichs with unique morphological and ontogenetic features (e.g. the oral primordium develops in a deep pouch) that, however, did not provide a definite phylogenetic signal. Thus, we studied the 18S rRNA gene of Urospinula succisa (Müller 1786) Esteban et al., 2001 as well as the morphology and ontogenesis of Psilotrichides hawaiiensis, a new genus and species from an ephemeral swamp on Oahu Island, Hawaii. The molecular data classify the psilotrichids into the oxytrichids but without clear branching position. A brief revision, using the structure of the oral apparatus, the location of the contractile vacuole, and three ontogenetic features, showed four distinct genera: Psilotricha Stein, 1859; Urospinula Corliss, 1960; Hemiholosticha Gelei, 1954; and Psilotrichides nov. gen., which differs from the confamilials mainly by the obliquely oriented buccal cavity and the shape of the undulating membranes as well as by a distinct ridge along the right buccal margin. The pyriform species, P. hawaiiensis, is about 65 × 45 µm in size and is easily recognized by the table tennis racket-shaped appearance due to the elongated last cirrus of the left marginal row. Refined diagnoses are provided for the family Psilotrichidae Bütschli, 1889 and the genera contained.

Typification of the genus Dileptus Dujardin, 1841 (Ciliophora, Rhynchostomatia).

In their monograph of the dileptids, Vdacný and Foissner (2012) could not clarify the type species of the genus DileptusDujardin, 1841. Thus, they suggested that the problem be referred to the International Commission on Zoological Nomenclature. However, recently we discovered that Dujardin (1841) has originally typified Dileptus with Amphileptus anser sensu Ehrenberg (1838) which is in fact a misidentified Amphileptus margaritiferEhrenberg, 1833, a common species also originally classified in Dileptus. Under Article 70.3.2 of the Code, Dileptus margaritifer (Ehrenberg, 1833) Dujardin, 1841, thoroughly redescribed by Foissner et al. (1995), is now the type of Dileptus. This has the great advantages of historical continuity and that new combinations (names) are not required.

An update of 'basic light and scanning electron microscopic methods for taxonomic studies of ciliated protozoa'.

This is an update of the review by Foissner (1991). Since then, I have improved some methods considerably. The following methods are described in detail: live observation, supravital staining with methyl green-pyronin, dry silver nitrate impregnation, wet silver nitrate impregnation, silver carbonate impregnation, protargol impregnation (three procedures), scanning electron microscopy, and deciliation. Familiarity with these methods (or modifications) is a prerequisite for successful taxonomic work. No staining method is equally appropriate to all kinds of ciliates. A table is provided which indicates those procedures which work best for certain groups of ciliates. A second table relates to the structures revealed by the procedures. Good descriptions usually demand at least live observation, silver nitrate and protargol or silver carbonate impregnation. Some instructions are provided for distinguishing mono- and dikinetids as well as ciliated and non-ciliated basal bodies in silvered ciliates. Furthermore, I added a section on 'Deposition and Labeling of Preparations'. All methods work not only with ciliates but also with many other heterotrophic and autotrophic flagellated and amoeboid protists. The brilliancy of silver preparations has tempted some taxonomists to neglect live observation. However, many important species characteristics cannot be seen or are changed in silvered specimens. I thus consider all species descriptions based exclusively on silver slides as incomplete and of doubtful value for both α-taxonomists and ecologists.

Schmidingerothrix salinarum nov. spec. is the molecular sister of the large oxytrichid clade (ciliophora, hypotricha).

In 2012, Foissner described a curious hypotrich: Schmidingerothrix extraordinaria. This ciliate, which he discovered in hypersaline soils (~100‰) from Namibia, had a frayed buccal lip, three-rowed adoral membranelles, only one frontal cirrus, and a miniaturized first frontal membranelle, while a paroral membrane, dorsal bristle rows and buccal, transverse and caudal cirri were absent. All opisthe structures developed de novo, while parental structures were involved in the proter. When Foissner's study became available, we discovered a similar species in a Portuguese solar saltern, differing from S. extraordinaria mainly by the number of frontoventral cirral rows (3 vs. 1). Furthermore, parental structures were involved in the ontogenesis of both proter and opisthe. The small subunit (SSU) rDNA shows Schmidingerothrix as sister of a large clade containing most classical oxytrichids (e.g. Sterkiella, Oxytricha, Steinia) and many related taxa (e.g. Pattersoniella, Bistichella, Uroleptus). This clade shows a bifurcation named "Oxytricha subclade" and "Uroleptus subclade". Foissner () interpreted the peculiarities of Schmidingerothrix as a reduction caused by the extreme habitat. However, the molecular data do not exclude that Schmidingerothrix presents an ancient state. A morphology-based scheme is presented, showing how the subclades might have evolved from a Schmidingerothrix-like ancestor.

New SSU-rDNA sequences for eleven colpodeans (Ciliophora, Colpodea) and description of Apocyrtolophosis nov. gen.

Using 11 new SSU-rDNA sequences, we analyze relationships within the class Colpodea, especially of some uncommon taxa, such as Kalometopia duplicata, Cyrtolophosis minor, and Jaroschia sumptuosa. The sequences do not change the basic structure of the molecular Colpodea tree, i.e., all belong to one of the four molecular clades recognized by Foissner et al. (2011): Colpodida, Cyrtolophosidida, Bursariomorphida, and Platyophryida. The addition of three Colpoda sequences strengthens the observation that species of this genus are distributed over the whole molecular Colpodea tree. Very likely, this is caused by a fast radiation of Colpoda, several species of which then evolved independently, forming new genera and families. Cyrtolophosis minor, which belongs to the molecular Pseudocyrtolophosis clade, is referred to a new genus, Apocyrtolophosis nov. gen., characterized by a comparably large, deltoid oral opening, an unciliated posterior region, and the absence of an oblique kinety in the left oral polykinetid. Bryometopus triquetrus does not erase the paraphyly of its genus. Platyophrya vorax, P. spumacola, and P. bromelicola form a highly supported clade in the order Platyophryida. Platyophryides and Ottowphrya are close genetically but differ in the silverline pattern (colpodid vs. platyophryid).