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.

Published by the International Society of Protistologists

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Eukaryotic Microbiology

Journal of Eukaryotic Microbiology ISSN 1066-5234

ORIGINAL ARTICLE

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 Jae-Ho Jung1, Kyung-Min Park & Gi-Sik Min Department of Biological Sciences, Inha University, Incheon, 402-751, South Korea

Keywords Brackish water ciliate; King George Island; new genus; South Korea. Correspondence G.-S. Min, Department of Biological Sciences, Inha University, Incheon 402-751, South Korea Telephone number: +82-32-860-7692; FAX number: +82-32-874-6737; e-mail: [email protected] Received: 22 December 2013; revised 18 August 2014; accepted August 18, 2014. doi:10.1111/jeu.12179

ABSTRACT We discovered a new brackish water oxytrichid Pseudocyrtohymena koreana n. g., n. sp. in South Korea and investigated the new species on the basis of morphology, ontogenesis, and 18S rRNA gene sequences. The new genus has the 18 frontal-ventral-transverse cirri of typical oxytrichids with flexible body, cortical granules, Cyrtohymena undulating membranes (UM), and one left and one right marginal cirral row. Ontogenesis of the new species indicated that dorsal kinety anlage 3 stretches within the parental row without any fragmentations (Urosomoida pattern) and exclusively forms all caudal cirri. The new genus is morphologically similar to Cyrtohymena Foissner, 1989, but has the following distinctive features: (i) caudal cirri absent in dorsal kineties 1 and 2 (vs. present in Cyrtohymena); and (ii) dorsal kinety 3 nonfragmented (vs. fragmented in Cyrtohymena). Further, we collected an additional species Neokeronopsis asiatica Foissner et al. 2010, from King George Island, Antarctica, and the species shares the morphology of UM with Cyrtohymena. Herein, we describe the previously unidentified characteristics of N. asiatica (i.e., cortical granules, body flexibility, contractile vacuole, and 18S rRNA gene sequence). In addition, we obtained two 18S rRNA gene sequences from Cyrtohymena muscorum and Parasterkiella thompsoni to expand samples for phylogenetic analysis. Our 18S rRNA gene tree supports the hypothesis that the Cyrtohymena UM pattern might have evolved several times in hypotrichs (e.g., Neokeronopsidae, Oxytrichinae, and Stylonychinae).

OXYTRICHIDS represent a highly evolved and diverse group of Ciliophora (for a review, see Berger 1999). Oxytricha Bory de Saint-Vincent in Lamouroux, Bory de SaintVincent and Deslongchamps, 1824—one of the oldest taxa in Ciliophora—is the type genus of Oxytrichidae Ehrenberg, 1838, and the melting pot for the typical flexible 18-cirri hypotrichs with caudal cirri (Berger 1999; Shao et al. 2011). However, because of their morphological convergent evolution, the phylogenetic relationships of oxytrichids remain to be elucidated. Thus far, several hypotheses and revisions have been proposed (Berger 1999, 2006, 2011; Foissner et al. 2004; Shao et al. 2011). Of these hypotheses, the convergent evolution of urostylids and uroleptids hypothesis (CEUU) proposed by Foiss-

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ner et al. (2004) suggests that the evolution of hypotrichs is characterized by numerous homoplasies and emphasizes the need for more detailed evolutionary evaluation, including sufficient morphological, ontogenetic, and molecular studies (Foissner and Stoeck 2006, 2008). The CEUU hypothesis holds true for Neokeronopsis Warren et al. 2002; which was previously assigned to urostylids. Neokeronopsis has the typical zigzag pattern of ventral cirri found in urostylids; however, all congeners in Neokeronopsis possess the morphological features of typical oxytrichids, that is, fragmented dorsal kinety and dorsomarginal kineties (Foissner et al. 2010; Warren et al. 2002). In addition, the oral apparatus—the Cyrtohymena undulating membranes (UM) pattern—was previously considered to

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 280–297

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be an apomorphic character of Cyrtohymena (for a review, see Berger 1999). However, Berger (2011) established Rigidohymena in Stylonychinae and denoted that the oral apparatus might have evolved twice or have existed in the most recent common ancestor. Neokeronopsidae Foissner and Stoeck 2008 has recently been established. The genera assigned to Neokeronopsidae are as follows: Neokeronopsis Warren et al. 2002; Afrokeronopsis Foissner and Stoeck 2008; and Pattersoniella Foissner, 1987. The type genus of Neokeronopsidae, Neokeronopsis, is an interesting taxon because having the dorsal kinety fragmentation of typical oxytrichids and the zigzag midventral cirral pattern of typical urostylids (Foissner et al. 2010; Wang et al. 2007; Warren et al. 2002). Foissner et al. (2010) provided a detailed description of Neokeronopsis asiatica on the basis of protargol-impregnated specimens. However, the live observation of N. asiatica has not yet been reported. Further, genetic materials are unavailable. Herein, we discuss the phylogenetic relationships of Neokeronopsis with Pseudocyrtohymena and other hypotrichs having the Cyrtohymena UM pattern on the basis of 18S rDNA sequence similarity. We investigated the new species on the basis of morphology, ontogeny, and molecular phylogeny. Further, we describe N. asiatica on the basis of previously unidentified characteristics by using live observation and 18S rRNA gene phylogeny. To expand the number of taxon samples for our 18S rDNA gene tree, we collected two additional oxytrichids—Cyrtohymena muscorum (from South Korea) and Parasterkiella thompsoni (from King George Island). MATERIALS AND METHODS Sample collection and identification We collected four benthic ciliates—two species from South Korea (C. muscorum (Kahl 1932) Foissner, 1989, Pseudocyrtohymena koreana n. g., n. sp.) and two species from King George Island, Antarctica (N. asiatica Foissner €ppers et al. 2010 and P. thompsoni (Foissner, 1996) Ku et al. 2011). Detailed information regarding sample collection is shown in Table 1. Clonal cultures were maintained in Petri dishes and also in 50-ml tissue culture flasks (Greiner Bio-One, Frickenhausen, Germany). The two species collected from South Korea were cultured at 18 °C, whereas those collected from King George Island were cultured at 10 °C. Rice grains were added to the Petri dishes to promote the growth of bacteria and bacteriovorous flagellates. Living specimens were observed under a light microscope (Leica DM2500; Wetzlar, Hesse, Germany) at magnifications ranging from X50 to X1,000. Protargol impregnation was performed to observe the infraciliature (Foissner 1991). The parental structures in the ontogenetic stages are shown by contours; the newly formed structures are shaded black. Terminology and classification are according to Berger (1999) and Lynn (2008).

Pseudocyrtohymena koreana n. g., n. sp. and Neokeronopsis asiatica

Polymerase chain reaction amplification and sequencing Specimens were washed several times with distilled water of saline water to isolate a single cell. Genomic DNA was extracted using a RED-Extract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO), according to the manufacturer’s protocol. A modified EukA (50 -CTG GTT GAT YCT GCC AGT-30 ) forward primer (Jung et al. 2012) and LSU rev3 (50 -GCA TAG TTC ACC ATC TTT CG-30 ) (Sonnenberg et al. 2007) reverse primer were used for polymerase chain reaction (PCR) amplification of the nearly complete 18S rRNA gene. The optimized PCR conditions were as follows: denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 4 min, and a final extension step at 72 °C for 7 min. The PCR products were purified using a QIAquickâ PCR Purification Kit (Qiagen, Hilden, Germany). The following two internal primers were used for sequencing: 18S + 810 (50 -GCC GGA ATA CAT TAG CAT GG-30 ) and 18S-300 (50 -CAT GGT AGT CCA ATA CAC TAC-30 ) (Jung et al. 2011). DNA sequencing was performed using an ABI 3700 sequencer (Applied Biosystems, Foster City, CA). Phylogenetic analysis Sequences were assembled using Geneious v6.1.6 (Biomatters Ltd., Auckland, New Zealand). The genetic distance was calculated using MEGA 4.0 (Tamura et al. 2007) with the Kimura 2-parameter distance option (Kimura 1980). To confirm phylogenetic relationships, we compared the sequences of the newly discovered species with those retrieved from GenBank, including the sequences analyzed by Foissner and Stoeck (2008). The gene tree constructed by Foissner and Stoeck (2008) included most of the oxytrichids, which showed strong sequence similarity with our newly discovered species. The sequences were aligned using M-Coffee (Di Tommaso et al. 2011). We used Pseudourostyla cristata (JerkaDziadosz, 1964) Borror, 1972 and Pseudourostyla cristatoides Jung et al. 2012 as an outgroup. We evaluated phylogenetic relationships using maximum likelihood (ML) analysis and Bayesian inference (BI). To determine the appropriate DNA substitution model for ML and BI analyses, we used the Akaike information criterion to identify the best-fit model according to the jModelTest 2.1.1 (Darriba et al. 2012). The model selected was GTR + I (0.7490) + G (0.4980). The ML analysis was conducted using PhyML version 3.1 (Guindon and Gascuel 2003). Confidence in the resulting relationship was assessed using the bootstrap procedure, with 1,000 replications for ML. BI assessment was performed using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) by simulating a Markov chain Monte Carlo for 1,000,000 generations. Trees were sampled every 100 generations, of which 30% were discarded as burn-in. We repeated the phylogenetic analysis to confirm the results and they were almost identical.


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Table 1. List of specimens, together with information regarding sampling locality, and length and accession number of the 18S rRNA gene sequences

Species

Accession no.

Length (base pairs)

Sampling locality

Pseudocyrtohymena koreana n. g., n. sp. Cyrtohymena muscoruma

KM061385

1,576

KM061384

1,576

Neokeronopsis asiatica

KM061386

1,575

Parasterkiella thompsonib

KM0613847

1,577

Afrokeronopsis aurea

EU124669

1,768

Cyrtohymena citrina Cyrtohymena shii

AY498653 JQ513386

1,767 1,770

Gastrostyla steinii Histriculus histrio

AF508758 FM209294

1,771 1,723

Hemiurosoma terricola Hypotrichidium paraconicum

AY498651 JQ918371

1,773 1,798

Laurentiella strenua Notohymena apoaustralis

AJ310487 KC430934

1,767 1,769

Onychodromopsis flexilis Onychodromus grandis Orthoamphisiella breviseries Oxytricha acidotolerans

AY498652 AJ310486 AY498654 FN429123

1,774 1,768 1,773 1,723

Oxytricha elegans

AM412767

1,727

Oxytricha granulifera Oxytricha lanceolata Parasterkiella thompsoni

AM412768 AM412773 HM569264

1,726 1,732 1,621

Paraurostyla weissei Paruroleptus lepisma

AY294648 AF164132

1,723 1,772

Brackish water (5.0 psu); Incheon, South Korea (37°260 N, 126°370 E) Freshwater marsh; Jangdo Island, South Korea (34°400 N, 125°220 E) Fresh water; King George Island, Antarctica (62°140 S, 58°450 W) Fresh water; King George Island, Antarctica (62°130 S, 58°470 W) Floodplain soil from the Matjula River, Republic of South Africa (25°200 N, 31°280 E) Soil from Ghost Forest, Namibia Water catchment area close to the entry point of the Barsey Rhododendron Sanctuary (27°150 –27°270 N, 88°010 –88°230 E), Sikkim, India N/A Water sample from the Botanical Garden of the University of Salzburg, Austria Soil from floodplain of Zambezi River, Botswana Surface of intertidal gravel in the Mipu mangrove forest, Hong Kong (22°290 N, 114°020 E) Darwin, Australia Freshwater pond in the southwestern part of Zhongshan Park, Qingdao (Tsingtao, 36°080 N, 120°430 E), China Watts Lake (obtained from J. Laybourn-Parry), Antarctica Pet shop Aquarium, BO, USA Mud and soil from water hole, Namibia Pelagial of the smallest of three acidic mining lakes, north-northeast of the village of Langau in Lower Austria Isolated from soil from the margin of a Mangrove forest in Dominican Republic Soil in Baumgarten, Austria Soil of Namibia Rancho Hambre peat bog, Tierra del Fuego Province, Argentina Lakes and soils in Princeton, NJ, USA N/A

Pattersoniella vitiphila Pleurotricha lanceolata

AJ310495 FJ748886

1,768 1,743

Jo~ ao Pessoa, Brazil N/A

Ponturostyla enigmatica

KC896649

1,576

Pseudouroleptus caudatus

DQ910904

1,717

Pseudouroleptus jejuensis

KF471024

1,561

Pseudourostyla cristata Pseudourostyla cristatoides Rigidohymena candens

FJ598608 JN887467 KC414885

1,774 1,674 1,766

Rubrioxytricha ferruginea Steinia sphagnicola

AF370027 AJ310494

1,769 1,768

Sea water (34.1 psu); Dongbaek Island, South Korea (35°090 N, 129°090 E) Hydrated mud from the margins of the Alma River, Tocantins, Brazil Soil sample of Jeju Island, South Korea (33°180 N, 126°150 E) Lake Biwa, Shiga Prefecture, Japan Songjiho lagoon in South Korea Upper soil layer of a small garden on the main campus of the Ocean University of China (36°30 N, 120°200 E), Qingdao, China N/A Murrey River flood plain, Australia

Data source This study This study This study This study Foissner and Stoeck (2008) Foissner et al. (2004) Singh et al. (2013)

Hewitt et al. (2003) Schmidt et al. (2008) Foissner et al. (2004) Chen et al. (2013a,b) Bernhard et al. (2001) Lv et al. (2013) Foissner et al. (2004) Bernhard et al. (2001) Foissner et al. (2004) Weisse et al. (2006)

Schmidt et al. (2007) Schmidt et al. (2007) Schmidt et al. (2007) €ppers et al. (2011) Ku Chang et al. (2005) Unpublished GenBank data Bernhard et al. (2001) Unpublished GenBank data Kim et al. (2013) Paiva et al. (2009) Jung et al. (2014) Chen et al. (2010) Jung et al. (2012) Chen et al. (2013a,b)

Gong et al. (2006) Bernhard et al. (2001)

(continued)

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Table 1 (continued)

Species Sterkiella histriomuscorum Sterkiella nova Stylonychia ammermanni Stylonychia lemnae Stylonychia mytilus Stylonychia notophora Styxophrya quadricornuta Tetmemena bifaria Tetmemena pustulata Uroleptoides magnigranulosus Uroleptus gallina Uroleptus piscis Urosomoida longa Urosomoida sp.

Accession no.

Length (base pairs)

AF508770 AF508771 FM209295 AM260994 AM086666 FM209297 X53485 FM209296 X03947 AM412774

1,771 3,629 1,723 1,723 1,723 1,723 1,771 1,723 1,771 1,729

AF508779 AF508780 AF508763 FN429124

3,617 3,599 3,621 1,723

Sampling locality Jordan River, Bloomington, IN, USA Pond in Burlington, NC, USA N/A Pretoria, South Africa Sydney, Australia N/A Boulder, CO, USA N/A N/A Dune soil in the surroundings of Nijmegen, the Netherlands Teller Lake, Boulder County, CO, USA Teller Lake, Boulder County, CO, USA Ten Mile Creek, CO, USA AML lake 111 located in Lusatia, East Germany

Data source Hewitt et al. (2003) Hewitt et al. (2003) Schmidt et al. (2008) Haentzsch et al. (2006) Schmidt et al. (2006) Schmidt et al. (2008) Schlegel et al. (1991) Schmidt et al. (2008) Elwood et al. (1985) Schmidt et al. (2007) Hewitt et al. (2003) Hewitt et al. (2003) Hewitt et al. (2003) Weisse et al. (2013)

N/A, not available. a The morphology of the species matched with the description of monograph by Berger (1999). Thus, this species is not discussed in the text. b €ppers et al. (2011) recently described this species in detail. Additionally, our gene tree The locus classicus of this species is Antarctica, and Ku €ppers et al. (2011) and the populations examined in the present study. Thus, this indicates conspecificity between the populations studied by Ku species is not discussed in the text.

RESULTS Description of P. koreana n. sp. (Tables 1, 2; Fig. 1–4, 11) Description Size in vivo 100–145 lm 9 30–60 lm (Fig. 1A, 2A–E); on average 112 lm 9 47 lm in protargol preparations (Table 2; Fig. 1C, D, 2F, G). Body slender to ellipsoidal shape, flexible and slightly contractile; cell color grayish to yellowish under low magnification. Invariably two elongate elliptical macronuclear nodules left of mid-body, each nodule usually 18.2 lm 9 9 lm in protargol preparations; 1–5 oval to spherical micronuclei near macronuclear nodules, usually 2.4 lm 9 2 lm in protargol preparations. A single contractile vacuole in mid-body near left margin of cell, approximately 6 lm in diam. during diastole (Fig. 1A, 2A). Cortical granules spherical, distributed over entire cortex, more densely along dorsal kineties, yellowish, approximately 1 lm in diam. in vivo (Fig. 1B, 2B, E). All cirri in vivo relatively fine, mostly 10–15 lm long; frontal and transverse cirri 15 lm long; other cirri 10 lm long (Fig. 1A, 2A, C, D, G). Usually, 3 frontal, 4 frontoventral, 1 buccal, 3 postoral ventral, 2 pretransverse ventral, and 5 transverse cirri. Most specimens showed the typical oxytrichid pattern, namely, 18 frontal-ventral-transverse (FVT) cirri with 22–39 left and 25–37 right marginal cirri. A few cells showed variation in the number of FVT cirri, ranging from 18 to 22 due to the presence of extra postoral ventral and transverse cirri. The extra cirri very likely originated from extra anlage (see Ontogenesis). Three dorsal kineties extending from anterior to posterior pole without fragmentation; two shorter dorsomarginal

kineties on right cell margin, right kinety distinctly shorter than left dorsomarginal kinety and all dorsal kineties. Dorsal cilia approximately 2.5 lm long in vivo (Fig. 2D, E). One to three caudal cirri in the posterior part of dorsal kinety 3. Adoral zone of membranelles approximately 42% of body length in protargol preparations; base of the largest membranelles approximately 6 lm long, cilia of membranelles approximately 13 lm long. Paroral membrane (PM) and endoral membrane (EM) in typical Cyrtohymena pattern, namely, distinctly curved anterior part of PM in the leftward direction; the UM intersect in mid-buccal cavity (Fig. 1C, 2G). Ontogenesis Stomatogenesis and development of the FVT cirral anlagen occur as follows: the formation of the oral primordium begins with the de novo development of two fields of basal bodies—one left of the postoral ventral cirrus IV/2 and the other left of the leftmost transverse cirrus II/1 (Fig. 3A, arrows); the basal bodies increase in number and the patches elongate toward each other, forming a single slender anarchic field (Fig. 3A–C). Buccal cirrus begins to dedifferentiate (Fig. 3C, arrowhead), and cirri IV/2, V/3, V/4 either resorb or involve in anlagen formation. Posterior frontoventral cirri (III/2, IV/3) likely dedifferentiate (Fig. 3D). Anterior basal body streaks with the dedifferentiated basal bodies likely contribute to proter’s FVT. FVT anlagen and the UM anlage of the opisthe split from the anterior part of the oral primordium (Fig. 3D, OP). The anterior field of the opisthe’s oral primordium forms adoral membranelles in a backward direction. Three frontal, anterior two frontoventral (VI/3, 4), and pretransverse ventral and transverse


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Table 2. Morphometric data for the protargol-impregnated specimens of Pseudocyrtohymena koreana n. sp. (pk) and Cyrtohymena muscorum (cm)

Body, length Body, width Adoral zone of membranelles, length Percentage of body length occupied by adoral zone Longest adoral membranelles, length Adoral membranelles, number Frontal cirri, number Ventral cirri, number Transverse cirri, number Posterior end of cell to rearmost transverse cirri, distance Left marginal cirri, number Right marginal cirri, number Caudal cirri, number Dorsal kineties, number Bristles in dorsal kinety 1, number Bristles in dorsal kinety 2, number Bristles in dorsal kinety 3, number Bristles in dorsal kinety 4, number Bristles in dorsomarginal row 1, number Bristles in dorsomarginal row 2, number Bristles in dorsomarginal row 3, number Macronucleus nodules, number Macronucleus nodules, length Macronucleus nodules, width Micronuclei, number Micronuclei, length Micronuclei, width

pk cm pk cm pk cm pk cm pk cm pk cm pk cm pk cm pk cm pk cm pk cm pk cm pk cm pk cm pk cm pk cm pk cm cm pk cm pk cm cm pk cm pk cm pk cm pk cm pk cm pk cm

Min

Max

Mean

SD

CV

n

96 95.4 32.4 35.5 41.2 40.8 38.3 34.6 5.5 5.5 38 33 8 8 5 4 5 5 4.4 1.2 22 24 25 22 1 3 5 6 18 24 20 23 21 15 12 17 16 1 8 4 2 2 12.9 13.1 6.3 7.4 1 1 1.3 1.5 1.2 1.4

136.8 136.2 64.9 60.5 59.2 50.9 44.4 51.4 7.6 8.6 53 39 9 8 7 5 6 6 13.6 6.3 39 30 37 26 3 3 5 7 29 29 29 26 31 19 19 26 22 3 15 4 2 2 24.5 20.6 13.1 11.9 5 4 4.5 3.3 3 3.2

112.4 117.9 46.9 45.5 47.5 46.2 42.2 39.5 6.3 7.2 45.2 36.1 8.1 8 5.3 4.9 5.2 5 10.2 3.5 30.9 27.4 32.2 23.7 1.8 3 5 6 23.4 26.2 25.3 24.9 24.9 16.9 15 20.7 17.8 1.9 10.9 – 2 2 18.2 16.9 9 9.4 1.9 2.1 2.4 2.4 2 2.1

9.9 11.6 7.1 7.3 4.4 3 1.7 4.2 0.5 0.7 3.9 1.7 0.4 0 0.6 0.3 0.4 0.2 2.4 1.2 3.8 1.8 3.1 1.2 0.5 0 0 0.2 2.7 1.7 2.6 1 3.1 1.2 1.5 2.4 1.5 0.7 1.9 – 0 0 3.2 1.9 1.7 1.3 1.1 0.8 0.7 0.4 0.5 0.3

8.8 9.9 15.2 16.1 9.3 6.5 4 10.6 8.3 9 8.6 4.6 4.4 0 10.8 6.1 8.3 4.3 23 35.8 12.2 6.7 9.8 5.2 28.3 0 0 3.6 11.6 6.5 10.3 4 12.5 7.1 9.8 11.7 8.6 36.8 17.3 – 0 0 17.8 11 18.4 14.4 57.2 37.5 28.9 17.3 24.6 16.8

21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 1 21 21 21 21 21 21 21 20 21 20 21 20

All measurements in lm. CV = coefficient of variation (%); Max = maximum; Min = minimum; n = number of specimens investigated; SD = standard deviation.

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Figure 1 Pseudocyrtohymena koreana n. sp. (A, B) living specimens and (C, D) after protargol impregnation. (A, B) Ventral (A) and dorsal (B) views of a representative specimen; arrow denotes contractile vacuole. (C, D) Ventral (C) and dorsal (D) views of the holotype specimen. AZM = adoral zone of membranelles; CC = caudal cirri; DK = dorsal kineties; EM = endoral membrane; G = cortical granules; LMR = left marginal cirral row; Ma = macronuclear nodules; Mi = micronuclei; PM = paroral membrane; RMR = right marginal cirral row; TC = transverse cirri. Scale bars, 50 lm.

cirri usually remain intact up to the middle stage (Fig. 3) and are subsequently resorbed (Fig. 4). Usually six longitudinal streaks develop for each daughter cell (Fig. 3D, E). The streaks become separate and migrate to the proter and opisthe, respectively. The UM anlage of the proter develops from the dedifferentiated parental UM that later reorganize to form the UM of the proter (Fig. 3E). In general, five streaks develop at the right of the proter’s and opisthe’s UM anlagen (Fig. 3G). The leftmost anterior frontal cirrus develops from anterior part of the UM anlage (Fig. 3G, 4A). A few specimens have an extra streak, which very likely forms one transverse cirrus and two postoral ventral cirri (the proter in Fig. 3G). The opisthe’s adoral membranelles differentiate in the posterior direction, and the anterior part of AZM arches slightly to the right (Fig. 3D, E, G). In late dividers, the new adoral membranelles are completely developed in the opisthe, while the parental membranelles are completely retained during ontogenesis (Fig. 4). The six streaks form three frontal, four frontoventral, one buccal, three postoral ventral, two pretransverse ventral, and five transverse cirri; in other words, these anlagen generate the following number of cirri: 1, 3, 3, 3, 4, and 4, respectively (Fig. 4). The newly formed pretransverse ventral and transverse cirri migrate posteriorly in both daughter cells (Fig. 4C), whereas the parental cirri are resorbed. Marginal and dorsal anlagen: in the early stage, a few cirri of the anterior and middle part of the marginal rows begin to dedifferentiate, and parental cirri are incorporated into the primordium (Fig. 3E, G). Simultaneously, the dorsal kinety anlagen develop intrakinetally within parental dorsal kineties 1–3 (Fig. 3F, double-arrowheads); thus, two


dorsal kinety primordia develop within a single dorsal kinety (Fig. 3F, H). Each marginal cirral primordium subsequently elongates posteriorly and replaces the parental cirri. The dorsal kinety anlagen stretch in both directions, and the parental dorsal kineties are very likely incorporated or resorbed. Dorsal kinety anlage 3 forms a single row without any fragmentations (Fig. 4; Urosomoida pattern). Additionally, a second group of dorsal kineties—the dorsomarginal kineties—develops at the right anterior marginal cirral row anlage in the proter and opisthe (Fig. 4, arrows). The dorsomarginal kineties invariably consist of two kineties and migrate to the dorsal side of the right margin. The dorsomarginal kineties are shorter and have fewer bristles than dorsal kineties (Table 2). Division of nuclear apparatus: the division is in the typical pattern of hypotrichs. The two macronuclei fuse to form a single mass in the middle stage, and then elongate and split twice (Fig. 3H, 4B, D). The micronuclei divide at a late stage (Fig. 4D). Molecular analysis Pseudocyrtohymena showed a close relationship with soft-body oxytrichids possessing the Cyrtohymena UM pattern and nested in the clade Cyrtohymena–Notohymena–Paraurostyla–Ponturostyla–Rubrioxytricha. The new genus clustered away from Neokeronopsidae and Rigidohymena, although these genera also possess the Cyrtohymena UM pattern. The genus Cyrtohymena on the gene tree consisted of three species, and they were not clustered together. Even the species Cyrtohymena shii showed a sister relationship with Neokeronopsis and nested in Neokeronopsidae. The soft-body oxytrichids showed slightly lower supporting values on interior branches than the rigid-body oxytrichids Stylonychinae. Description of N. asiatica Foissner et al. 2010 (Tables 1, 3; Fig. 5–11) Description Size in vivo 240–400 lm 9 90–160 lm (Fig. 5A–D, 9A, B, E, F); on average 308 lm 9 140 lm in protargol preparations (Table 3; Fig. 6, 7, 10A, B). Body slender to ellipsoidal in shape with slight indentation near caudal cirri (Fig. 6B); slightly flexible; cell color, yellowish to greenish under low magnification. Crystals in cytoplasm—usually located on left and posterior sides of cell; yellowish in color (Fig. 5H). Invariably 2 elongate elliptical macronuclear nodules, elongate elliptical, in mid-body at left margin; 2–5 micronuclei, spherical to elliptical, usually 7.7 lm 9 6.2 lm in size. A single contractile vacuole on left side of cell above mid-body, approximately 20 lm in diam. during diastole (Fig. 5D, 9A, B). Cortical granules spherical, yellow-greenish to greenish, approximately 1–2 lm in diam. in vivo, distributed along dorsal kineties and cirri (Fig. 5B, C, E, F, 9E–H, J, K). All cirri in vivo relatively fine, mostly 15–20 lm long; frontal and transverse cirri 20 lm long; remaining cirri 15 lm long (Fig. 5A). Cirral pattern urostylid, namely, 13–19 frontal cirri as bicorona, 2 frontoterminal, 2 pretransverse ventral,


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Figure 2 Pseudocyrtohymena koreana n. sp., (A–E) living specimens and (F–K) after protargol impregnation. (A, B, D, E) Dorsal views of representative specimens. (C) Ventral view showing Cyrtohymena undulating membranes pattern. (F–K) Dorsal (F, I, K) and ventral (G, H, J) views of the holotype (F, G) and other specimen (H–K). Cyrtohymena undulating membranes pattern (H), nuclear apparatus (I), postoral ventral cirri (J), and caudal cirri at posterior end of dorsal kinety 3. AZM = adoral zone of membranelles; CC = caudal cirri; CV = contractile vacuole; DB = dorsal bristle; EM = endoral membrane; G = cortical granules; K1–K3 = dorsal kineties 1–3; LMR = left marginal cirral row; Ma = macronuclear nodules; Mi = micronuclei; PM = paroral membrane; RMR = right marginal cirral row; TC = transverse cirri; VC = ventral cirri. Scale bars, 50 lm.

15–23 transverse, and 48–71 left and 36–65 right marginal cirri. Twelve to eighteen dorsal kineties (Fig. 6B). Dorsal kinety 1 either fragmented or not: in more than half of cells examined (10/18) fragmented at posterior position (Fig. 7, asterisks). Dorsal cilia approximately 4 lm long in vivo (Fig. 9G), encaged by long fiber (Fig. 5F); fiber distinct in vivo, variable shape, that is, nearly straight or highly curved

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(Fig. 5G). Invariably, 3 caudal cirral rows consisting of 8–15 cirri in total. Adoral zone of membranelles approximately 43% of body length in protargol preparations, base of largest membranelles approximately 20 lm long, cilia of membranelles approximately 20 lm long. PM and EM in typical Cyrtohymena pattern, namely, distinctly curved anterior


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streaks, opisthe’s UM anlage, opisthe’s oral primordium, and right marginal cirral anlage. Parental adoral membranelles very likely retained the shape in that stage, and the opisthe’s oral primordium formed adoral membranelles at the anterior part of the primordium. Between the two cirral anlagen groups (=longitudinal streaks), parental cirri were not observed. These cirri were very likely resorbed or involved in primordia formation. Two micronuclei near the posterior macronuclear nodule were inflated showing ellipsoidal shape (Fig. 8B). The reorganizer was in the late stage and formed almost all cirri except for marginal cirri. Some adoral membranelles were very likely altered by newly formed ones because anarchic basal bodies were observed at the left proximal end of the AZM. Five dorsomarginal rows formed from the anterior part of the right marginal anlage (Fig. 10G). Two macronuclear nodules were very likely fused as a single mass. Dorsal kinety 3 showed multiple fragmentations (Fig. 10H).

Figure 3 Pseudocyrtohymena koreana n. sp.—dividers after protargol impregnation. (A–F) Ventral (A–E) and dorsal (F) views of early dividers. Arrows denote basal body patches; arrowhead indicates the dedifferentiating buccal cirrus. Double-arrowheads denote dorsal kinety anlagen. (G, H) Ventral (G) and dorsal (H) views of middle divider, showing the fusing macronucleus. FC = frontal cirrus; L = left marginal anlagen; Ma = macronuclear nodules; Mi = micronuclei; OP = oral primordium; R = right marginal anlagen. Scale bars, 50 lm.

Molecular analysis Neokeronopsis asiatica was nested in the clade Afrokeronopsis aurea–C. shii with high supporting values (BI–1.00, ML–88%), and the clade showed a sister relationship with Hypotrichidium paraconicum. Cyrtohymena shii showed 99.8% of SSU rRNA gene sequence similarity with N. asiatica and was not clustered with other congeners (Fig. 11). DISCUSSION Pseudocyrtohymena koreana n. g., n. sp

Figure 4 Pseudocyrtohymena koreana n. sp.—dividers after protargol impregnation. (A–D) Ventral (A, C) and dorsal (B, D) views of late dividers. Arrows denote dorsomarginal rows. CC = caudal cirri; Ma = macronuclear nodules; Mi = micronuclei. Scale bars, 50 lm.

part of PM in leftward direction; buccal horn at distal end of UM, distinct in vivo (Fig. 5A, 9C, F); the UM intersect in posterior portion of buccal cavity (Fig. 5A, 6A, 10A, D). Divider and reorganizer Only a single specimen in mid-division (Fig. 8, 10E, F) and reorganization (Fig. 10G, H) stage was protargol-impregnated. The mid-divider showed two groups of longitudinal

Pseudocyrtohymena is a typical oxytrichid, with 18 FVT cirri, flexible body, cortical granules, and the Cyrtohymena UM pattern. The new genus is similar to Cyrtohymena Foissner, 1989, Rigidohymena Berger 2011; and Rubrioxytricha Berger 1999; but is characterized by the following features: (i) Cyrtohymena UM pattern; (ii) dorsal kinety 3 nonfragmented; (iii) caudal cirri lacking in dorsal kinety anlagen 1, 2; (iv) colorless cytoplasm; (v) cortical granules present; (vi) possibly involvement of postoral ventral cirrus V/3 in primordia formation; and (vii) body flexible (for details, see Table 4). Cyrtohymena differs from Pseudocyrtohymena by dorsal morphogenesis. Cyrtohymena muscorum, type species of the genus, has a fragmented dorsal kinety anlage (vs. nonfragmented; i.e., Oxytricha pattern vs. Urosomoida pattern), and 3 caudal cirri developed from dorsal kinety anlagen 1–3 (vs. 1–3 caudal cirri from dorsal kinety anlage 3) (Berger 1999). Rigidohymena can be separated from Pseudocyrtohymena by rigid body (vs. flexible), cortical granules lacking (vs. present), and postoral cirrus V/3 not involved in primordia formation (vs. very likely involved) (Berger 2011). Rubrioxytricha is distinguished from Pseudocyrtohymena by colored cytoplasm (vs. colorless) (Berger 1999). The colored cytoplasm is a key character of the genus Rubrioxytricha. However, Rubrioxytricha indica has colorless cytoplasm, unlike that found in other congeners, and


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Table 3. Morphometric data for the protargol-impregnated specimens of Neokeronopsis asiatica Characteristics Body, length Body, width Anterior body end to left end of adoral zone, distance Percentage of body length occupied by adoral zone Anterior body end to right end of adoral zone, distance Left and right end of adoral zone, distance in main body axis Longest adoral membranelles, length Proximal end of adoral zone to uppermost transverse cirrus, distance Adoral membranelles, number Frontal cirri, numbera Frontoterminal cirri, number Transverse cirri, number Left marginal cirri, number Right marginal cirri, number Caudal cirri, number Caudal cirri in dorsal kinety 1, number Caudal cirri in dorsal kinety 2, number Caudal cirri in dorsal kinety 3, number Dorsal kineties, number Bristles in dorsal kinety 1, number Bristles in dorsal kinety 2, number Left and right end of marginal cirri, distance between Macronucleus nodules, number Macronucleus nodules, length Macronucleus nodules, width Micronuclei, number Micronuclei, length Micronuclei, width

Min

Max

Mean

SD

CV

n

225.0 107.5 108.5 36.0 37.0 53.5 16.5 36.0

375.0 175.0 161.0 51.1 86.0 84 24 116.0

308.3 140.3 132.0 43.3 62.8 69.2 20.4 72.2

46.1 19.1 12.8 4.2 10.4 7.4 1.9 22.8

15.0 13.6 9.7 9.7 16.5 10.6 9.1 31.6

21 21 21 21 21 21 21 21

77.0 13.0 2.0 15.0 48.0 36.0 8.0 3.0 2.0 1.0 12.0 66.0 47.0 2.5 2.0 36.0 16.0 2.0 5.5 5.0

110.0 19.0 2.0 23.0 71.0 65.0 15.0 6.0 5.0 4.0 18.0 119.0 84.0 18.5 2.0 63.0 27.0 5.0 11.0 8.0

89.5 16.2 2.0 18.9 58.5 50.1 10.6 4.2 3.7 2.8 14.4 90.6 66.4 8.0 2.0 47.3 20.7 3.1 7.7 6.2

8.8 1.4 0.0 2.1 6.9 8.3 1.9 0.8 0.7 0.8 1.6 14.1 10.2 4.3 0.0 6.7 2.7 1.1 1.1 0.8

9.8 8.7 0.0 11.1 11.8 16.6 17.8 20.0 20.3 28.2 10.9 15.6 15.4 54.1 0.0 14.2 13.2 35.3 14.6 13.1

21 21 21 21 21 21 19 19 19 19 21 21 21 21 21 21 21 21 21 21

All measurements in lm. CV = coefficient of variation (%); Max = maximum; Min = minimum; n = number of specimens investigated; SD = standard deviation; SE = standard error of arithmetic mean. a Including 1 buccal cirrus.

shares similar morphology to P. koreana (for morphological comparison, see Table 5), implying that there might be congenericity between R. indica and P. koreana. Ontogenesis During the ontogenesis of P. koreana, we missed some stages to clarify these features: (i) participation of cirrus V/ 3 in anlage formation; (ii) involvement of parental cirri III/2, IV/2, IV/3, and V/4 in anlagen development of the proter; and (iii) de novo formation of anlagen V and VI of the proter. The two species C. muscorum and Notohymena rubescens, relatives of the genus Pseudocyrtohymena, show the following features that primordia II–VI develop from parental cirri II/2 (primordium II), III/3 (primordium III), IV/3 (primordium IV), and de novo (primordia V, VI). Pseudocyrtohymena koreana, as a relative of the species mentioned above, may have similar ontogenetic features. They are typical flexible oxytrichids with Cyrtohymena UM. On the basis of the characteristics of P. koreana, the ontogenesis is similar to that of C. muscorum—the type species of Cyrtohymena—except for the formation of caudal cirri and the nonfragmented dorsal kinety (Berger 1999). Pseudocyrtohymena has fewer caudal cirri because dorsal

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kineties 1, 2 do not form caudal cirri; in P. koreana, dorsal kinety 3 forms 1–3 caudal cirri. Naqvi et al. (2006) established a new freshwater species, R. indica, which is highly similar to P. koreana with regard to the interphasic cirral pattern, colorless cytoplasm, and nonfragmented dorsal kinety 3. However, R. indica has the following distinctive feature: cirrus V/3 is involved in the late ontogenetic stage (vs. early stage in Pseudocyrtohymena) that merges with posterior part of UM anlage of the opisthe. In vivo characteristics and comparison of N. asiatica with similar species Neokeronopsis asiatica was described by Wang et al. (2007) as N. spectabilis. Foissner et al. (2010) re-examined the slides described by Wang et al. (2007) and by Shi and He (1990) and established a new species, N. asiatica. Unfortunately, live observations of N. asiatica are unavailable. On the basis of the protargol-impregnated specimens described by Foissner et al. (2010), N. asiatica has cortical granules. However, the other features—color of the cortical granules, as well as that of the cytoplasm, body flexibility, and contractile vacuole—are unavailable from the


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Figure 5 Neokeronopsis asiatica, (A–F, H) living specimens and (G) after protargol impregnation. (A) Ventral view of a representative specimen. (B, C) Dorsal (B) and ventral (C) views showing cortical granules. (D) Dorsal views showing the formation of contractile vacuole. (E) Cortical granules on the lateral side of the cell. (F, G) Fiber system along dorsal kineties in living specimens (F) and after protargol impregnation (G). The fiber system (G) has a variable pattern. (H) Crystals in the cytoplasm; usually these structures are located on the left and posterior sides of the cell (see also Fig. 9E, F, shaded parts showing these crystals). AZM = adoral zone of membranelles; CV = contractile vacuole; DB = dorsal bristle; F = fibers; G = cortical granules; Ma = macronuclear nodules; Mi = micronuclei. Scale bars, 200 lm.

original description. On the basis of the Antarctic population, we observed these previously unidentified characteristics (for morphological comparison, see Table 6). Neokeronopsis spectabilis (Kahl 1932) Warren et al. (2002) differs from N. asiatica on the basis of the posterior body end (broadly rounded without indentation vs. indentation at the site of caudal cirri) and posterior ends of the marginal rows (different vs. at the same level). Foissner et al. (2010) considered that the broadly rounded body at the posterior end might have resulted from the inflated type specimens of N. spectabilis. Thus, further investigations, especially of N. spectabilis, are necessary to clarify the intact body shape. In terms of morphology, the Antarctic population shows an intermediate status between N. spectabilis and the Chinese population of N. asiatica. According to Foissner et al. (2010), these two species are distinguished mainly on the basis of the width of the adoral membranelles (15–20 lm in N. asiatica vs.


Figure 6 Neokeronopsis asiatica, infraciliature after protargol impregnation. (A, B) Ventral (A) and dorsal (B) views of a representative specimen. Asterisk in (B) denotes anterior fragmentation of dorsal kinety 1. (C) Ventral views showing a variation of undulating membranes; usually endoral membrane extends to the right and posterior directions, unlike the paroral membrane, as shown in the uppermost illustration. AZM = adoral zone of membranelles; CC = caudal cirri; EM = endoral membrane; K1 = dorsal kinety 1; K2 = dorsal kinety 2; LMR = left marginal cirral row; Ma = macronucleus nodules; Mi = micronuclei; RMR = right marginal cirral row; TC = transverse cirri. Scale bars, 200 lm.

22–35 lm in N. spectabilis; Table 6). However, the width of the adoral membranelles in the Antarctic population is 16.5–24 lm, which slightly overlaps with that of N. spectabilis. Unfortunately, the 18S rRNA gene sequence of N. spectabilis is unavailable, and further investigations of this species are required to confirm whether the midventral cirri contribute to the formation of cirral anlagen in the early stage, or are resorbed in late dividers (vs. resorbed in the early stage in N. asiatica; Fig. 8). Moreover, in the Antarctic population, half of the examined specimens showed fragmented dorsal kinety 1 (Fig. 7), which is also present in N. spectabilis. Thus, the conspecificity of these two species cannot be entirely excluded. Further studies are required to validate their identity. Cyrtohymena UM pattern Previously, the oral apparatus, namely the Cyrtohymena UM pattern, was a main character key, and appeared to be an apomorphic feature of the genus Cyrtohymena (Berger 1999). Recently, however, several new species with this oral apparatus but belonging to other genera or even other families, for example, Neokeronopsidae Foissner and Stoeck 2008; Rigidohymena Berger 2011; and Saudithrix Foissner et al., 2006, have been established (Berger et al. 2006; Chen et al. 2013a,b; Foissner and Stoeck 2008; Foissner et al. 2010; Wang et al. 2007; Warren et al. 2002).


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Figure 7 Neokeronopsis asiatica, dorsal kineties of interphasic specimens after protargol impregnation. Asterisks denote fragmentations in dorsal kinety 1. More than half of the cells examined (10/18) were fragmented at the posterior position.

In the 18S rRNA gene tree (Fig. 11), we combined oxytrichid representatives with species possessing the Cyrtohymena UM pattern. Our results showed that species possessing the Cyrtohymena UM pattern clustered sparsely and split into three clades: (i) Cyrtohymena– Notohymena–Paraurostyla–Ponturostyla–Pseudocyrtohymena– Rubrioxytricha; (ii) Neokeronopsidae; and (iii) Rigidohymena. Our results indicate that Cyrtohymena UM pattern might have evolved several times, or existed in the most recent common ancestor (Berger 2011). However, on the basis of a parsimonious point of view, Oxytricha UM pattern seems to have evolved in a stem-line of hypotrichs because most of the hypotrichs have the Oxytricha oral apparatus (Berger 1999). According to this assumption, the Cyrtohymena UM pattern might have evolved several times in hypotrichs and, in particular, in Rigidohymena, which supports the parallel/convergent evolution of the oral apparatus in hypotrichs. The genus Rigidohymena belongs to Stylonychinae, not Oxytrichinae, and has the Cyrtohymena UM pattern with a rigid body. In the gene tree, Rigidohymena candens is nested with Stylonychinae, and not with the soft-bodied oxytrichids; this result is consistent with that of Chen et al. (2013a,b). Further, Cyrtohymena (Cyrtohymenides) shii is nested with Neokeronopsidae, not with Cyrtohymena. This result is in accordance with that of Singh et al. (2013), who also noted similarity between these taxa. Another member of the subgenus Cyrtohymenides, Cyrtohymena (Cyrtohymenides) aspoecki, also appears to be closely related to Neokeronopsidae on the basis of the following common features: (i) Cyrtohymena oral apparatus and (ii) multiple kinety fragmentation.

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Figure 8 Neokeronopsis asiatica, divider after protargol impregnation. (A, B) Ventral (A) and dorsal (B) views of a middle divider. Parental midventral cirri between the cirral anlagen of the proter and the opisthe are resorbed. Asterisk(s) denote right marginal cirral row anlage in (A) and dorsal kinety anlagen in (B), respectively. CC = caudal cirri; EM = endoral membrane; FTC = frontoterminal cirri; K1–K3 = dorsal kineties 1–3; OP = oral primordium; PM = paroral membrane. Scale bars, 200 lm.

Hypotrichs are one of the most diverse groups in Ciliophora, and new taxa are constantly being described. However, their phylogenetic relationships are still problematic in the group possessing Cyrtohymena UM pattern. Saudithrix terricola Foissner et al., 2006 has the Cyrtohymena oral apparatus; however, Berger et al. (2006) classified the species as incertae sedis in Stichotrichia, and the genus still belongs to an unknown position in nondorsomaginalian Hypotricha (Berger 2011). In addition, convergent evolution between hypotrichs (e.g., CEUU hypothesis by Foissner and Stoeck 2008) emphasizes further detailed character sampling, especially on the basis of ontogenesis and molecular data. The new genus Pseudocyrtohymena showed close relationships with Cyrtohymena, Notohymena, Paraurostyla, Ponturostyla, Rubrioxytricha, and Neokeronopsidae. However, the supporting values were unstable, despite the distinctive morphological features. Thus, as discussed above, further investigations and more extensive sampling are required to elucidate their true relationships. TAXONOMIC SUMMARY Pseudocyrtohymena n. g Diagnosis. Oxytrichidae with Cyrtohymena UM; body, flexible, colorless cytoplasm; cortical granules present;


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Figure 9 Neokeronopsis asiatica living specimens. (A, B) Dorsal views showing contractile vacuole (CV) and entire body shape. (C) Ventral view of the anterior body. The buccal horn (BH) is visible between the distal end of the paroral membrane and the adoral membranelles. (D) Dorsal view of the posterior body end. Eight to fifteen caudal cirri are located at the indented body end. (E–H) Dorsal (E, G), ventral (F), and lateral (H) views showing cortical granules. Usually the cortical granules are distributed along the cirri and dorsal kineties. (I) Macronuclear nodules (Ma) and micronuclei (Mi). This nuclear apparatus is distinct and readily recognized in vivo. (J, K) Fiber system related to the dorsal bristles and right marginal cirri. These structures are readily identified in vivo. AZM = adoral zone of membranelles; CC = caudal cirri; DB = dorsal bristles; F = fiber system; G = cortical granules; PM = paroral membrane; RMR = right marginal cirral row. Scale bars, 200 lm.


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Figure 10 Neokeronopsis asiatica, (A–D) protargol-impregnated interphasic specimen, (E, F) mid-divider, and (G, H) late reorganizer. (A–D) Ventral (A, D) and dorsal (B, C) views of a representative specimen showing infraciliature. (E, F) Ventral (E) and dorsal (F) views of a mid-divider. At this stage, parental midventral cirri between the cirral anlagen of the proter and opisthe are already resorbed. (G, H) Ventral (G) and dorsal (H) views of a late reorganizer. Asterisk denotes leftmost frontal cirrus; arrowhead indicates right pretransverse cirrus. Fragmentation of dorsal kinety 3 is observed at this stage. AZM = adoral zone of membranelles; CC = caudal cirri; EM = endoral membrane; FTC = frontoterminal cirri; K1–K3 = dorsal kineties 1–3; LMR = left marginal cirral row; Ma = macronucleus nodules; Mi = micronuclei; MVR = midventral rows; OP = oral primordium; PM = paroral membrane; RMR = right marginal cirral row; TC = transverse cirri. Scale bars, 200 lm.

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Figure 11 Phylogenetic tree of 18S rRNA gene sequences, showing the position of Pseudocyrtohymena koreana n. sp. and Neokeronopsis asiatica on the basis of Bayesian inference (BI) and maximum likelihood (ML). Bootstrap values for ML and posterior probability values for BI are represented on the nodes. Species names in bold denote that the gene sequences were obtained from the present study. The illustration of Rigidohymena candens is from Chen et al. (2013a,b).

caudal cirri, when present, at end of dorsal kinety 3; dorsal kinety 3 nonfragmented (Urosomoida pattern). Type species. Pseudocyrtohymena koreana n. sp. Etymology. Pseudocyrtohymena is a composite of the Greek adjective pseudo- (wrong, lying) and the name of the genus Cyrtohymena Foissner, 1989. The name Pseudocyrtohymena indicates that the new species is similar but not identical to Cyrtohymena. Zoobank registration. urn:lsid:zoobank.org:act:D328CF 38-2989-4334-B0C3-1B1E064204E7.

Pseudocyrtohymena koreana n. sp Diagnosis. Size in vivo 100–145 lm 9 30–60 lm; slender to ellipsoidal in shape, grayish to slightly yellowish under low magnification. Single contractile at left margin of midbody. Two macronuclear nodules and usually 1–5 micronuclei. Cortical granules spherical in shape, yellowish with 38– 53 adoral membranelles, 18 FVT cirri, and 22–39 left and 25–37 right marginal cirri. Five dorsal kineties; 3 bipolar and 2 dorsomarginal kineties. Dorsal kinety 3 nonfragmented. One to three caudal cirri at end of dorsal kinety 3.


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Table 4. Comparison of morphological features in Pseudocyrtohymena with those of closely related genera Characteristicsa

Pseudocyrtohymena. n. g.

Cyrtohymena

Rigidohymena

Rubrioxytricha

Oral apparatus Dorsal kinety 3 fragmentation Caudal cirri originated from dorsal kinety anlagen 1, 2 Color of cytoplasm Cortical granules Involvement of postoral ventral cirrus V/3 in primordia formation Body flexibility Data source

Cyrtohymena UM pattern Nonfragmented Absent

Cyrtohymena UM pattern Fragmented Present

Cyrtohymena UM pattern Fragmented Present

Oxytricha UM patternb Nonfragmenteda Absenta

Colorless Present Yesc

Colorless Present Yes

Colorless Absent No

Coloredb Present Yesc

Flexible Original

Flexible Berger (1999)

Rigid Berger (2011)

Flexible Berger (1999)

a

Rubrioxytricha ferruginea is unclear about the features mentioned above (Berger 1999). Rubrioxytricha indica is the sole species possessing colorless cytoplasm and Cyrtohymena UM pattern (Naqvi et al. 2006). c The cirri V/3 either resolves or involves in primordia formation. d Rubrioxytricha haematoplasma is needed to confirm whether the cirrus V/3 involves in primordia formation or not (Berger 1999). If the cirrus V/3 dose not involved in the formation, the species has more close relationship with Stylonychinae, not Oxytrichinae. b

Table 5. Comparison of morphological features in Pseudocyrtohymena with those of closely related species

Characteristicsa Body, size Cytoplasm, color Cortical granules, color Contractile vacuole, number Adoral membranelles, number Caudal cirri, number Dorsal kineties, numberc Fragmentation of dorsal kinety 3 Habitat Data source

Pseudocyrtohymena. koreana n. sp.

Rubrioxytricha indica

112 9 47 Colorless Yellowish 1

69 9 27 Colorless Dark greenish 3

38–53

27–31

1–3b 5 Absent

1b 5 Absent

Brackish water Original

Freshwater Naqvi et al. (2006)

All measurements in lm. Data based on protargol-impregnated specimens. b Only from dorsal kinety 3. c Including dorsomarginal rows. a

Type locality. Estuarine brackish water in the Yellow Sea near Incheon, South Korea (Table 1). The sample was collected in April, 2011. Type material. The holotype (NIBRPR0000104259) and 3 paratype slides (NIBRPR0000104260–NIBRPR0000104263) with protargol-impregnated specimens, including dividing individuals, were deposited in the National Institute of Biological Resources (NIBR), South Korea. The holotype (Fig. 1C, D, 2F, G) and other relevant specimens were marked using circles on the bottom of the slides. Etymology. The species name “koreana” is derived from the name of the country where the species was discovered. Gene sequence. The 18S rRNA gene sequence of P. koreana has been deposited in the GenBank database with the accession number KM061385.

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Zoobank registration. urn:lsid:zoobank.org:act:953180F2BCE0-443B-99DC-A80381E0CB27. Neokeronopsis asiatica Foissner et al. 2010 Improved diagnosis. Size in vivo 240–400 lm 9 90– 160 lm; body slightly flexible, slender to ellipsoidal in shape, yellowish under low magnification. Two macronuclear nodules with 2–5 micronuclei. Contractile vacuole above mid-body at level of proximal curve of adoral zone with distinct collecting canals. Cortical granules elliptical to spherical in shape, yellow-greenish to greenish, approximately 1–2 lm in diam. with 77–110 adoral membranelles, 13–19 frontal, 2 frontoterminal, 2 pretransverse ventral, 15–23 transverse, 48–71 left marginal, and 36–65 right marginal cirri. Twelve to eighteen dorsal kineties consisting of dorsal and dorsomarginal kineties, with 8–15 caudal cirri. Examined materials. Fresh water, King George Island (the Antarctic) (Table 1). The slides (NIBRPR0000104340– NIBRPR0000104345) with protargol-impregnated specimens were deposited at the NIBR, South Korea. The sample was collected in January, 2012. Gene sequence. The 18S rRNA gene sequence of N. asiatica has been deposited in the GenBank database with the accession number KM061386. ACKNOWLEDGMENTS This study was supported by research grants PE99193 from the Korea Institute of Ocean Science and Technology (KIOST), the program on Management of Marine Organisms causing Ecological Disturbance and Harmful Effects funded by KIMST/MOF, and the National Institute of Biological Resources (NIBR) of the Ministry of Environment, Korea, as a part of the Discovery of Korean Indigenous Species Project 2013, and Graduate Program for the Undiscovered Taxa of Korea (1834-302).


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Table 6. Comparison of morphological features in Neokeronopsis asiatica (Antarctic and Chinese populations), N. spectabilis, and Afrokeronopsis aurea Characteristicsa

N. asiatica (Antarctic)

N. asiatica (Chinese)

N. spectabilis

A. aurea

Body, size Body, color

308 9 140 Yellowish

288 9 119 N/A

442 9 176 N/A

Cytoplasm, color Cortical granules, color Body, elasticity

Colorless Yellow-greenish to greenish Slightly flexible, but not contractile Left of proximal end of AZM Present 16.5–24

N/A N/A, but present N/A N/A N/A 15–20

Colorless Lemon-yellowish to yellowishb Distinctly flexible, but only slightly contractile Left of proximal end of AZM Present 22–35

36–116

48–100

70–200

9.2c

77–110

66–93

72–90

95–104

15–23 8–15 36–65 12–18 Absent Almost abutting



22–27 3 43–57 12–17 Present Distinctly separate

Rounded with slight indentation Fragmented in half of examined cells, usually at posterior position N/A Resorbed

Acute with distinct indentation Usually absent

Broadly rounded

Narrowly rounded to acute

Posterior fragmentation

Anterior fragmentation

Indistinct Resorbed

Indistinct (?) Maintained (?)

Distinct Maintained

Original

Foissner et al. (2010), Wang et al. (2007)

Berger (2006), Foissner et al. (2010), Kahl (1932), Warren et al. (2002)

Foissner and Stoeck (2008)

Contractile vacuole, position Buccal horn Base of largest adoral membranelles, width Proximal end of AZM to uppermost transverse cirrus, distance Adoral membranelles, number Transverse cirri, number Caudal cirri, number Right marginal cirri, number Dorsal kineties, numberd Buccal depression Anterior end of paroral and endoral membrane Posterior body end, shape Fragmentation of dorsal kinety 1 Dorsal whirl in mid-dividers Midventral cirri between anlagen in early dividers Data source

306 9 112 Golden-yellowish to brown-orange

Semi-rigid

Absent 16–20

All measurements in lm. AZM = adoral zone of membranelles; N/A = unavailable. a Data based on protargol-impregnated specimens. b Kahl (1932) noted that the yellowish cortical granules were linearly arranged on the dorsal side and along the marginal rows. c Data from Foissner et al. (2010). d Dorsal kineties consisting of 1–3 kinetids, and fragments of dorsal kineties 1–3, are not included.

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Present address: South Sea Institute, Korea Institute of Ocean Science and Technology, Geoje, 656-830, South Korea


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

Reconsideration of the 'well-known' hypotrichous ciliate Pleurotricha curdsi (Shi et al., 2002) Gupta et al., 2003 (Ciliophora, Sporadotrichida), with notes on its morphology, morphogenesis and molecular phylogeny.

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

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

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).

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.

Molecular phylogeny of an Indian population of Kleinstyla dorsicirrata (Foissner, 1982) Foissner et al., 2002. comb. nov. (Hypotrichia, Oxytrichidae): an oxytrichid with incomplete dorsal kinety fragmentation.

Morphology of three species of Amphileptus (Protozoa, Ciliophora, Pleurostomatida) from the South China Sea, with note on phylogeny of A. dragescoi sp. n.

Morphology and molecular phylogeny of two colepid species from China, Coleps amphacanthus Ehrenberg, 1833 and Levicoleps biwae jejuensis Chen et al., 2016 (Ciliophora, Prostomatida).

Morphology of Clapsiella magnifica gen. n., sp. n., a new hypotrichous ciliate with a curious dorsal ciliary pattern.

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).

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

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

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

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

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

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

Reply to S. Barni et Al, K.R. Dearing et al, and N. Murray.