Morphology and molecular phylogeny of a new hypotrich ciliate, Anteholosticha songi nov. spec., and an American population of Holosticha pullaster (Müller, 1773) Foissner et al., 1991 (Ciliophora, Hypotrichia)

Journal Pre-proof Morphology and molecular phylogeny of a new hypotrich ciliate, Anteholosticha songi nov. spec., and an American population of Holosticha pullaster (Muller, ¨ 1773) Foissner et al., 1991 (Ciliophora, Hypotrichia) Lingyun Chen, Jingyi Dong, Weining Wu, Yuanyuan Xin, Alan Warren, Yingzhi Ning, Yan Zhao

PII:

S0932-4739(19)30083-5

DOI:

https://doi.org/10.1016/j.ejop.2019.125646

Reference:

EJOP 125646

To appear in:

European Journal of Protistology

Received Date:

29 August 2019

Revised Date:

19 October 2019

Accepted Date:

21 October 2019

Please cite this article as: Lingyun C, Jingyi D, Weining W, Yuanyuan X, Alan W, Yingzhi N, Yan Z, Morphology and molecular phylogeny of a new hypotrich ciliate, Anteholosticha songi nov. spec., and an American population of Holosticha pullaster (Muller, ¨ 1773) Foissner et al., 1991 (Ciliophora, Hypotrichia), European Journal of Protistology (2019), doi: https://doi.org/10.1016/j.ejop.2019.125646

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Morphology and molecular phylogeny of a new hypotrich ciliate, Anteholosticha songi nov. spec., and an American population of Holosticha pullaster (Müller, 1773) Foissner et al., 1991 (Ciliophora, Hypotrichia)

Lingyun Chena,1, Jingyi Dongb,1, Weining Wuc,1, Yuanyuan Xina, Alan Warrend, Yingzhi Ninga,*, Yan Zhaoe,*

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Laboratory of Microbiota, College of Life Science, Northwest Normal University, Lanzhou 730070,

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China b

Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China

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College of Resources and Environmental Sciences, Gansu Agricultural University, Lanzhou 730070,

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China

Department of Life Sciences, Natural History Museum, London SW7 5BD, UK

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College of Life Sciences, Capital Normal University, Beijing 100048, China

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*Correspondence: Laboratory of Microbiota, College of Life Science, Northwest Normal University, Lanzhou 730070, China; College of Life Sciences, Capital Normal University, Beijing 100048, China.

These three authors contributed equally.

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Abstract

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E-mail: [email protected] (Y. Ning), [email protected] (Y. Zhao).

A new urostylid ciliate, Anteholosticha songi nov. spec., isolated from forest soil in Tibet, and an American population of Holosticha pullaster (Müller, 1773) Foissner et al., 1991, isolated from a freshwater pond in the USA, are investigated in terms of their morphology, ontogenesis, and molecular biology. Anteholosticha songi nov. spec. is characterized by a slender to ellipsoidal body measuring 160–205 × 40–55 μm in vivo; rod-shaped yellowish cortical granules arranged in irregular short rows; four dorsal kineties; adoral zone consisting of 35–40 membranelles; three frontal, one 1

buccal, one parabuccal, two frontoterminal, two pretransverse, and four to six transverse cirri and 14– 25 midventral pairs; 12–22 ellipsoidal macronuclear nodules longitudinally arranged in pairs left of cell mid-line. Supplemental information on morphogenesis in Holosticha pullaster is also presented. The phylogenetic relationship of Anteholosticha and Holosticha inferred from SSU rDNA sequence data are concordant with previous studies and showing that Holosticha is monophyletic whereas Anteholosticha is polyphyletic and should be split into two or more genera.

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Keywords: China; Morphogenesis; SSU rDNA; Taxonomy; Urostylida

Introduction

About 200 hypotrich species have been reported in the past decade (e.g., Bai et al. 2018; Berger

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2001, 2006, 2008, 2011; Deng et al. 2018; Foissner 2016; Jung and Berger 2019; Kumar et al. 2018; Li et al. 2018; Lu et al. 2018; Luo et al. 2017; Lynn 2008; Méndez-Sánchez et al. 2018; Shao et al.

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2007, 2019; Wang et al. 2019a; Zhang et al. 2018; Zhu et al. 2019). Nevertheless, recent studies have indicated that there are many more taxa awaiting discovery (e.g., Li et al. 2017, 2019; Luo et al. 2018;

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Lyu et al. 2018a, b; Shao et al. 2013, 2015; Song and Shao 2017; Song et al. 2009). Furthermore, the classification, validity, and delimitation of some of these hypotrich species have been problematic and

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have been resolved differently by various authors (Gao et al. 2016, 2017; Huang et al. 2010, 2016; Lyu et al. 2018c; Sheng et al. 2018). Among them, the taxonomic history and complexity of

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Holosticha and its allies are rather complicated and have been debated by many authors (Berger 2003, 2006; Chen et al. 2018; Fan et al. 2016; Zhao et al. 2015).

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Holosticha was established by Wrześniowski (1877) and over the following ca. 125 years numerous nominal species were described from marine, limnetic and terrestrial habitats (for details, see Berger 2003, 2006; Gong et al. 2001; Hu et al. 2001, 2003; Kim et al. 2017; Lei et al. 2005; Luo et al. 2015; Song and Wilbert 1997, 2002; Song et al. 2002; Wilbert and Song 2008). Holosticha kessleri (Wrześniowski, 1877), a subjective junior synonym of H. gibba (Müller, 1786), is the type species by subsequent designation (Borror 1972). Holosticha was taxonomically revised by Berger (2003, 2006, 2008), who divided its members into four genera, namely Holosticha Wrześniowski, 1877, 2

Anteholosticha Berger, 2003, Caudiholosticha Berger, 2003 and Biholosticha Berger, 2003. To date, there are eight valid species of Holosticha, however phylogenetic relationships among these are largely unresolved due to the lack of molecular data for most of them (Berger 2003, 2006, 2008; Luo et al. 2015). Anteholosticha was established for species previously assigned to Holosticha sensu Kahl (1932) and Borror (1972) that lack appropriate apomorphies (for details, see Berger 2003, 2006). Analyses of the molecular phylogeny and patterns of morphogenesis have revealed that this genus is extremely divergent (Berger 2006, 2008; Chen et al. 2018; Huang et al. 2014; Lv et al. 2015; Park et al. 2012,

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2013; Zhao et al. 2015). Consequently, in order to gain a better understanding of the systematics and taxonomy of Anteholosticha, the 27 constituent species need to be reinvestigated using modern methods including molecular phylogenetic analyses.

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In the present paper, we describe a new species of Anteholosticha, i.e., A. songi nov. spec., and

reinvestigate an American population of Holosticha pullaster (Müller, 1773) Foissner et al., 1991. We

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also present their phylogenetic relationships inferred from SSU rDNA sequence data and describe the

Material and methods

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morphogenesis of Holosticha pullaster in order to evaluate their systematic relationships.

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Sampling, morphology and morphogenesis

Anteholosticha songi nov. spec. was isolated from the upper 5 cm layer of soil in the Motuo Virgin

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Forest (29°25′41′′N, 95°24′14′′E), Tibet, most of which is over 2000 m above sea level. The mean annual temperature is 12–16 °C and annual precipitation is 2300 mm. The vegetation is dominated by

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Lagerstroemia minuticarpa, Schefflera heptaphylla, Sambucus williamsii and Senecio scandens. Four soil samples (about 500 g each) were collected in December 2016 when the air temperature was 15 °C and soil pH 6.5. The samples were air-dried for one month and then sealed in a large paper envelope until they were processed during May to October 2018. The non-flooded Petri dish method was used to stimulate ciliates to excyst (Foissner et al. 2002). Clonal cultures were established at room temperature (about 25 °C) in Petri dishes using mineral water (Nongfu Spring) with rice grains to

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enrich the growth of bacteria as a food resource for the ciliates (Foissner et al. 2002). Other hypotrichs such as Pattersoniella sp., Hemiurosoma sp. and Gonostomum sp. were also found in samples. Holosticha pullaster was collected from the surface water (0–10 cm) of a small freshwater pond near the Duke Garden in the center of Duke University campus (36°00′02″N, 78°56′08″W), North Carolina, USA on 11 January 2015 (for details of location, see URL: https://gardens.duke.edu/visit/duke-gardens-map and Fig. 1). The water temperature was 10 °C and pH 7.0. Specimens were isolated and a clonal culture was maintained using the same methods as for A. songi nov. spec.

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Living and stained specimens were observed by bright field and differential interference contrast

microscopy (Leica DM5000). Protargol staining was utilized to reveal the infraciliature and nuclear

apparatus (Wilbert 1975). The protargol powder was made according to Pan et al. (2013). Counts and

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measurements of cellular structures in stained specimens were performed at a magnification of 1000×. Drawings of stained specimens were performed with the help of a drawing device. Terminology and

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DNA extraction and PCR amplification

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classification are mainly according to Berger (2006) and Lynn (2008).

A single cell of each species was isolated, washed and transferred into a microfuge tube (1.5 ml).

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DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen, CA) according to manufacturer instructions and Huang et al. (2018). The SSU rDNA sequence was amplified using the universal

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primers EukA and EukB (Medlin et al. 1988). PCR conditions, cycling parameters and sequencing

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were according to Wang et al. (2019b).

Phylogenetic analyses The newly sequenced SSU rDNA of Anteholosticha songi nov. spec. and Holosticha pullaster, plus

another 100 hypotrich ciliate sequences obtained from GenBank database (for accession numbers, see Fig. 7), were used to infer phylogenetic relationships of Anteholosticha and Holosticha. The GenBank accession numbers of the seven species of Oxytrichidae are as follows: Gastrostyla steinii AF508758, Sterkiella nova AF508771, Pleurotricha lanceolata AF508768, Sterkiella histriomuscorum 4

AF508770, Stylonychia mytilus AF508774, Cyrtohymena citrina AF164135, Paraurostyla weissei AF508767. Six euplotids, namely Uronychia setigera, U. multicirrus, Diophrys scutum, Paradiophrys zhangi, Apodiophrys ovalis and Pseudodiophrys nigricans, were selected as the outgroup taxa. The sequences were aligned using the MUSCLE program implemented in the GUIDANCE 2 web server (http://guidance.tau.ac.il/ver2). Subsequently, the sequences were manually edited to achieve a better alignment. The final alignment was used to construct phylogenetic trees by maximum likelihood (ML) and Bayesian inference (BI) analyses via the CIPRES Science Gateway web server (http://www.phylo.org) (Miller et al. 2010). ML analysis was carried out using RAxML-HPC2 on

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XSEDE (8.2.10) employing the GTRGAMMA model with 1000 bootstrap replicates (Stamatakis 2014). BI analysis was implemented in the program MrBayes (Ronquist and Huelsenbeck 2003)

under the best-fit model GTR+I+G selected by Akaike Information Criterion (AIC) in MrModeltest

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v2 (Nylander 2004). Markov chain Monte Carlo simulations were run with two sets of four chains for 10,000,000 generations and trees were sampled every 100 generations. The first 25% of each run were

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discarded as burn-in and the remaining trees were used to calculate posterior probabilities for nodes in

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the majority rule consensus tree. Trees were visualized using MEGA 6.0 (Tamura et al. 2013).

ZooBank LSID of Anteholosticha songi nov. spec.: urn:lsid:zoobank.org:pub:3629FDBC-970B-

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4F9A-927C-2CBDE2720365

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Results

Subclass Hypotrichia Stein, 1859

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Order Urostylida Jankowski, 1979 Family Urostylidae Bütschli, 1889 Genus Anteholosticha Berger, 2003 Anteholosticha songi nov. spec. Diagnosis: Body 160–205 × 40–55 μm in vivo, slender-ellipsoidal, colorless. 12–22 macronuclear nodules. Contractile vacuole located at about mid-body. Cortical granules yellowish, rod-shaped, 2 ×

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1 µm in vivo, arranged in irregular short rows on ventral side and in sparse clusters on dorsal side. 35–40 adoral membranelles and 40 right marginal, 41 left marginal, five transverse cirri and 20 midventral pairs. Midventral complex extends about 90% down length of body. One buccal, one parabuccal, three frontal, two frontoterminal and two pretransverse cirri. Four bipolar dorsal kineties. Soil habitat. Type locality: Surface soil of Motuo Virgin Forest (29°25′41′′N, 95°24′14′′E), Tibet, China. For details, see Material and Methods. Material deposited: The slide containing the holotype (Fig. 2H, I; registration number

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CLY2016120602A) and one paratype (registration number CLY2016120602B) slide have been

deposited in the Laboratory of Protozoology, Ocean University of China. Another paratype slide

(registration number NHMUK 2019.10.15.1) has been deposited in the Natural History Museum, UK.

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All slides are protargol preparations.

Etymology: This species is named after the eminent ciliatologist Prof. Dr. Weibo Song, Ocean

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University of China, Qingdao, for his contributions to the taxonomy of ciliates in China.

songi nov. spec. is MK713372.

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Gene sequence: The GenBank accession number for the SSU rDNA sequence of Anteholosticha

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Morphological description of Anteholosticha songi nov. spec. (Figs. 2A–I, 3A–K; Table 1) Body 160–205 × 40–55 μm in vivo, length to width ratio approximately 3.5–4:1 in living cells,

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3.7:1 in stained cells. Body non-contractile but flexible, usually slender to ellipsoidal in shape, anterior end more or less narrowed, posterior broadly rounded; left margin slightly convex, right

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margin slightly concave in normal cells (Figs. 2A, 3A–C). Dorsoventrally flattened about 1.5:1 (Fig. 3F). Cytoplasm colourless containing numerous refringent globules measuring 5–10 μm in diameter, colorless globules (about 1–5 μm across) and ingested green algae (about 5–25 μm across) in midbody and posterior region (Figs. 2A, 3A–C, E). Cortical granules yellowish, rod-shaped, about 2 × 1 µm in vivo, arranged in short irregular rows on ventral side and in sparse clusters on dorsal side (Figs. 2B, C, 3G). Contractile vacuole about 18 µm across, located slightly anterior of mid-body near left

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cell margin, with anterior and posterior collecting canals, pulsating at intervals of 10 s (Figs. 2D, E, 3C, D). Approximately 16 (12–22) ellipsoidal macronuclear nodules, sometimes arranged in pairs, longitudinally aligned at left of mid-line; micronuclei not observed in vivo and protargol preparations (Figs. 2G, I, 3E, J, Table 1). Locomotion by moderate-to-rapid crawling on bottom of Petri dish or among debris, or by swimming while rotating about its longitudinal axis. Adoral zone occupies 28–35% of cell length in vivo, 25–30% in protargol-stained cells, composed of 35–40 membranelles (Figs. 2A, F, H, 3A, H). Bases of largest membranelles about 18 μm long (Figs. 2A, 3A, B). Distal portion of adoral zone extends dorsally and slightly toward the right side,

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with a DE-value of 0.12 in the holotype specimen (Figs. 2 F, H, 3H; for explanation of DE-value, see Berger 2006, p. 18). Endoral and paroral relatively short, latter extends slightly further anteriorly and posteriorly than the former (Figs. 2F, H, 3H). Frontal cirri approximately 15 μm long in vivo, other

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somatic cirri 10–15 μm long. Frontal cirri inconspicuous, form oblique pseudorow close behind distal portion of adoral zone, buccal cirrus located at level of anterior end of endoral, parabuccal (= III/2)

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cirrus behind rightmost frontal cirrus (= III/3). Two frontoterminal cirri near anterior end of midventral complex. Midventral complex composed of 14–25 pairs of cirri arranged in a zig-zag

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pattern, extending about 90% down length of body (Figs. 2F, H, 3H, I). Single left and right marginal row composed of 35–48 and 33–46 cirri, respectively; marginal rows overlap at posterior end of cell

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(Fig. 2H). Four to six transverse cirri, each protrudes slightly beyond posterior cell margin. One or two (due to lack of ontogenetic data, we cannot be precise) pretransverse cirri situated right of

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transverse cirri (Figs. 2H, 3I). Four bipolar dorsal kineties, comprising 17–28, 17–25, 17–26, and 22–

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31 dikinetids, respectively (Figs. 2I, 3K).

Morphology of American population of Holosticha pullaster (Müller, 1773) Foissner et al., 1991 (Figs. 4A–F, 5A–G; Table 1) Body 70–80 × 30–40 μm in vivo, 53–81 × 22–34 μm in protargol-preparations; long elliptical to broadly fusiform, widest at about mid-body, anterior body portion usually more or less curved leftwards (Figs. 4A, B, 5A, B). Pellicle flexible. Cytoplasm colourless, usually with numerous refringent globules ca. 5 µm in diameter and food vacuoles (Figs. 4A, B, 5A, B). Cortical granules 7

lacking. Contractile vacuole situated approximately 70% down body length, approximately 12 μm in diameter (Figs. 4C, D, 5B, D). Invariably two macronuclear nodules located right of body midline (Figs. 4B, F, 5E–G). Locomotion by slowly crawling on substrate or on bottom of Petri dish, occasionally jerking back and forth (twitching movement). Somatic ciliature as shown in Fig. 4E, F. Adoral zone occupies about 40% of body length in protargol-stained specimens, bipartite with conspicuous gap between distal and proximal part which comprise 6–10 (on average 8) and 13–16 (on average 14) membranelles, respectively (Figs. 4E, 5E, F). Distal end of adoral zone extends down right-ventral side of cell with DE-value of 0.37 (Fig. 4E).

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Paroral and endoral almost equal in length, slightly curved leftward and optically intersect in midregion (Figs. 4E, 5E, F).

Cilia of apical membranelles and ventral cirri roughly 10–12 μm long in vivo, those of transverse

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cirri about 14 μm long. Constantly three slightly enlarged frontal cirri and one buccal cirrus distinctly ahead of undulating membranes (Figs. 4E, 5E, F). One parabuccal cirrus in frontal portion and two

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relatively fine frontoterminal cirri positioned behind rightmost frontal cirrus (= III/3). Midventral complex ends about at level of anteriormost transverse cirri, composed of 6–8 (on average 7) pairs of

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cirri arranged in a typical zigzag pattern. Usually 8–10 (on average 9) transverse cirri, although in one divider 11 were present in opisthe (Fig. 6G), well developed, arranged in a J-shaped pseudorow (Figs.

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4E, 5E, F). Posterior ends of marginal rows separated by transverse cirri. Left marginal row with 9–16 cirri, anterior portion (three cirri) distinctly curved rightwards. Right marginal row with 10–15 cirri,

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adjacent to midventral complex (Figs. 4E, 5E, F). Four more or less bipolar dorsal kineties comprising

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in total about 53 dikinetids (Figs. 4F, 5G; Table 1).

Notes on morphogenesis of Holosticha pullaster (Müller, 1773) Foissner et al., 1991 (Figs. 5H–

M, 6A–H)

Only middle and middle-late stages of binary fission were found. Stomatogenesis

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The very first stage in the dedifferentiation of the parental adoral zone of membranelles was not observed. During the middle stage, new membranelles begin to organize in a posteriad direction formed from the oral primordium in the opisthe (Figs. 5H, 6A). In the middle-late stage, the anterior end of the newly built adoral zone for the opisthe bends to the right and the differentiation of membranelles is completed (Figs. 5J, L, 6C, E, G). The parental adoral zone is retained completely intact by the proter. The undulating membranes anlage (anlage I) appears independently in both proter and opisthe since there is no connection with the oral primordium. The leftmost frontal cirrus is generated from the anterior end of anlage I in each daughter (Figs. 5H, 6A). Each undulating

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membrane anlage splits longitudinally to form two streaks from which the paroral and endoral are originate (Figs. 5J, L, 6C, E, G).

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Somatic ciliature

In middle-stage dividers, two axial groups of frontal-ventral-transverse cirral anlagen (FVT-

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anlagen) are formed, each consisting of nine or 10 short diagonal streaks (Figs. 5H, 6A). The posterior field, which will form the cirri of the opisthe, lies mostly to the right of the parental midventral

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complex at about the same level as the oral primordium; the anterior field will form the cirri of the proter (Figs. 5H, 6A). It is unknown whether each group remains united where it crosses the

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midventral row in earlier stages of division. In addition, we cannot confirm whether the cirri of the parental midventral complex contribute to the formation of the FVT-anlagen.

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In the next stage, the FVT cirri differentiate and begin to migrate to their final positions. The frontoterminal cirri are the two anteriormost cirri of the last FVT cirral streak which subsequently

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move anteriad (Figs. 5H, J, L, 6C, E, G). Streak I becomes the leftmost frontal cirrus, streak II provides the middle frontal and the buccal cirrus, streak III produces the rightmost frontal and parabuccal cirrus, streaks IV to n-2 each develops a midventral cirral pair and a transverse cirrus, streak n-1 provides a midventral cirral pair, a pretransverse ventral and a transverse cirrus, and the last streak (streak n) develops the two frontoterminal cirri, a pretransverse ventral cirrus and one transverse cirrus (Figs. 5H, J, L, 6C, E, G).

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Finally, in the opisthe, new marginal cirral anlagen develop within the parental structures on each side whereas in the proter they develop beside the parental structures, ahead of the parental row and intrakinetally on the left side. All these anlagen move further apart to replace the old structures, which are now being resorbed (Figs. 5H, J, L, 6C, E, G). The formation of the dorsal kineties follows the Holosticha pattern, i.e. the four dorsal kinety anlagen are formed beside the old kineties in both dividers. The anlage of dorsal kinety 2 develops de novo to the right and left of parental dorsal kinety 2 in the proter and opisthe, respectively (double arrowheads in Fig. 6). Thus, four dorsal kinety anlagen are formed in total. No caudal cirri develop at

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the end of the dorsal kineties.

Development of macronuclear apparatus

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The macronuclear apparatus develops in the usual way, i.e., during the middle-late stage, the two

macronuclear nodules fuse to a single mass; the division of the micronucleus was not observed (Figs.

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5I, K, M, 6B, D, F, H).

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Deposition of voucher slides of Holosticha pullaster

Eight protargol slides have been deposited as voucher slides (registration numbers

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CLY2015011102/A–H) in the Laboratory of Microbiota, College of Life Science, Northwest Normal

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University, Lanzhou, China.

SSU rDNA sequence data and phylogenetic analyses (Fig. 7)

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The SSU rDNA sequences of Holosticha pullaster (American population) and Anteholosticha songi nov. spec. have been deposited in GenBank. The length, GC content and GenBank accession number of these sequences are: 1650 bp, 45.82% and MK713373 for Holosticha pullaster, and 1648 bp, 45.57% and MK713372 for Anteholosticha songi nov. spec. Topologies of the SSU rDNA tree inferred from ML and BI methods are nearly congruent, therefore only the ML tree is shown here (Fig. 7). The genus Holosticha is monophyletic (ML/BI, 100/1.00) and is the sister group to the genus Uncinata. The new population of H. pullaster (MK713373) 10

clusters with two populations of H. diademata (KF306396 and DQ059583) and H. heterofoissneri (DQ09582 and KP717082) with low support (less than 50/0.50). Anteholosticha songi nov. spec. clusters tightly with two Pseudourostyla species (P. cristatoides JN88767 and P. nova KJ645976) with high support values (ML/BI, 99/1.00). This clade is the sister group to Anteholosticha monilata (HM568416) (ML/BI, 96/1.00).

Discussion Anteholosticha songi nov. spec.

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Morphological comparison with related taxa

Anteholosticha is a speciose genus with more than 40 nominal species (Berger 2003, 2006, 2008; Chen et al. 2018; Fan et al. 2014, 2016; Jung et al. 2016; Kumar et al. 2010; Li et al. 2007, 2008,

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2011; Park et al. 2012, 2013; Xu et al. 2011). However, it is not a monophyletic group and lacks

morphological apomorphies (Berger 2003, 2006; Fan et al. 2014; Li et al. 2009; Lv et al. 2015; Lyu et

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al. 2018c; Zhao et al. 2015).

Anteholosticha songi nov. spec. matches the diagnosis of the genus Anteholosticha (Berger 2003,

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2006, 2008). The key diagnostic features of A. songi nov. spec. are that it has fewer than 30 macronuclear nodules, four dorsal kineties, and the midventral complex extends more or less to the

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posterior end of the cell. Thus it can be easily separated from its congeners (Table 2). In terms of body shape and size, somatic ciliature, possession of cortical granules, and habitat, two

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congeners, namely A. australis (Blatterer and Foissner, 1988) Berger, 2003 and A. sigmoidea (Foissner, 1982) Berger, 2003, should be compared with A. songi nov. spec. in detail.

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With reference to the general infraciliature, Anteholosticha australis resembles A. songi nov. spec. However, the former can be distinguished from the latter by having fewer adoral membranelles (27– 33 vs. 35–40) and midventral pairs (7–11 vs. 14–25), a shorter adoral zone relative to the body length in vivo (27% vs. 28–35%) and the appearance of the left marginal row which is almost confluent posteriorly with the right marginal in A. australis (vs. the posterior part of the left marginal row is bent to the right side of the body in A. songi nov. spec.) (for revision, see Berger 2006).

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Anteholosticha songi nov. spec. differs from A. sigmoidea by its larger body size in vivo (160–205 × 40–55 μm vs. 90–130 × 20–30 μm) and having more adoral membranelles (35–40 vs. 16–28), midventral pairs (14–25 vs. 10) and macronuclear nodules (12–22 vs. 5–12) (for revision, see Berger 2006). Patterns of morphogenesis have long been used to infer evolutionary relationships among ciliates in general and urostylids in particular (Berger 2006; Foissner 1996). Even though morphogenetic data of Anteholosticha are only available for ten species, i.e., A. monilata, A. multistilata, A. manca, A. marimonilata, A. multicirrata, A. heterocirrata, A. pulchra, A. paramanca, A. randani, and A.

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intermedia (Berger 2006, 2008; Chen et al. 2018; Fan et al. 2014, 2016; Jung et al. 2016; Kumar et al. 2010; Li et al. 2007, 2008, 2011; Park et al. 2012, 2013; Xu et al. 2011), members of this genus are

known to be highly diverse in terms of their modes of ontogenesis. For example, the parental adoral

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zone can be inherited intact by the proter or be completely resorbed; the undulating membranes can be inherited intact by the proter or the anlage can dedifferentiate; and the FVT-anlagen can be generated

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in one of two ways, i.e. from the primary primordia or form separately (Berger 2006, 2008; Chen et al. 2018). Therefore, as morphogenetic data become available for more nominal species of

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Anteholosticha, a taxonomic revision of the genus will likely become necessary.

Identification

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Holosticha pullaster (Müller, 1773) Foissner et al., 1991

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Currently there are eight valid species of Holosticha Wrześniowski, 1877: H. gibba (Müller, 1786) Wrześniowski, 1877, H. diademata (Rees, 1884) Kahl, 1932, H. pullaster (Müller, 1773) Foissner et

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al., 1991, H. foissneri Petz et al., 1995, H. spindleri Petz et al., 1995, H. heterofoissneri Hu and Song, 2001, H. hamulata Lei et al., 2005, and H. muuiensis Kim et al., 2017. Holosticha pullaster can be easily separated from its congeners by the pattern of its infraciliature and the position of its contractile vacuole (Berger 2003, 2006). The identity of the American population of H. pullaster is therefore not in doubt (Fig. 4A–F, 5A–D; Table 1).

Comparison with other populations of Holosticha pullaster 12

Holosticha pullaster was originally discovered by Müller (1773) in an infusion of plant material and was subsequently redescribed by numerous researchers (for details, see Berger 2006). The most detailed redescriptions were provided by Wang and Nie (1932), Foissner et al. (1991) and Petz et al. (1995). The American population of H. pullaster closely resembles these descriptions although it shows slight differences in terms of body size in vivo (70–80 × 30–40 µm vs. 50–70 × 20–26 µm) and the numbers of adoral membranelles (21–24, on average 22 vs. 10–21, on average 19), midventral cirral pairs (6–8, on average 7 vs. 10–11, on average 10), transvers cirri (8–10, on average 9 vs. 6–8, on average 7), left (9–16, on average 11 vs. 9–12, on average 9) and right (10–15, on average 12 vs.

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9–11, on average 10) marginal cirri. These differences are likely due to their widely separated habitats and habitat types, i.e., freshwater habitats in Denmark, Germany, France, Romania, and Switzerland, a littoral area in Hungary, and marine habitats in Antarctica (Wedell Sea) and China (Bay of Amoy)

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(Foissner et al. 1991; Gellért and Tamás 1959; Müller 1773, 1786; Petz et al. 1995; Sterki 1878;

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Vuxanovici 1963; Wang and Nie 1932).

Morphogenesis

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Hemberger (1982) reported the morphogenetic process of a European population of H. pullaster, which is basically similar to that of the American population reported here. Some early stages are

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lacking in our study, therefore it remains unclear whether the formation of FVT-anlagen is by way of primary primordia, i.e. common anlagen for both dividers. To date, primary primordia have only been

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recognized for H. heterofoissneri (Hu and Song 2001). Here we report that the formation of the frontoterminal cirri in H. pullaster is plesiomorphic, i.e. they are formed from the last streak of the

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FVT-anlage (streak n). This clearly differs from Hemberger’s illustration (Berger 2006, Fig. 28e), so we consider Hemberger’s observation to be a misinterpretation. The present study reveals that the anlage for the left marginal row of the proter originates de novo, a feature that is also known for H. heterofoissneri and H. diademata (Berger 2006; Hu and Song 2001) indicating that this is an apomorphy for Holosticha (Berger 2003). Additionally, the formation of the dorsal kineties in H. pullaster is in the typical pattern for Holosticha whereby each of the four dorsal kinety anlagen is formed beside a parental dorsal kinety. In the present study, the anlage of dorsal kinety 2 develops de 13

novo to the right and left of parental dorsal kinety 2 in the proter and opisthe, respectively. This information on the dorsomarginal kinety largely substantiates Hemberger’s original observations, which were once thought to be questionable due to their insufficiently detailed description (Berger 2006; Hemberger 1982).

Phylogenetic analyses The genera Anteholosticha and Holosticha were established with the following combination of characters: three distinct frontal cirri, a zig-zag midventral complex composed only of cirral pairs,

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transverse cirri, single left and right marginal rows and without caudal cirri (Berger 2003, 2006; Hu and Song 2001; Kim et al. 2017; Li et al. 2009; Lv et al. 2015). Phylogenetic relationships within

Anteholosticha and Holosticha inferred from SSU rDNA sequence data (Fig. 7) are consistent with

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previous studies and show that Holosticha is monophyletic whereas Anteholosticha is polyphyletic

(Gao et al. 2010; Kim et al. 2017; Luo et al. 2015; Lv et al. 2015; Park et al. 2013; Shao et al. 2011;

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Xu et al. 2011; Yi et al. 2010; Zhao et al. 2015).

In the present study, all populations of the genus Holosticha form a clade, albeit with low support

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(Fig. 7). An environmental sequence of an indeterminate species of Holosticha (AY462947) clusters with members of the Oxytrichidae rather than with Holosticha, suggesting that its taxonomic identity

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is doubtful. It is noteworthy that three populations of H. heterofoissneri were scattered within the genus Holosticha. However, morphological information is only available for one of these (KP717082)

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and the identity of the other two remains uncertain. In addition, the tight cluster formed by two populations of Holosticha cf. heterofoissneri (ML/BI, 95/0.98) is distinctly separated from the three

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populations of H. heterofoissneri, suggesting that “Holosticha cf. heterofoissneri” might be a distinct species (Luo et al. 2015). The diagnostic characters that distinguish Anteholosticha from Holosticha are not supported by

SSU rDNA phylogenies (Luo et al. 2015; Lv et al. 2015; Park et al. 2013; Shao et al. 2011; Xu et al. 2011; Zhao et al. 2015). The present SSU rDNA tree shows that Anteholosticha is non-monophyletic since A. songi nov. spec. groups with two species of Pseudourostyla (P. cristatoides and P. nova), and other Anteholosticha species are scattered among different branches of the urostylid assemblage (Fig. 14

7). This is consistent with previous findings and supports the assertion that Anteholosticha is a heterogeneous collection of species that lacks morphological synapomorphies and should probably be divided into separate genera (Berger 2003, 2006; Gao et al. 2010; Li et al. 2009; Lv et al. 2015; Kim et al. 2017). The first such step was recently taken with the establishment of the genus Caudiholosticha by Li et al. (2017).

Author contributions Y.N designed and supervised the research study. L.C, J.D and Y.Z wrote the manuscript. W.W and

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Y.X collected samples and performed staining. A.W revised and improved the manuscript.

Acknowledgements

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This work was supported by the National Natural Science Foundation of China (project numbers:

41761056 to Y. Ning, 31702009 to J. Li), the Project for Enhancing the Research Capability of Young

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Teachers and undergraduate students in Northwest Normal University (NWNU-LKQN-16-11, NWNU-LKQN-18-9 and NWNU2019KT159) and the Project for Enhancing the Innovation

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Capability of Higher School in Gansu province (2019A-001). Our thanks are also given to Dr. Helmut

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Berger and anonymous reviewers for improving this manuscript.

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Wang, Y., Wang, C., Jiang, Y., Katz, L.A., Gao, F., Yan, Y., 2019b. Further analyses of variation of ribosome DNA copy number and polymorphism in ciliates provide insights relevant to studies of both molecular ecology and phylogeny. Sci. China Life Sci. 62, 203–214. Wilbert, N., Song, W.B., 2008. A further study on littoral ciliates (Protozoa, Ciliophora) near King George Island, Antarctica, with description of a new genus and seven new species. J. Nat. Hist. 42, 979–1012. Wilbert, N., 1975. Eine verbesserte Technik der Protargolimprägnation für Ciliaten. Mikrokosmos 64, 171–179.

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Yi, Z., Lin, X., Warren, A., Al-Rasheid, K.A.S., Song, W.B., 2010. Molecular phylogeny of

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Nothoholosticha (Protozoa, Ciliophora, Urostylida) and systematic relationships of the Holosticha-complex. Syst. Biodivers. 81, 149–155.

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Zhang, T.Y., Qi, H., Zhang, T.T., Sheng, Y., Warren, A., Shao, C., 2018. Morphology, morphogenesis and molecular phylogeny of a new brackish water subspecies, Neourostylopsis flava paraflava

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nov. subsp. (Ciliophora, Hypotrichia, Urostylidae), with redefinition of the genus Neourostylopsis. Eur. J. Protistol. 66, 48–62.

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Zhao, X., Gao, S., Fan, Y., Strueder-Kypke, M., Huang, J., 2015. Phylogenetic framework of the systematically confused Anteholosticha-Holosticha complex (Ciliophora, Hypotrichia) based on

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multigene analysis. Mol. Phylogenet. Evol. 91, 238–247. Zhu, E., Ba, S., Lyu, Z., Li, J., Shao, C., 2019. Morphogenesis and molecular phylogeny of the soil ciliate Holostichides chardezi (Ciliophora, Hypotrichia, Bakuellidae), with redefinition of Holostichides Foissner, 1987 and establishment of a new genus Anteholostichides. J. Eukaryot. Microbiol. 66, 730–739.

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Figure legends

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Fig. 1. A–D. Locations of the sample sites. (A) Map showing the location of Motuo, Tibet Autonomous Region. (B) Location where Anteholosticha songi nov. spec. was collected. (C, D)

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Location where Holosticha pullaster was collected.

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Fig. 2. A–I. Morphology of Anteholosticha songi nov. spec. from life (A–E) and after staining with

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protargol (F–I). (A) Ventral view of a representative individual. (B) Anterior body portion in dorsal view, showing the distribution of cortical granules. (C) Cortical granules in top view. (D, E) Ventral views of other individuals showing contractile vacuole; arrows in E denote the collecting canals. (F)

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Detailed ventral view of the anterior region, showing the frontal cirri, parabuccal cirrus, frontoterminal cirri and buccal cirrus (arrow). (G) Ventral view showing macronuclear nodules mainly

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arranged in pairs. (H, I) Ciliature of ventral and dorsal side and macronuclear nodules of holotype specimen; arrow depicts the frontoterminal cirri; arrowheads depict the overlapping marginal rows.

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AZM, adoral zone of membranelles; FC, frontal cirri; FT, frontoterminal cirri; LMR, left marginal row; Ma, macronuclear nodules; MP, midventral pairs; P, paroral; PBC, parabuccal cirrus; PTVC, pretransverse ventral cirri; RMR, right marginal row; TC, transverse cirri; 1–4, dorsal kineties. Scale bars = 70 μm.

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Fig. 3. A–K. Photomicrographs of Anteholosticha songi nov. spec. from life (A–G) and after protargol preparation (H–K). (A) Ventral view of a typical specimen. (B, C) Showing the midventral cirri

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(arrows in B) and the green algae (arrows in C). (D) Showing the collecting canal (arrows). (E) showing the green algae (arrows). (F) Lateral view. (G) Ventral surface showing the irregularly

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arranged cortical granules (arrows). (H) Infraciliature of anterior body portion showing the buccal cirrus (arrowhead) and midventral pairs (in circle). (I) Infraciliature of posterior region in ventral view

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showing the pseudopairs of the midventral complex (arrowheads). (J) Ventral view of central body region showing the infraciliature and macronuclear nodules. (K) Dorsal view of anterior body portion showing dorsal kineties (1–4). AZM, adoral zone of membranelles; CV, contractile vacuoles; FC, frontal cirri; FT, frontoterminal cirri; LMR, left marginal row; Ma, macronuclear nodules; MP, midventral pairs; PC, parabuccal cirrus; PTVC, pretransverse ventral cirri; RMR, right marginal row; TC, transverse cirri; 1–4, dorsal kineties. Scale bars = 75 μm (A–C); 15 μm (H, J–K). 27

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Fig. 4. A–F. Morphology of Holosticha pullaster from life (A–D) and after staining with protargol (E,

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F). (A) Ventral view of a typical cell. (B–D) Ventral view of other individuals showing different body shapes. (E, F) Ciliature of ventral and dorsal side and macronuclear nodules of same specimen, arrow

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depicts buccal cirrus, arrowhead marks the conspicuous gap of the adoral zone, double arrowhead indicates the transversely oriented left marginal cirri. AZM1, distal part of adoral zone of membranelles; AZM2, proximal part of adoral zone of membranelles; E, endoral; FC, frontal cirri;

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FTC, frontoterminal cirri; LMR, left marginal row; Ma, macronuclear nodules; MP, midventral pairs; P, paroral; PBC, parabuccal cirrus; PTVC, pretransverse ventral cirri; RMR, right marginal row; TC,

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transverse cirri; 1–4, dorsal kineties. Scale bars = 30 μm.

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Fig. 5. A–M. Photomicrographs of Holosticha pullaster in vivo (A–D) and after protargol staining

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(E–M). (A) Ventral view of a representative cell. (B–D) Ventral views of different individuals

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showing more or less strongly squeezed cells, arrowheads demonstrate the dorsal cilia. (E, F) Ciliature of ventral side, arrow indicates the distinct gap in the AZM, arrowhead marks the buccal

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cirrus. (G) Dorsal view showing dorsal kineties (1–4). (H, I) Ventral and dorsal views of an earlymiddle stage of division, double arrowhead marks the buccal cirrus, arrowheads indicate the right marginal anlagen, arrow marks the de novo anlage of left marginal row for proter. (J, K) Ventral and

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dorsal views of a slightly later divider. (L, M) Ventral and dorsal views of a later divider, arrowheads indicate the migrating frontoterminal cirri in proter. AZM, adoral zone of membranelles; AZM1, distal

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part of adoral zone of membranelles; AZM2, proximal part of adoral zone of membranelles; CV, contractile vacuole; FC, frontal cirri; FTC, frontoterminal cirri; LMR, left marginal row; Ma,

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macronuclear nodules; MP, midventral pairs; PBC, parabuccal cirrus; PTVC, pretransverse ventral cirri; RMR, right marginal row; TC, transverse cirri; 1–4, dorsal kineties. Scale bars = 30 μm.

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Fig. 6. A–H. Morphogenesis of Holosticha pullaster after protargol preparation (A–H). (A, B) Ventral and dorsal views of an early-middle stage of division. Arrowheads show the leftmost frontal cirrus

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generated from the anterior end of anlage I in both proter and opisthe, arrow marks the de novo anlage of left marginal row for proter, double arrowheads depict the anlage of dorsal kinety 2 developing de novo. (C, D) Ventral and dorsal views of a middle divider, double arrowheads depict the anlage of dorsal kinety 2 developing de novo. (E, F) Ventral and dorsal views of a slightly later divider, double arrowheads depict the anlage of dorsal kinety 2 developing de novo. (G, H) Ventral and dorsal views of a later divider. Double arrowheads depict the anlage of dorsal kinety 2 developing de novo. Broken 30

lines show the migrating frontoterminal cirri in both proter and opisthe. LMA, left marginal anlagen; Ma, macronuclear nodules; RMA, right marginal anlage; 1–4, new dorsal kineties. Scale bars = 40

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μm.

Fig. 7. Maximum likelihood (ML) tree inferred from the SSU rDNA sequences showing the

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representative taxa and newly sequenced species (in red). “–” indicates that the topologies differ between the BI and ML analyses. Fully supported (ML/BI, 100/1.00) branches are marked with solid

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circles. The scale bar corresponds to 0.02 expected substitutions per site.

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Table 1. Morphometric characterization of Anteholosticha songi nov. spec. (upper line) and Holosticha pullaster (Müller, 1773) Foissner et al., 1991 (lower line). Min

Max

Mean

M

SD

CV

n

Body length

168

223

191.5

186.5

22.7

11.9

8

53

81

64.0

65.0

6.7

10.5

20

40

70

53.0

53.5

10.7

20.1

8

22

34

27.4

29.0

4.0

14.5

20

49

62

55.5

56.0

5.0

9.0

8

22

30

26.1

27.0

2.1

8.2

19

35

40

36.6

36.5

1.6

4.4

8

21

24

21.9

22.0

1.1

5.0

20

-

-

-

6

10

-

Adoral zone, length

Adoral membranelles, total number

Membranelles in AZM1, number

Membranelles in AZM2, number

Buccal cirri, number

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Midventral pairs, number

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Midventral cirri, number

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Left marginal cirri, number

Right marginal cirri, number

Pretransverse ventral cirri, number

Transvers cirri, number

-

-

7.5

8.0

0.9

12.0

20

-

-

-

-

-

-

13

16

14.4

15.0

0.9

6.3

20

1

1

1.0

1.0

0

0

10

1

1

1.0

1.0

0

0

20

1

1

1.0

1.0

0

0

8

1

1

1.0

1.0

0

0

20

3

3

3.0

3.0

0

0

10

3

3

3.0

3.0

0

0

20

2

2

2.0

2.0

0

0

10

2

2

2.0

2.0

0

0

20

14

25

19.5

19.0

4.1

20.9

8

6

8

7.0

7.0

0.5

6.8

19

29

51

40.0

39.0

8.1

20.4

8

13

17

15.0

15.0

1.0

6.3

19

35

48

40.7

40.0

4.8

11.7

7

9

16

11.3

11.5

1.6

13.8

19

33

46

40.0

41.0

4.8

12.0

8

10

15

12.4

13.0

1.2

9.4

20

2

2

2.0

2.0

0

0

8

2

2

2.0

2.0

0

0

20

5

5

5.0

5.0

0

0

8

8

10

8.9

9.0

1.0

10.9

20

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Frontal cirri, number

Frontoterminal cirri, number

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Parabuccal cirri, number

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Body width

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Characteristica

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Dorsal kinety 1, number of bristles

Dorsal kinety 2, number of bristles

Dorsal kinety 3, number of bristles

Dorsal kinety 4, number of bristles

Macronuclear nodules, number

a

4

4

4.0

4.0

0

0

8

4

4

4.0

4.0

0

0

20

17

28

21.6

20.5

3.8

17.5

8

10

15

12.7

13.0

1.0

7.8

15

17

25

21.0

20.5

2.6

12.5

8

12

16

14.1

14.5

1.0

7.0

14

17

26

21.6

22.5

3.3

15.2

8

11

16

13.8

14.0

1.6

11.2

11

22

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25.1

25.0

2.9

11.3

8

9

15

12.2

12.0

1.7

14.2

13

12

22

15.6

15.0

3.0

19.3

8

2

2

2.0

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Dorsal kineties, number

2.0

0

0

20

All data are based on protargol-prepared specimens. Measurements in µm. Abbreviations: AZM1,

distal portion of adoral zone of membranelles; AZM2, proximal portion of adoral zone of

membranelles; CV, coefficient of variation in %; M, median; Max, maximum; Mean, arithmetic mean;

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Min, minimum; n, sample number; SD, standard deviation. “-” data unavailable.

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Table 2. Comparison of morphological features of Anteholosticha songi nov. spec. with those of other Anteholosticha species. Character A. songi nov. spec. A. australis A. sigmoidea Body, length in vivo (µm) 160–205 139–190 90–130 Body, width in vivo (µm) 40–55 30–40 20–30 Length:width ratio in vivo 3.5–4:1 4:1a Body, length (protargol, µm) 168–223 105–163 50–98 Body, width ( protargol, µm) 40–70 20–31 12–30 Length:width ratio(protargol) 3.7:1 5.2:1 3.9–5:1 Cortical granules Yellowish Colourless or Colourless yellowish Position of contractile vacuole Anterior 40% Anterior 46%a Anterior 44%a Collecting canals present present absent Ratio of adoral zone of membranelles in 28–35% 27% 25–33% vivo Adoral zone of membranelles, length 49–62 30–41 (µm) Adoral zone of membranelles, number 35–40 27–33 16–28 Buccal cirri, number 1 1 1 Frontal cirri, number 3 3 1–3 Parabuccal cirri, number 1 1 Frontoterminal cirri, number 2 2 2 Left marginal cirri, number 35–48 33–48 16–35 Right marginal cirri, number 33–46 34–43 15–33 Midventral pairs, number 14–25 7–11 9–12 Pretransverse cirri, number 2 1–2 1 or 2 Transverse cirri, number 4–6 3–6 3–6 Dorsal kineties, number 4 4 4 a Dikinetids in dorsal kinety 1, number 17–28 16 9–16a a Dikinetids in dorsal kinety 2, number 17–25 20 14–15a a Dikinetids in dorsal kinety 3, number 17–26 16 15–18a a Dikinetids in dorsal kinety 4, number 22–31 20 9–16a Macronuclear nodules, number 12–22 10–16 5–12 Micronuclei, number 1–4 2 or 3 Habitat terrestrial terrestrial terrestrial Data source Present work Berger (2006), Foissner (1982), Blatterer and Foissner (1984) Foissner (1988) a Data from drawings.

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