Morphology and molecular phylogeny of Apoterritricha lutea n. g., n. sp. (Ciliophora, Spirotrichea, Hypotrichia): a putative missing link connecting Cyrtohymena and Afrokeronopsis.

Published by the International Society of Protistologists

The Journal of

Eukaryotic Microbiology

Journal of Eukaryotic Microbiology ISSN 1066-5234

ORIGINAL ARTICLE

Morphology and Molecular Phylogeny of Apoterritricha lutea n. g., n. sp. (Ciliophora, Spirotrichea, Hypotrichia): A Putative Missing Link Connecting Cyrtohymena and Afrokeronopsis Ji Hye Kima, Peter Vd’a cn yb, Shahed Uddin Ahmed Shaziba & Mann Kyoon Shina a Department of Biological Science, College of Natural Sciences, University of Ulsan, Ulsan 680-749, South Korea b Department of Zoology, Faculty of Natural Sciences, Comenius University, Mlynsk a dolina B-1, SK-84215 Bratislava, Slovakia

Keywords 18S rRNA gene; Korea; midventral cirral pattern; Neokeronopsidae; Oxytrichidae. Correspondence M. K. Shin, Department of Biological Science, College of Natural Sciences, University of Ulsan, Ulsan 680-749, South Korea Telephone number: +82 52 259 2396; FAX number: +82 52 259 1694; e-mail: [email protected] Received: 14 November 2013; revised 27 March 2014; accepted April 1, 2014. doi:10.1111/jeu.12131

ABSTRACT A new hypotrichous ciliate, Apoterritricha lutea n. g., n. sp., was discovered in a sample of a terrestrial liverwort from Korea. Its morphology was studied using detailed in vivo observation and protargol impregnation. Its phylogenetic relationships were revealed by analyses of the 18S rRNA gene. This new taxon is characterized by a combination of the following traits: (i) ellipsoidal to narrowly ellipsoidal body with an average size of 230 9 85 lm; (ii) two macronuclear nodules and two to five micronuclei; (iii) golden yellow cortical granules, forming small groups along the microtubular appendages of cirri, adoral membranelles, and dorsal kineties; (iv) typically three frontal cirri, one buccal cirrus, four frontoventral cirri, seven midventral cirri, two pretransverse cirri, seven transverse cirri, ca. 38 left, and ca. 36 right marginal cirri; and (v) on average six dorsal kineties, three dorsomarginal kineties, and three caudal cirri. In molecular phylogenies, A. lutea clusters with strong support within a clade containing Afrokeronopsis aurea and several “typical” oxytrichids having golden yellow to brown cortical granules. In this light we propose a hypothesis that is not unambiguously rejected by the present phylogenetic analyses, which shows how the Afrokeronopsis-like pattern could have evolved from a Rubrioxytrichalike ancestor via an Apoterritricha-like stage by cirri-multiplication.

HYPOTRICHS are a highly diverse group of ciliates living in a variety of habitats all around the world. They exhibit very distinct cirral patterns that have been traditionally used for their classification and for inferring their phylogenetic relationships (for reviews, see Berger 1999, 2006, 2008, 2011). However, morphology-based frameworks only poorly harmonize with molecular phylogenies, indicating that several cirral patterns evolved convergently and/or are inherited plesiomorphies (e.g., Foissner et al. 2004; Paiva et al. 2009). One of the most conspicuous discrepancies between classical and molecular classifications concerns the placement of some hypotrichs with midventral cirral complex (e.g., Uroleptus Ehrenberg, 1831) within the oxytrichid clade which usually includes species with 18 fronto-ventral-transverse cirral pattern. This fundamental disagreement between the morphological and molecular classifications was reconciled by the CEUU (Convergent Evolution of Urostylids and Uroleptids) hypothesis proposed by Foissner et al. (2004). Later, Foissner and Stoeck (2006, 2008) found two further taxa, Rigidothrix

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goiseri and Afrokeronopsis aurea, which not only corroborated the CEUU hypothesis but also showed that the midventral cirral pattern evolved at least two times independently among oxytrichids. In Korea, we have discovered a new key hypotrich whose morphology displays features that interconnect typical oxytrichids, such as Rubrioxytricha ferruginea or Cyrtohymena citrina, with the morphologically highly complex A. aurea having a midventral pattern. This has stimulated us to propose and statistically test an evolutionary scenario that shows how neokeronopsids could have evolved from a Rubrioxytricha-like ancestor via an Apoterritrichalike stage by cirri- and anlagen-multiplication. MATERIALS AND METHODS Sample collection and cultivation Apoterritricha lutea n. g., n. sp. was discovered in a sample of a terrestrial liverwort (Conocephalum conicum (L.)

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 61, 520–536

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Underw.) that was collected from the province of Chungcheongnam-do, South Korea (N 36°390 57″, E 126°370 09″) on 14 May 2010. After transportation of the sample to the laboratory, the collected fresh material was transferred to a Petri dish with a diam. of 8 cm and a height of 2 cm. Subsequently, it was liberally flooded with distilled water to stimulate ciliates to excyst. A few cells of the new taxon were then transferred to a new Petri dish, where they were cultured in commercial mineral water (Pulmuone Co., South Korea) and some wheat grains. Apoterritricha lutea grew well in this culture at room temperature, especially, when water and wheat grains were changed once a week. About 3 wk later, spontaneously, also resting cysts appeared in the culture. Other three Petri dishes containing A. lutea were prepared and maintained for about 6 mo to confirm the morphological distinctness of the studied species. Morphological and morphogenetic methods Living and protargol-impregnated cells were observed under a stereo microscope (Nikon SMZ800) and under an optical microscope (Axio Imager A1; Carl Zeiss, Oberkochen, Germany) at low (50–400X) and high (1,000X; immersion oil) magnifications using a DIC device. Protargol impregnation followed the protocols described by Wilbert (1975) and Foissner (1991). Counts and measurements on impregnated specimens were performed at a magnification of 1,000X. In vivo drawings are based on hand sketches of free-swimming cells, while drawings of impregnated specimens were made with the aid of a drawing device or micrographs. To demonstrate the changes occurring during morphogenesis, parental structures are depicted by contour, whereas new ones are shaded black. The images were captured using a CCD camera (Axio Cam MRc; Carl Zeiss). Terminology follows Berger (1999, 2006). DNA extraction, polymerase chain reaction amplification, and sequencing To be sure that morphological and molecular data are linked with the same species, 10 cells of A. lutea were picked up from two Petri dishes that contained specimens derived from the initial clonal culture. The collected individuals from each Petri dish were washed several times to remove contaminants and then were separately transferred to a 1.5 ml micro-tube. Genomic DNA was separately extracted from each tube using the RED Extract-NAmp Tissue PCR Kit (Sigma, St. Louis, MO), following the manufacturer’s instruction except for the reduction of each reaction volume to one-tenth (Gong et al. 2007). The universal forward and reverse eukaryotic primers EukA (50 AAC CTG GTT GAT CCT GCC AG-30 ) and EukB (50 -CAC TTG GAC GTC TTC CTA GT-30 ) (Medlin et al. 1988) were used for the amplification of the 18S rRNA gene by the means of a polymerase chain reaction (PCR), using the TaKaRa ExTaq DNA polymerase Kit (TaKaRa Bio-medicals, Otsu, Japan). PCR cycling parameters followed the

Apoterritricha lutea, a New Hypotrichous Ciliate from Korea

procedure described in Chen and Song (2001). The purified PCR product was sequenced on an ABI 3730 automatic sequencer (Macrogen Inc., Seoul, Korea), with PCR primers serving as sequencing ones. The sequence fragments were checked and assembled into contigs using the Geneious ver. 6.1.6 program created by Biomatters (available from http://www.geneious.com/). Sequences from both samples were analyzed to find polymorphisms using the DnaSP ver. 5.0 (Librado and Rozas 2009). No polymorphic sites were found between specimens from different clonal cultures. Phylogenetic analyses To determine the phylogenetic position of A. lutea, we analyzed an alignment of 18S rRNA gene sequences of 47 hypotrichous taxa. All sequences, except that of A. lutea, were downloaded from NCBI and were aligned by the computer program Mafft ver. 7.0 using the Q-INS-I algorithm that considers the secondary structure of the 18S rRNA molecule (Katoh and Toh 2008). The resulting alignment was checked by eye and masked according to the column scores calculated by G-blocks ver. 0.91b (Castresana 2000; Talavera and Castresana 2007). GTR+I+Г was the best fitted evolutionary model for phylogenetic analyses selected by jModeltest ver. 2.0.1 under the Akaike Information Criterion (Guindon and Gascuel 2003; Posada 2008). The final alignment, containing 1,660 nucleotide characters, was used to construct phylogenetic trees and neighbor-net networks as well as to perform statistical tree topology tests. Phylogenetic trees were constructed using three algorithms: Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP). Bayesian analysis was run with four MCMC chains (one cold and three heated) for 5 million generations with a sample frequency of 100 generations under the selected GTR+I+Г model, using the computer program MrBayes ver. 3.2.1 (Ronquist and Huelsenbeck 2003). The first 25% sampled trees were discarded as burn-in prior to constructing a 50% majorityrule consensus tree and calculating its branch lengths and posterior probabilities (PP). ML trees were constructed under the GTR+I+Г model in the computer program PhyML (Guindon et al. 2010). MP trees were computed in PAUP* ver. 4.0 (Swofford 2003). The reliability of the internal branches for both the ML and the MP trees was assessed by nonparametric bootstrapping with 1,000 replicates. Phylogenetic networks were computed with the neighbor-net algorithm and uncorrected distances using SplitsTree ver. 4 (Bryant and Moulton 2004; Huson 1998). Their reliability was also assessed by bootstrapping with 1,000 replicates. The approximately unbiased, the weighted Shimodaira– Hasegawa, and the weighted Kishino–Hasegawa statistical tests, as implemented in CONSEL ver. 0.1j, were carried out in order to assess significant differences in log likelihoods between the best and alternative tree topologies (Shimodaira 2002, 2008; Shimodaira and Hasegawa 2001). The per-site log likelihoods needed for these tests were


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calculated in PAUP*. Trees representing alternative hypotheses were generated in the ML framework with a heuristic search under the selected GTR+I+Γ model, with the TBR swapping algorithm and 10 random sequence addition replicates. RESULTS Morphological description of A. lutea n. g., n. sp. (Table 1; Fig. 1–4) Size in vivo spans a range of about 200–275 9 70– 105 lm, with an average of 230 9 85 lm. The body length/width ratio of living cells is about 2.7:1, becoming approximately 1.8:1 in protargol preparations due the shrinkage of body length by about 10% and the inflation of body width by about 35% (Table 1). The body shape of living specimens is usually ellipsoidal to narrowly ellipsoidal, but some cells are slightly sigmoidal (Fig. 1A, D–H,

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2B, C, 3A–C). The ventral side is flat while the dorsal side is convex; the body is dorsoventrally flattened by about 1.5:1 (Fig. 1I). Invariably there are two macronuclear nodules localized near mid-body left of cell’s main axis. The individual nodules are about 34 9 17 lm in size (Table 1), ellipsoidal to reniform, and often with an irregular outline after protargol impregnation. The nucleoli are small, globular and more or less evenly scattered over the macronuclear surface (Fig. 1A, C, 4A). There are two to five micronuclei near or attached to the macronuclear nodules. They are globular and about 9.5 lm in diam. after protargol impregnation (Fig. 1A, C, 4A–C; Table 1). The single contractile vacuole is localized slightly above mid-body near the left margin, exhibiting an anterior and a posterior collecting canal during diastole (Fig. 1A, D–I, 3D). The cortex is flexible and caries very conspicuous golden yellow to brown cortical granules that are oblong, i.e., have a size of about 1.2–1.5 9 1.5–2.0 lm in the lateral view. The cortical granules are unevenly distributed over the ventral

Table 1. Morphometric data on Apoterritricha lutea n. g., n. sp. Characteristic

Mean

M

SD

SE

CV

Min

Max

n

Body, length (in vivo) Body, length Body, width (in vivo) Body, width Body, length/width ratio (in vivo) Body, length/width ratio Macronuclear nodules, number Anterior macronuclear nodule, length Anterior macronuclear nodule, width Micronucleus, number Micronucleus, largest diameter Anterior body end to proximal end of adoral zone, distance (in vivo) Anterior body end to proximal end of adoral zone, distance Percentage of body length occupied by AZM (in vivo) Percentage of body length occupied by AZM (%) Adoral membranelles, number Left marginal cirri, number Right marginal cirri, number Frontal cirri, numbera Frontoventral cirri, number Buccal cirri, numberb Midventral cirri, number Pretransverse cirri, numberc Transverse cirri, number Caudal cirri, numberd Posterior most transverse cirrus to posterior body end, distance Dorsal and dorsomarginal kineties, total number Dorsal kineties, number Dorsomarginal kineties, number

232.3 210.9 86.6 115.7 2.7 1.8 2.0 34.5 17.7 2.7 9.5 87.4 83.9 37.7 39.9 56.8 38.2 35.8 3.0 4.0 1.0 7.8 2.0 7.5 2.8 14.6 8.9 5.8 3.2

233.0 194.0 84.0 106.0 2.7 1.8 2.0 34.0 17.0 3.0 9.6 87.0 75.0 37.3 40.1 56.0 38.0 36.0 3.0 4.0 1.0 7.0 2.0 7.0 3.0 14.0 9.0 6.0 3.0

20.4 46.1 8.9 33.2 0.2 0.2 0.0 3.7 2.6 0.8 1.3 8.2 19.7 3.3 3.9 4.6 3.4 2.7 0.2 0.2 0.2 1.2 0.2 0.6 0.4 5.6 0.7 0.4 0.4

4.1 9.6 1.8 7.2 0.0 0.1 0.0 0.8 0.5 0.2 0.3 1.6 4.1 0.7 0.8 1.0 0.8 0.6 0.0 0.0 0.0 0.3 0.0 0.1 0.1 1.4 0.2 0.1 0.1

8.8 21.9 10.3 28.7 5.8 12.5 0.0 10.7 14.9 29.1 13.6 9.4 23.4 8.7 9.7 8.1 9.0 7.7 7.2 5.4 22.3 15.7 11.2 7.9 14.0 38.1 7.9 7.0 12.7

201.0 164.0 71.0 73.0 2.4 1.5 2.0 27.0 13.0 2.0 7.4 73.0 65.0 32.3 33.4 51.0 33.0 32.0 2.0 3.0 0.0 6.0 1.0 7.0 2.0 7.0 8.0 5.0 3.0

277.0 311.0 106.0 200.0 3.0 2.2 2.0 43.0 25.0 5.0 12.0 101.0 137.0 43.8 47.2 72.0 44.0 43.0 3.0 4.0 1.0 11.0 2.0 9.0 3.0 26.0 10.0 6.0 4.0

25 23 25 21 25 21 23 23 23 22 23 25 23 25 23 22 20 21 22 22 22 21 21 23 22 15 11 11 11

Data are based on protargol-impregnated specimens (Wilbert’s method), if not stated otherwise. Measurements are in lm. AZM = adoral zone of membranelles; CV = coefficient of variation in %; Max = maximum; Mean = arithmetic mean; M = median; Min = minimum; n = number of specimens investigated; SD = standard deviation; SE = standard error of arithmetic mean. a One out of 22 specimens had only two frontal cirri. b One out of 22 specimens lacked buccal cirrus. c One out of 21 specimens had only one pretransverse cirrus. d Four out of 22 specimens had only two caudal cirri.

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Figure 1 Apoterritricha lutea n. g., n. sp., from life (A, D–J) and after protargol impregnation (B, C). A. Ventral view of a representative specimen. B. Ciliature of the ventral side of holotype specimen. Arrowhead indicates the special fiber close to the distal end of the endoral. C. Ciliature of the dorsal side and nuclear apparatus. D–H. Shape variants. I. Left lateral view of a living specimen. The contractile vacuole has two collecting canals and the cytoplasm is filled with fat globules and food vacuoles containing diatoms. J. Resting cyst. 1–9, dorsal kineties (1–6) including also dorsomarginal ones (7–9). AZM = adoral zone of membranelles; CC = caudal cirri; CV = contractile vacuole; CW = cyst wall; E = endoral; FC = frontal cirri; FVC = frontoventral cirri; LMR = left marginal row; MA = macronuclear nodules; MI = micronuclei; MVP = midventral pairs; P = paroral; PTC = pretransverse cirri; RMR = right marginal row; S = scutum; TC = transverse cirri. Scale bars, 50 lm in (A, B, C, J).

and dorsal surfaces, forming small groups near cirri, between adoral membranelles and along fibrilar associates of dorsal dikinetids (Fig. 2B–E, 3A–C, F–H, 4E). The cytoplasm is colorless and filled up with various granules,

crystals, fat globules, and food vacuoles containing bacteria, algae as well as some small ciliates (Fig. 1A, I). They glide moderately fast on the bottom of the Petri dish and slowly climb on the mud particles.


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Figure 2 Schematic drawings of Apoterritricha lutea n. g., n. sp. from life (B–D) and after protargol impregnation (A, E–J). A. Infraciliature in anterior body region. Arrowhead indicates the special fiber close to the distal end of the endoral. B, C. Cortical granulation of the ventral and dorsal side. Cortical granules are unevenly distributed over the cortex, usually forming small groups along the microtubular appendages of cirri, adoral membranelles and dorsal kineties. D. Cortical granules are oblong in lateral view. E. Microtubular fibers are lined across each dorsal kinetid and cortical granules are lined along the fibers. F. Microtubular associates of pretransverse and transverse cirri. G–I. Variability in the undulating membrane pattern. Arrowheads note the special fiber of the endoral. J. Microtubular associates of the caudal cirri. AL = anterior longitudinal fiber; AR = anterior microtubular bundle; CG = cortical granules; E = endoral; LPR = left posterior microtubular bundle; MF = microtubular fiber; RPR = right posterior microtubular bundle; P = paroral; PCMT = postciliary microtubular ribbon; PR = posterior microtubular bundle; PTC = pretransverse cirri; SSR = small sub-ectoplasmic rootlet; TR = transverse rootlet. Scale bar, 5 lm in (D).

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Figure 3 Apoterritricha lutea n. g., n. sp., interphase specimens (A–H) and resting cyst (I) from life. A–C. Ventral view showing the ellipsoidal body and the conspicuous golden yellow to brown cortex. The right side of the oral cavity is delimited by a conspicuous paroral (C). D. The single contractile vacuole is localized slightly above the mid-body near the left margin. E. Lateral view of a living specimen, showing the flat ventral side and the vaulted dorsal side. F. Detail of the oral apparatus, showing the paroral and endoral as well as the adoral zone of membranelles. The arrow denotes the transparent scutum at the anterior body end. G. Cortical granulation of the dorsal side. Arrowheads point to the clustered cortical granules. H. Lateral view showing the oblong cortical granules perpendicularly arranged under the pellicule. I. Resting cyst. AZM = adoral zone of membranelles; BL = buccal lip; CG = cortical granules; CV = contractile vacuole; CW = cyst wall; E = endoral; FC = frontal cirri; FV = food vacuole; FVC = frontoventral cirrus; LMR = left marginal row; MA = macronuclear nodule; OC = oral cavity; P = paroral; PVC = postoral ventral cirri; RMR = right marginal row; TC = transverse cirrus. Scale bars, 5 lm in (G); 10 lm in (H); 50 lm in (A, B, E, I).

The cirral pattern is usually as shown in Fig. 1B. Typically there are three 24–28 lm long frontal cirri whose bases are enlarged and each bears a shorter anterior and a longer posterior microtubular bundle. The left and middle two frontal cirri are located slightly posterior to the anterior end of adoral membranelles, while the right frontal

-cirrus is situated at the level of distal end of adoral zone of membranelles (Fig. 1B, 2A, 4A–C). The single about 23 lm long buccal cirrus, which carries only a posterior microtubular bundle, is usually positioned right of the most convex point of the paroral (Fig. 1A, 2A, 4A, B). There are four about 26 lm long frontoventral cirri arranged in a


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Figure 4 Apoterritricha lutea n. g., n. sp., infraciliature and nuclear apparatus after protargol impregnation. A, B. Ventral view showing the increased number of postoral ventral and transverse cirri. C. Detail of the anterior body portion. The undulating membranes form a Cyrtohymena-like pattern. A special fiber (arrowhead) is localized at the anterior end of the endoral. There are many pharyngeal fibers originating from the paroral and endoral. D, E, G. Ciliary pattern of the dorsal side. Asterisks mark the fragmented dorsal kinety 3. Arrows indicate microtubular appendages of dorsal dikinetids. Arrowheads show cortical granules clustered along microtubules of dorsal kineties. F. There are three caudal cirri each having an anterior microtubular bundle (arrow). BC = buccal cirrus; CC = caudal cirri; CP = cytopharynx; DK = dorsal kinety; E = endoral; F = pharyngeal fibers; FC = frontal cirrus; FVC = frontoventral cirri; LMR = left marginal row; MA = macronuclear nodules; MI = micronuclei; MVP = midventral pairs; P = paroral; PTC = pretransverse cirri; RMR = right marginal row; TC = transverse cirrus; UM = undulating membranes. Scale bars, 50 lm in (A, B, D).

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V-shaped pattern in the right of the undulating membranes, as is typical for oxytrichids. Each frontoventral cirrus is equipped with a shorter anterior and a longer posterior microtubular bundle (Fig. 2A). The number of postoral ventral (midventral) cirri is 6–11. These cirri are 18–25 lm long and form three to five pseudo-pairs, except for the last midventral cirrus that usually remains unpaired (Fig. 1B, 4A, B). Each midventral cirrus is associated with three microtubular appendages: an anterior, a right posterior, and a left posterior bundle. The anterior and the right posterior bundles are similar in length, while the left posterior one is distinctly shorter and extends obliquely leftwards (Fig. 2A). Usually, there are two about 20 lm long pretransverse cirri, each having an anterior and a posterior microtubular bundle (Fig. 1B, 2F, 4A, B). There are 7–9 transverse cirri arranged in a hook-shaped pattern. They are 28–35 lm long and their bases are distinctly enlarged and displaced anteriorly so that only the posteriormost transverse cirri slightly protrude beyond the rear body end (Fig. 1A, B, 4A, B). The distance between the posterior most transverse cirrus and posterior body end spans a range of 7–26 lm (Table 1). Each transverse cirrus bears a strongly developed anterior bundle and a very fine transverse fiber (Fig. 2F). The right marginal row is composed of an average of 36 cirri that are 15–20 lm long; commences near the level of the first frontoventral cirrus and posteriorly is distinctly separated from the left row (Fig. 1B, 4A, B; Table 1). Each right marginal cirrus carries an anterior longitudinal fiber, a small sub-ectoplasmic rootlet, and a postciliary microtubular ribbon (Fig. 2A). The left marginal row begins near the level of the buccal vertex and usually consists of 38 cirri, bearing a transverse rootlet in addition to the three microtubular appendages associated also with the right marginal cirri (Fig. 1B, 2A, 4A, B; Table 1). The left marginal cirri are about 17–20 lm long. The dorsal bristles are 3–4 lm long in vivo. The dorsal ciliature consists of an average of six dorsal (labeled as dorsal rows 1–6) and three dorsomarginal (labeled as dorsal rows 7–9) kineties (Fig. 1C, 4D; Table 1). According to the ontogenetical data, dorsal kineties 4–6 are very likely the remainder of the multiple fragmentation of dorsal kinety 3 (Fig. 1C, 4D, G, asterisks). Dikinetids of the dorsal kineties are associated with long sigmoidal fibers extending obliquely anteriorly and posteriorly (Fig. 2E, 4G). Usually there are three caudal cirri that are 21–24 lm long, each one situated at the posterior end of dorsal kineties 1, 2 and 6 (Fig. 1C, 4D, F). Each caudal cirrus bears a longer anterior and a shorter posterior bundle (Fig. 2J), and hence are clearly distinguished from marginal cirri. The oral apparatus is basically in a Cyrtohymena-like pattern and occupies about 40% of body length on average. The adoral zone usually consists of 56 membranelles of a typical oxytrichid structure (Table 1). The anterior end of cell is roofed by a transparent scutum that is about 5–7 lm long in vivo (Fig. 3F, arrow). The adoral membranelles have three fibrilar associates: (i) a short bundle at the medial end of the membranelles; (ii) a long submembranellar bundle extending from the anteriormost membranelles to the


proximal half of the adoral zone; and (iii) a fine straight fiber in between membranelles; on the left margin of the adoral zone, these fine fibers curve posteriorward to assemble into a conspicuous bundle that traverses the left side of the adoral zone to sink into the cytopharynx (Fig. 2A). The buccal cavity is of ordinary width and depth. The buccal lip overlaps half of the paroral and covers the proximal end of the adoral zone of membranelles (Fig. 3B). Both endoral and paroral have a similar length and optically intersect in the posterior half of the buccal cavity. The paroral is distinctly curved, especially in its anterior half as shown in Fig. 2A. The endoral is less distinctly curved (Fig. 1B, 2A, 4A–C), and its anterior end is constantly associated with a special comparatively thick and short fiber that extends obliquely backwards (Fig. 1B, 2A, G–I, arrowheads). The right wall of the buccal cavity is lined by paroral fibers, while the bottom of the cavity by endoral ones. Both the paroral and endoral fibers form bundles that posteriorly assemble to cytopharyngeal fibers running obliquely rightwards deep into the cytoplasm (Fig. 2A). The resting cysts are spherical with a diam. of about 80 lm in vivo. They are golden yellow to brownish at higher magnifications. The cyst wall is laminated and approximately 3 lm thick. There are two oblong macronuclear nodules in the cyst center. The cytoplasm is studded with many small granules and some fat globules (Fig. 1J, 3I). Late dividing stage of A. lutea (Fig. 5) We found only one late divider in the protargol preparations (Fig. 5A–D). Development of the new oral apparatus and ventral cirral pattern is almost complete at this stage except for the formation of paroral and endoral which are still arranged in parallel. The three parental frontal and two frontoventral cirri are still present in the proter, while the parental pretransverse and transverse cirri are still present in the opisthe. In the proter, new marginal rows develop at the anterior end of the parental marginal rows, while in the opisthe they are formed within the parental rows stretching between the old marginal cirri and the new adoral zone of membranelles (Fig. 5A, C). Formation of the dorsal ciliature basically follows the processes described in Cyrtohymena (Cyrtohymenides) shii (Singh et al. 2013). Specifically, (i) the new dorsal kinety 3 is multiple fragmented; (ii) the new caudal cirri are formed at the posterior end of dorsal kineties 1, 2, and 6, respectively; and (iii) the new dorsomarginal rows develop at the right anterior margin of the proter and the opisthe. The parental dorsal kineties become segregated by the newly formed ones and are very likely resorbed later (Fig. 5A–D). The nuclear apparatus divides as typical for hypotrichs having two macronuclear nodules. Thus, the nodules fuse into a mass that splits into an elongated structure both in the proter and the opisthe. Later, in each daughter cell, this mass divides into two macronuclear nodules. Daughter micronuclei are broadly fusiform and still connected at this late stage (Fig. 5B, C).


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Figure 5 Apoterritricha lutea n. g., n. sp., a late divider after protargol impregnation. A, C. Ventral view showing the organization of new and parental ciliature in the proter and opisthe. The endoral and paroral are not completely separated at this stage. Arrows indicate new dorsomarginal kineties. B. Dorsal view showing the dorsal kineties and the origin of caudal cirri. Arrowheads indicate the parental dorsal dikinetids. D. Dorsal view of the proter to show the caudal cirri (arrowheads) and fragmentation of dorsal kinety 3. AZM = adoral zone of membranelles; BC = buccal cirrus; CC = caudal cirri; E = endoral; FC = frontal cirri; FVC = frontoventral cirri; MA = macronuclear nodules; MI = micronuclei; LMR = left marginal row; MVP = midventral pairs; P = paroral; PTC = pretransverse cirri; RMR = right marginal row; TC = transverse cirrus. Scale bars, 100 lm in (B, C).

Phylogenetic analyses (Table 2; Fig. 6, 7) All phylogenetic analyses (BI, ML, MP) depicted the family Oxytrichidae as a paraphyletic assemblage that contains members of the subfamilies Oxytrichinae and Stylonychinae as well as of the families Kahliellidae, Neokeronopsidae, Rigidotrichidae, and Uroleptidae. Apoterritricha lutea was classified within a comparatively well supported cluster (designated here as clade A), containing R. ferruginea, Ponturostyla enigmatica, several C. citrina isolates, C. (Cyrtohymenides) shii, Paraurostyla weissei, Paraurosomoida indiensis, Notohymena apoaustralis, and A. aurea (81% ML/1.00 PP/70% MP). Within this clade, A. lutea clustered together with two C. citrina isolates (GenBank accession numbers AF508755 and AF164135), C. (Cyrtohymenides) shii and A. aurea with strong (95% ML/1.00 PP) to

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moderate (84% MP) support. However, phylogenetic interrelationships of these five taxa were only very poorly resolved (Fig. 6). Statistical tree topology tests firmly rejected a close relationship between A. lutea and R. goiseri, another taxon with midventral pattern. Likewise, the monophyletic origin of A. lutea and all other “oxytrichids” with midventral pattern (i.e., Uroleptus + Rigidotrix + Pattersoniella + Apoterritricha + Afrokeronopsis) is consistently excluded by all statistical tests at the significance level of 0.001 (Table 2). This indicates that the midventral organization independently evolved at least four times, viz., in the Oxytrichidae (Pattersoniella), Uroleptidae (Uroleptus), Rigidotrichidae (Rigidothrix), and Neokeronopsidae (Apoterritricha and Afrokeronopsis). The same applies also to the paraurostylid ventral pattern that has formed at least three times



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Table 2. Log likelihoods and p-values of AU (approximately unbiased), WSH (weighted Shimodaira–Hasegawa), and WKH (weighted Kishino– Hasegawa) tests for tree comparisons considering different topological scenarios

Topology Best maximum likelihood tree (unconstrained) Monophyly of Rigidothrix and Apoterritricha Monophyly of Afrokeronopsis and Pattersoniella Monophyly of “oxytrichids” with midventral patternb Monophyly of Paraurostyla weissei and P. viridis Monophyly of P. weissei and Ponturostyla enigmatica Monophyly of P. weissei, Apoterritricha and Afrokeronopsis Monophyly of P. enigmatica, P. weissei, Apoterritricha and Afrokeronopsis Monophyly of the Cyrtohymena citrina isolates Basal position of Rubrioxytricha within the clade A and sister relationship of Afrokeronopsis and Apoterritricha

Log likelihood (ln L)

D (ln L)a

7204.6021 7325.9939 7300.9629 7417.1915 7353.1006 7228.7621 7295.4168

AU

WSH

WKH

Conclusion

– 121.39 96.36 212.59 148.50 24.16 90.81

0.991 2e–33 0.001 2e–51 1e–05 0.014 2e–04

1.000 0.000 2e–04 0.000 0.000 0.098 0.000

0.858 0.000 0.000 0.000 0.000 0.020 0.000

– Rejected Rejected Rejected Rejected Rejected Rejected

7312.1766

107.57

4e–04

0.000

0.000

Rejected

7297.9077 7210.7158

93.31 6.11

5e–06 0.027

0.002 0.571

3e–04 0.142

Rejected Not rejected

Significant differences (p < 0.05) between the best unconstrained and constrained topologies are in bold. Difference between log likelihoods of constrained and best (unconstrained) tree. b Excluding urostylids, i.e., monophyly of Uroleptus + Rigidothrix + Pattersoniella + Apoterritricha + Afrokeronopsis. a

convergently, as the monophyletic origin of P. weissei and Paraurostyla viridis as well as a sister relationship between P. weissei and P. enigmatica are rejected by all statistical tests at the significance level of 0.001 (Table 2). Although the basal position of R. ferruginea within the clade A and a sister relationship between A. lutea and A. aurea are not depicted in phylogenetic trees, they cannot be excluded according to the two statistical tree topology tests (pvalue >> 0.05 for the WKH and WSH test). Concerning the monophyly of the C. citrina isolates, it is consistently rejected. As two C. citrina isolates (GenBank accession numbers AF508755 and AF164135) are genetically highly similar to C. (Cyrtohymenides) shii (Fig. 6, 7), they are very likely misidentifications. On the other hand, due to the absence of morphological data on these two isolates, we also cannot exclude that they are a cryptic species, strongly resembling C. citrina. As any of these C. citrina sequences came from the type population, this problem cannot be solved unambiguously. Phylogenetic network analyses (Fig. 7) reveal much more complex and conflicting relationships among members of the subfamily Oxytrichinae as well as among the families Kahliellidae, Rigidotrichidae and Uroleptidae than shown in phylogenetic trees. On the other hand, the monophyletic origin of the lineage containing Plagiotoma lumbrici, Urosomoida longa, the subfamily Stylonychinae and the clade A is strongly supported with a distinct set of parallel edges and a bootstrap value of 99%. The monophyly of the subfamily Stylonychinae is strongly corroborated by a distinct set of comparatively long parallel edges and a bootstrap value of 92%, while the monophyly of the clade A is sustained only by comparatively short parallel edges and a bootstrap value of 57%. Rubrioxytricha ferruginea together with P. enigmatica branch off first (93% BS) within the clade A. As in phylogenetic trees, A. lutea, two C. citrina isolates (GenBank accession numbers

AF508755 and AF164135), C. (Cyrtohymenides) shii and A. aurea form a comparatively long and strongly supported split (97% BS). However, phylogenetic relationships among those taxa cannot be unambiguously resolved as indicated by several short parallel edges linking A. lutea + C. citrina isolates with A. aurea (Fig. 7). DISCUSSION Comparison of Apoterritricha with similar genera Morphologically, Apoterritricha is most similar to Territricha in having three frontal cirri, one buccal cirrus, four frontoventral cirri, more than three postoral ventral cirri forming a midventral pattern, two pretransverse cirri, more than five transverse cirri, and multiple fragmentation of kinety 3. However, Apoterritricha differs from Territricha by the pattern of the undulating membranes (Cyrtohymena-like vs. Oxytricha-like) (Berger 1999; Berger and Foissner 1988). Due to the absence of sequence data for Territricha, it is not known whether Apoterritricha and Territricha are really closely related and whether their morphologies evolved convergently or not. This is indicated by classification of Territricha within the family Rigidotrichidae by Foissner and Stoeck (2006), while the monophyletic origin of Apoterritricha and Rigidothrix is firmly rejected by all statistical tree topology tests (Table 2). Moreover, Rigidothrix lacks dorsal kinety 3 fragmentation (Foissner and Stoeck 2006) and therefore a close relationship with Territricha and Apoterritricha is very unlikely. Apoterritricha displays some features that occur in Cyrtohymena, including the subgenus Cyrtohymenides, and also some traits that are properties of Neokeronopsis and Afrokeronopsis. Specifically, Apoterritricha and Cyrtohymena share the Cyrtohymena-like oral apparatus, three frontal cirri, one buccal cirrus, and four frontoventral cirri arranged


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Figure 6 Maximum likelihood phylogenetic tree based on 1,660 nucleotide characters from 45 hypotrichous taxa. The tree was constructed under the GTR+I+Γ evolutionary model and the gamma shape parameter at 0.5120, the proportion of invariable sites at 0.6520, and a rate matrix for the model as suggested by jModeltest. The nodal support is indicated as follows: maximum likelihood (ML) bootstraps/Bayesian inference posterior probabilities (PP)/maximum parsimony (MP) bootstraps. A dash indicates posterior probabilities below 0.50 and/or MP bootstraps below 50%. The scale bar indicates the fraction of substitutions per site. IS, incertae sedis.

in a V-shaped pattern (Berger 1999; Foissner 2004; Singh et al. 2013). However, these are very likely rather old plesiomorphies and therefore cannot be used to estimate phylogenetic relationships (Berger 2008). On the other hand, Apoterritricha, the subgenus Cyrtohymenides, Neokeronopsis and Afrokeronopsis share an important apomorphic feature, i.e., the multiple fragmentation of dorsal

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kinety 3 (Foissner 2004; Foissner and Stoeck 2008; Foissner et al. 2010; Singh et al. 2013; present study). However, Apoterritricha is distinguished from Cyrtohymena and subgen. Cyrtohymenides by the higher number of postoral ventral (6–11 vs. invariably 3) and transverse (7–9 vs. 5, rarely 4) cirri. Moreover, the postoral ventral cirri form a midventral complex in Apoterritricha, while they


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Figure 7 Phylogenetic network based on 1,660 nucleotide characters of 45 hypotrichous taxa. The split graph was constructed using the neighbor-net algorithm and the uncorrected distances. Numbers along edges are bootstrap values coming from 1,000 replicates. Values < 50% are not shown. The scale bar indicates the fraction of substitutions per site. IS = incertae sedis.

form a dense cluster behind the buccal vertex in Cyrtohymena and subgen. Cyrtohymenides. On the other hand, the midventral pattern is a common feature of Apoterritricha, Neokeronopsis and Afrokeronopsis. However, the former has one buccal and three frontal cirri, while the two latter genera exhibit a distinct corona of frontal and pseudobuccal cirri (Foissner and Stoeck 2008; Foissner et al. 2010; Wang et al. 2007; Warren et al. 2002).

Comparison of A. lutea with similar species Morphologically, A. lutea is most similar to Territricha stramenticola as originally described by Berger and Foissner (1988). However, A. lutea differs from T. stramenticola by (i) the undulating membrane pattern (Cyrtohymena-like vs. Oxytricha-like); (ii) the number of the adoral membranelles (51–72 vs. 39–50); (iii) the color of the cortical


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granules (yellow vs. colorless); (iv) the length:width ratio of the cortical granules (about 1.5:1 vs. about 3:1); (v) the position of transverse cirri (situated more posteriorly in A. lutea); and (vi) the length of the dorsal kinety 1 bristles (3–4 lm vs. up to 7 lm long). According to the present molecular phylogenies, A. lutea is most closely related to A. aurea on one hand and to C. citrina and C. (Cyrtohymenides) shii on the other one. Interestingly, all these species share several conspicuous morphological traits such as golden yellow to brown cortical granules and the Cyrtohymena-like oral apparatus. However, they are distinguished by the cirral pattern. Specifically, A. aurea has a distinct corona of frontal and pseudobuccal cirri, while A. lutea possesses one buccal and three frontal cirri. Cyrtohymena citrina and C. (Cyrtohymenides) shii display the typical oxytrichid 18 frontoventral-transverse cirral organization (Berger 1999; Singh et al. 2013), while A. lutea exhibits the midventral complex. Apoterritricha, a putative missing link between Cyrtohymena and Afrokeronopsis (Fig. 8) Although Afrokeronopsis clusters within the oxytrichid clade, its general appearance is very dissimilar from that of typical “18-cirri oxytrichids” (cp. Berger 1999 with Foissner and Stoeck 2008; Foissner et al. 2010; Wang et al. 2007; Warren et al. 2002). In the present study, we have discovered a new hypotrich, A. lutea, which very well interconnects typical oxytrichids, such as R. ferruginea, C. citrina or C. (Cyrtohymenides) shii, with morphologically highly complex A. aurea. This has stimulated us to propose phylogenetic relationships as shown in the cladogram on Fig. 8. To test the reliability of this evolutionary scenario, we constructed an ML tree in which R. ferruginea is basal within the clade A, and A. lutea and A. aurea form a monophylum. This constrained tree (ln L = 7210.7158) differs from the best unconstrained tree (ln L = 7204.6021; phylogenetic relationships as shown in Fig. 6) by a log likelihood of only 6.11, which is statistically not a significant difference according to the WSH and WKH topology tests. On the other hand, this evolutionary scenario is rejected by the AU test but only at the significance level of 0.05 (Table 2). Therefore, we hypothesize that Apoterritricha could have evolved from a Cyrtohymena-like ancestor by cirri-multiplication in correlation with the production of a midventral complex. Afrokeronopsis might have evolved from an Apoterritricha-like progenitor by anlagen-multiplication and further increase in the number of frontal, buccal, midventral and transverse cirri. In Afrokeronopsis, these multiplication processes were associated with the formation of (i) a frontal and a pseudobuccal corona, (ii) a very distinct midventral pattern, as well as of (iii) a long, J-shaped transverse cirral row almost reaching the buccal vertex. The present scenario is basically in agreement with Berger’s (2006) explanation of the classification of Neokeronopsis/Afrokeronopsis within the oxytrichids. Further, he already assumed a close relationship of Cyrtohymena and Neokeronopsis on the basis

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of the undulating membrane pattern and the colored cortical granules. Redefinition of the family Neokeronopsidae Foissner and Stoeck (2008) and Foissner et al. (2010) defined the family Neokeronopsidae, in particular by the cirral pattern and by the details of the dorsal kinety 3 fragmentation. Originally, three genus-group nominal taxa were assigned to the Neokeronopsidae: Neokeronopsis Warren et al. 2002; Neokeronopsis (Afrokeronopsis) Foissner and Stoeck 2008 (raised to generic level by Foissner et al. (2010)), and Pattersoniella Foissner, 1987. However, the present phylogenetic trees and neighbornet networks do not support classification of Pattersoniella within the Neokeronopsidae (Fig. 6, 7). Moreover, the present statistical tree topology tests firmly reject classification of Pattersoniella within the Neokeronopsidae even at the significance level of 0.001 (Table 2). Therefore, we suggest excluding Pattersoniella from this family and transferring it to the subfamily Stylonychinae, as already proposed by Berger (1999). On the other hand, the present phylogenetic analyses clearly show that several other taxa, viz., R. ferruginea, C. citrina isolates, C. (Cyrtohymenides) shii, N. apoaustralis, A. lutea, P. indiensis, P. enigmatica, and P. weissei, from a distinct monophylum with A. aurea (Fig. 6, 7). Although all these taxa exhibit comparatively different cirral patterns, they have similar 18S rRNA gene sequences and share colored cortical granules (Berger 1999; Foissner and Stoeck 2008; Foissner et al. 2010; Kim et al. 2013; Lv et al. 2013; Singh and Kamra 2013). On the basis of this evidence, we believe that they are closely related to the family Neokeronopsidae and the golden yellow to brown cortical granules are the most important morphological apomorphy of all these taxa. Morphological homoplasies within the clade A Various comparatively different cirral patterns are found within the clade A. However, this is nothing exceptional as a variety of cirral organizations also occur within the subfamilies Stylonychinae and Oxytrichinae (Berger 1999). Furthermore, all these patterns can be easily derived from an Oxytricha-like ancestor by cirri- and/or anlagen-multiplication, which may or may not be associated with the formation of a midventral complex (Berger 2006). In Apoterritricha and Afrokeronopsis, the cirri- and/or anlagen-multiplication is connected with the production of the midventral pattern (Foissner and Stoeck 2008; Foissner et al. 2010; Wang et al. 2007; Warren et al. 2002), while in P. weissei and P. enigmatica it is not (Song 2001; Wirnsberger et al. 1985). According to the present phylogenetic trees and statistical topology tests (Fig. 6; Table 2), the cirri- and/or anlagen-multiplication occurred at least three times independently within the clade A, viz., in P. enigmatica, P. weissei, and A. lutea + A. aurea. Moreover, phylogenetic trees and networks as well as topology tests show that the paraurostylid cirral pattern evolved also at


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Figure 8 Hypothesis for the evolution of Afrokeronopsis from a Rubrioxytricha-like ancestor via a Cyrtohymena- and Apoterritricha-like stage. Morphological apomorphies are shown above branches and come from diagnosis of the taxa under discussion. Asterisk denotes a possible convergence in the number of dorsal kineties increased to six between Cyrtohymena and many Stylonychinae. On the other hand, this pattern could be also a plesiomorphy of the clade A, as the number of dorsal kineties was reduced from six to four in Rubrioxytricha due to the loss of dorsal kinety 3 fragmentation. Question mark indicates a lack of distinct morphological apomorphies, at the present state of knowledge. BC = buccal cirrus; CC = caudal cirri; DK = dorsal kineties; FC = frontal cirri; FVC = frontoventral cirri; MVP = midventral pairs; MVR = midventral rows; PTC = pretransverse cirri; TC = transverse cirri.

least two times convergently within the oxytrichid clade, as P. weissei and P. viridis do not form a monophylum (Fig. 6, 7; Table 2). However, P. viridis is very likely an “illdefined” species and there is no information on morphology

of the population used for molecular studies. Possibly, it is a misidentified Oxytricha or a new oxytrichid genus. Dorsal morphogenesis seems to be also quite variable within the clade A. The dorsal kinety 3 is simply


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fragmented in the typical Cyrtohymena species (Berger 1999; Singh et al. 2013; Song 2004), in P. weissei (Wirnsberger et al. 1985) and P. enigmatica (Song 2001), while multiple fragmented in Apoterritricha (the present study), Neokeronopsis, Afrokeronopsis (Foissner and Stoeck 2008; Foissner et al. 2010), and in Cyrtohymena (Cyrtohymenides) (Foissner 2004; Singh et al. 2013). On the other hand, dorsal morphogenesis was simplified, i.e., the dorsal kinety 3 fragmentation was lost in P. indiensis (Singh and Kamra 2013) and at least in two Rubrioxytricha species, viz., in R. haematoplasma (Blatterer and Foissner 1990) and R. indica (Naqvi et al. 2006). Nonfragmenting dorsal kinety 3 was also found in some other members of the oxytrichid clade, for instance, in Parasterkiella thompsoni from the subfamily €ppers et al. 2011) and in the genus Stylonychinae (Ku Urosomoida which was therefore excluded from the subfamily Oxytrichinae (Berger 2008). This indicates that specialities of dorsal morphogenesis should be also taken with caution, when used to infer phylogenetic relationships within the oxytrichid clade. TAXONOMIC SUMMARY Neokeronopsidae Foissner and Stoeck 2008 Improved diagnosis. Semirigid or flexible Dorsomarginalia with colored cortical granules. Three to many frontal cirri, one to many buccal and pseudobuccal cirri, three to many postoral ventral cirri forming midventral pattern, usually two pretransverse cirri, and four to many transverse cirri. Oral apparatus in Oxytricha- or Cyrtohymena-like pattern, with or without buccal depression. Dorsal ciliature composed of one or several dorsomarginal kineties and three dorsal kineties, of which row 3 usually produces one or several kineties by multiple fragmentation; caudal cirri present. Type genus. Neokeronopsis Warren, Fyda and Song, 2002. Taxa assignable. Afrokeronopsis Foissner and Stoeck 2008; Apoterritricha gen. n.; Neokeronopsis Warren et al. 2002. Apoterritricha gen. n. Diagnosis. Neokeronopsidae with three frontal cirri, one buccal cirrus, four frontoventral cirri, more than three postoral ventral cirri forming a midventral pattern, two pretransverse cirri, and more than five transverse cirri. Oral apparatus Cyrtohymena-like, without buccal depression but with a special fiber positioned near the anterior end of the endoral. Dorsal kinety 3 multiple fragmented; dorsomarginal kineties and caudal cirri present. Type species. Apoterritricha lutea sp. n. Etymology. Composite of the Greek prefix apo- (derived from) and the generic name Territricha meaning Territricha-like ciliate. Feminine gender.

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Apoterritricha lutea sp. n. Diagnosis. Size about 230 9 85 lm in vivo. Body ellipsoidal to narrowly ellipsoidal, rarely slightly sigmoidal. Two macronuclear nodules and two to five micronuclei. Cortical granules golden yellow to brown, oblong, and unevenly distributed over ventral and dorsal side, forming small groups near cirri, between adoral membranelles, and along microtubular associates of dorsal dikinetids. Typically three frontal cirri, one buccal cirrus, four frontoventral cirri, seven postoral ventral cirri, two pretransverse cirri, seven transverse cirri, ca. 38 left marginal and 36 right marginal cirri. Adoral zone composed of an average of 56 membranelles occupying 40% of body length. On average six dorsal and three dorsomarginal kineties, and three caudal cirri. Type locality. A terrestrial liverwort from the Sudeoksa Temple, Sacheon-li, Deoksan-myon, Yesan-gun, Chungcheongnam-do Province, South Korea (N 36°390 57″ E 126°370 09″). Type material. One holotype slide and two paratype slides with protargol-impregnated specimens have been deposited in the Natural Institute of Biological Resources (NIBR), Incheon, South Korea and Department of Biology, University of Ulsan, Ulsan, South Korea with registration numbers NIBRIV0000103954, KJH-20131015-05-01, and KJH-20131015-05-02. Holotype specimen (Fig. 1B) and relevant paratype specimens are marked by black ink circles on the coverslip. Etymology. The Latin adjective luteus, -a, -um [m, f, n] (yellow) refers to the color of the cortical granules. Gene sequence. The 18S rRNA gene sequence has been deposited in GenBank with the registration number KJ619458. The sequence is 1661 nucleotides long and has a GC content of 45.33%. ACKNOWLEDGMENTS We are very grateful to Associate Editor and two anonymous reviewers for their valuable suggestions and thoughtful comments. Financial support was provided by the National Institute of Biological Resources (NIBR) of Ministry of Environment, Korea (project 1834-302 and Indigenous Species Discovery project 2010), the National Research Foundation of Korea funded by the Korean government (2012R1A1A2005751) and by a grant from the Slovak Scientific Grant Agency VEGA (Project no. 1/0248/13). LITERATURE CITED Berger, H. 1999. Monograph of the Oxytrichidae (Ciliophora, Hypotrichia). Monogr. Biol., 78:1–1080. Berger, H. 2006. Monograph of the Urostyloidea (Ciliophora, Hypotricha). Monogr. Biol., 85:1–1304. Berger, H. 2008. Monograph of the Amphisiellidae and Trachelostylidae (Ciliophora, Hypotricha). Monogr. Biol., 88:1– 737. Berger, H. 2011. Monograph of the Gonostomatidae and Kahliellidae (Ciliophora, Hypotricha). Monogr. Biol., 90:1–741.


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Morphology, morphogenesis and molecular phylogeny of a new marine ciliate, Trichototaxis marina n. sp. (Ciliophora, Urostylida).

Morphology and phylogeny of Bryophryoides ocellatus n. g., n. sp. (Ciliophora, Colpodea) from in situ soil percolates of Idaho, U.S.A.

Morphology, morphogenesis, and molecular phylogeny of Paraparentocirrus sibillinensis n. gen., n. sp., a "Stylonychine Oxytrichidae" (Ciliophora, Hypotrichida) without transverse cirri.

Morphology and Molecular Phylogeny of Pseudocyrtohymena koreana n. g., n. sp. and Antarctic Neokeronopsis asiatica Foissner et al., 2010 (Ciliophora, Sporadotrichida), with a Brief Discussion of the Cyrtohymena Undulating Membranes Pattern.

Morphology and Molecular Phylogeny of Three Cyrtophorid Ciliates (Protozoa, Ciliophora) from China, Including Two New Species, Chilodonella parauncinata sp. n. and Chlamydonella irregularis sp. n.

Morphology, morphogenesis, and molecular phylogeny of Sterkiella tetracirrata n. sp. (Ciliophora, Oxytrichidae), from the Silent Valley National Park, India.

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

Morphology and Phylogeny of Three Pleuronema Species (Ciliophora, Scuticociliatia) from Hangzhou Bay, China, with Description of Two New Species, P. binucleatum n. sp. and P. parawiackowskii n. sp.

Morphology and molecular phylogeny of an Indian population of Cyrtohymena citrina (Ciliophora, Hypotricha), including remarks on ontogenesis of Urosomoida-Notohymena-Cyrtohymena group.

Morphology and phylogenetic analysis of two oxytrichid soil ciliates from China, Oxytricha paragranulifera n. sp. and Oxytricha granulifera Foissner and Adam, 1983 (Protista, Ciliophora, Hypotrichia).

Morphology, morphogenesis and molecular phylogeny of a novel soil ciliate, Pseudouroleptus plestiensis n. sp. (Ciliophora, Oxytrichidae), from the uplands of Colfiorito, Italy.

The morphology of three Loxophyllum species (Ciliophora, Pleurostomatida) from southern China, L. lembum sp. n., L. vesiculosum sp. n. and L. perihoplophorum Buddenbrock, 1920, with notes on the molecular phylogeny of Loxophyllum.

Muscular sarcocystosis in two arctic foxes (Vulpes lagopus) due to Sarcocystis arctica n. sp.: sarcocyst morphology, molecular characteristics and phylogeny.

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 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).

Bozasella gracilis n. sp. (Ciliophora, Entodiniomorphida) from Asian elephant and phylogenetic analysis of entodiniomorphids and vestibuliferids.

Morphology, ontogenesis and molecular phylogeny of Platynematum salinarum nov. spec., a new scuticociliate (Ciliophora, Scuticociliatia) from a solar saltern.

Morphology and phylogeny of two species of Loxodes (Ciliophora, Karyorelictea), with description of a new subspecies, Loxodes striatus orientalis subsp. n.

Morphology, morphogenesis and molecular phylogeny of a soil ciliate, Pseudouroleptus caudatus caudatus Hemberger, 1985 (Ciliophora, Hypotricha), from Lhalu Wetland, Tibet.

Morphology and morphogenesis of a novel mangrove ciliate, Sterkiella subtropica sp. nov. (Protozoa, Ciliophora, Hypotrichia), with phylogenetic analyses based on small-subunit rDNA sequence data.

Morphology and molecular phylogeny of three marine Condylostoma species from China, including two new ones (Ciliophora, Heterotrichea).

Euduboscquella costata n. sp. (Dinoflagellata, Syndinea), an Intracellular Parasite of the Ciliate Schmidingerella arcuata: Morphology, Molecular Phylogeny, Life Cycle, Prevalence, and Infection Intensity.

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

Gymnodinium smaydae n. sp., a new planktonic phototrophic dinoflagellate from the coastal waters of Western Korea: morphology and molecular characterization.