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European Journal of Protistology 72 (2020) 125646

Morphology and molecular phylogeny of a new hypotrich , Anteholosticha songi nov. spec., and an American population of Holosticha pullaster (Müller, 1773) Foissner et al., 1991 (Ciliophora, ) Lingyun Chena,1, Jingyi Dongb,1, Weining Wuc,1, Yuanyuan Xina, Alan Warrend, Yingzhi Ninga,∗, Yan Zhaoe,∗ aLaboratory of Microbiota, College of Life Science, Northwest Normal University, Lanzhou 730070, China bInstitute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China cCollege of Resources and Environmental Sciences, Gansu Agricultural University, Lanzhou 730070, China dDepartment of Life Sciences, Natural History Museum, London SW7 5BD, UK eCollege of Life Sciences, Capital Normal University, Beijing 100048, China

Received 29 August 2019; received in revised form 19 October 2019; accepted 21 October 2019 Available online 31 October 2019

Abstract

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 ␮m 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 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. © 2019 Elsevier GmbH. All rights reserved.

Keywords: China; Morphogenesis; SSU rDNA; ; Urostylida

Introduction 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 About 200 hypotrich species have been reported in the et al. 2007, 2019; Wang et al. 2019a; Zhang et al. 2018; Zhu past decade (e.g., Bai et al. 2018; Berger 2001, 2006, 2008, et al. 2019). Nevertheless, recent studies have indicated that 2011; Deng et al. 2018; Foissner 2016; Jung and Berger there are many more taxa awaiting discovery (e.g., Li et al. 2017, 2019; Luo et al. 2018; Lyu et al. 2018a,b; Shao et al. 2013, 2015; Song and Shao 2017; Song et al. 2009). Further- ∗Corresponding authors. more, the classification, validity, and delimitation of some E-mail addresses: [email protected] (Y. Ning), [email protected] of these hypotrich species have been problematic and have (Y. Zhao). been resolved differently by various authors (Gao et al. 2016, 1 These three authors contributed equally. https://doi.org/10.1016/j.ejop.2019.125646 0932-4739/© 2019 Elsevier GmbH. All rights reserved. 2 L. Chen, J. Dong, W. Wu et al. / European Journal of Protistology 72 (2020) 125646

2017; Huang et al. 2010, 2016; Lyu et al. 2018c; Sheng et al. pH 6.5. The samples were air-dried for one month and then 2018). Among them, the taxonomic history and complexity sealed in a large paper envelope until they were processed of Holosticha and its allies are rather complicated and have during May to October 2018. The non-flooded Petri dish been debated by many authors (Berger 2003, 2006; Chen method was used to stimulate to excyst (Foissner et al. et al. 2018; Fan et al. 2016; Zhao et al. 2015). 2002). Clonal cultures were established at room temperature Holosticha was established by Wrzesniowski´ (1877) and (about 25 ◦C) in Petri dishes using mineral water (Nongfu over the following ca. 125 years numerous nominal species Spring) with rice grains to enrich the growth of bacteria as were described from marine, limnetic and terrestrial habi- a food resource for the ciliates (Foissner et al. 2002). Other tats (for details, see Berger 2003, 2006; Gong et al. 2001; hypotrichs such as Pattersoniella sp., Hemiurosoma sp. and Hu and Song, 2001, Hu et al., 2003; Kim et al. 2017; Lei Gonostomum sp. were also found in samples. et al. 2005; Luo et al. 2015; Song and Wilbert 1997, 2002; Holosticha pullaster was collected from the surface Song et al. 2002; Wilbert and Song 2008). Holosticha kess- water (0–10 cm) of a small freshwater pond near the leri (Wrzesniowski,´ 1877), a subjective junior synonym of H. Duke Garden in the center of Duke University cam- gibba (Müller, 1786), is the type species by subsequent desig- pus (36◦0002N, 78◦5608W), North Carolina, USA nation (Borror 1972). Holosticha was taxonomically revised on 11 January 2015 (for details of location, see URL: by Berger (2003, 2006, 2008), who divided its members https://gardens.duke.edu/visit/duke-gardens-map and Fig. 1). into four genera, namely Holosticha Wrzesniowski,´ 1877, The water temperature was 10 ◦C and pH 7.0. Specimens Anteholosticha Berger, 2003, Caudiholosticha Berger, 2003 were isolated and a clonal culture was maintained using the and Biholosticha Berger, 2003. To date, there are eight valid same methods as for A. songi nov. spec. species of Holosticha, however phylogenetic relationships Living and stained specimens were observed by bright among these are largely unresolved due to the lack of molec- field and differential interference contrast microscopy (Leica ular data for most of them (Berger 2003, 2006, 2008; Luo DM5000). Protargol staining was utilized to reveal the infra- et al. 2015). ciliature and nuclear apparatus (Wilbert 1975). The protargol Anteholosticha was established for species previously powder was made according to Pan et al. (2013). Counts assigned to Holosticha sensu Kahl (1932) and Borror (1972) and measurements of cellular structures in stained specimens that lack appropriate apomorphies (for details, see Berger were performed at a magnification of 1000×. Drawings of 2003, 2006). Analyses of the molecular phylogeny and pat- stained specimens were performed with the help of a drawing terns of morphogenesis have revealed that this genus is device. Terminology and classification are mainly according extremely divergent (Berger 2006, 2008; Chen et al. 2018; to Berger (2006) and Lynn (2008). Huang et al. 2014; Lv et al. 2015; Park et al. 2012, 2013; Zhao et al. 2015). Consequently, in order to gain a better under- DNA extraction and PCR amplification standing of the systematics and taxonomy of Anteholosticha, the 27 constituent species need to be reinvestigated using A single cell of each species was isolated, washed and modern methods including molecular phylogenetic analyses. transferred into a microfuge tube (1.5 ml). DNA was extracted In the present paper, we describe a new species of Ante- using DNeasy Blood & Tissue Kit (Qiagen, CA) accord- holosticha, i.e., A. songi nov. spec., and reinvestigate an ing to manufacturer instructions and Huang et al. (2018). American population of Holosticha pullaster (Müller, 1773) The SSU rDNA sequence was amplified using the universal Foissner et al., 1991. We also present their phylogenetic primers EukA and EukB (Medlin et al. 1988). PCR condi- relationships inferred from SSU rDNA sequence data and tions, cycling parameters and sequencing were according to describe the morphogenesis of Holosticha pullaster in order Wang et al. (2019b). to evaluate their systematic relationships.

Phylogenetic analyses Material and methods The newly sequenced SSU rDNA of Anteholosticha Sampling, morphology and morphogenesis songi nov. spec. and Holosticha pullaster, plus another 100 hypotrich ciliate sequences obtained from GenBank Anteholosticha songi nov. spec. was isolated from the database (for accession numbers, see Fig. 7), were used upper 5 cm layer of soil in the Motuo Virgin Forest to infer phylogenetic relationships of Anteholosticha and (29◦2541N, 95◦2414E), Tibet, most of which is over Holosticha. The GenBank accession numbers of the 2000 m above sea level. The mean annual temperature is seven species of are as follows: Gastrostyla 12–16 ◦C and annual precipitation is 2300 mm. The vegeta- steinii AF508758, Sterkiella nova AF508771, Pleu- tion is dominated by Lagerstroemia minuticarpa, Schefflera rotricha lanceolata AF508768, Sterkiella histriomuscorum heptaphylla, Sambucus williamsii and Senecio scandens. AF508770, mytilus AF508774, Cyrtohymena Four soil samples (about 500 g each) were collected in citrina AF164135, Paraurostyla weissei AF508767. Six December 2016 when the air temperature was 15 ◦C and soil euplotids, namely Uronychia setigera, U. multicirrus, Dio- L. Chen, J. Dong, W. Wu et al. / European Journal of Protistology 72 (2020) 125646 3

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) Location where Holosticha pullaster was collected. phrys scutum, Paradiophrys zhangi, Apodiophrys ovalis and Family Urostylidae Bütschli, 1889 Pseudodiophrys nigricans, were selected as the outgroup Genus Anteholosticha Berger, 2003 taxa. The sequences were aligned using the MUSCLE pro- gram implemented in the GUIDANCE 2 web server Anteholosticha songi nov. spec (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 Diagnosis: Body 160–205 × 40–55 ␮m in vivo, slender- by maximum likelihood (ML) and Bayesian inference (BI) ellipsoidal, colorless. 12–22 macronuclear nodules. Contrac-

analyses via the CIPRES Science Gateway web server tile vacuole located at about mid-body. Cortical granules

(http://www.phylo.org)(Miller et al. 2010). ML analysis yellowish, rod-shaped, 2 × 1 ␮m in vivo, arranged in irregu- was carried out using RAxML-HPC2 on XSEDE (8.2.10) lar short rows on ventral side and in sparse clusters on dorsal employing the GTRGAMMA model with 1000 bootstrap side. 35–40 adoral membranelles and 40 right marginal, 41 replicates (Stamatakis 2014). BI analysis was implemented left marginal, five transverse cirri and 20 midventral pairs. in the program MrBayes (Ronquist and Huelsenbeck 2003) Midventral complex extends about 90% down length of body. under the best-fit model GTR+I+G selected by Akaike One buccal, one parabuccal, three frontal, two frontoterminal Information Criterion (AIC) in MrModeltest v2 (Nylander and two pretransverse cirri. Four bipolar dorsal kineties. Soil 2004). Markov chain Monte Carlo simulations were run with habitat. two sets of four chains for 10,000,000 generations and trees Type locality: Surface soil of Motuo Virgin Forest were sampled every 100 generations. The first 25% of each (29◦2541N, 95◦2414E), Tibet, China. For details, see run were discarded as burn-in and the remaining trees were Material and methods. used to calculate posterior probabilities for nodes in the majority rule consensus tree. Trees were visualized using Type material: The slide containing the holotype (Fig. 2H, MEGA 6.0 (Tamura et al. 2013). I; registration number CLY2016120602A) and one paratype (registration number CLY2016120602B) slide have been ZooBank LSID of present work: urn:lsid:zoobank. deposited in the Laboratory of Protozoology, Ocean Univer- org:pub:3629FDBC-970B-4F9A-927C-2CBDE2720365 sity of China. Another paratype slide (registration number NHMUK 2019.10.15.1) has been deposited in the Natural History Museum, UK. All slides are protargol preparations. Results Etymology: This species is named after the eminent cilia- tologist Prof. Dr. Weibo Song, Ocean University of China, Subclass Hypotrichia Stein, 1859 Qingdao, for his contributions to the taxonomy of ciliates in China. Order Urostylida Jankowski, 1979 Gene sequence: The GenBank accession number for the 4 L. Chen, J. Dong, W. Wu et al. / European Journal of Protistology 72 (2020) 125646

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

SSU rDNA sequence of Anteholosticha songi nov. spec. is Approximately 16 (12–22) ellipsoidal macronuclear nodules, MK713372. sometimes arranged in pairs, longitudinally aligned at left of mid-line; micronuclei not observed in vivo and protar- gol preparations (Figs. 2G, I, 3 E, J, Table 1). Locomotion by Morphological description of Anteholosticha moderate-to-rapid crawling on bottom of Petri dish or among songi nov. spec. (Figs. 2A–I, 3 A–K; Table 1) debris, or by swimming while rotating about its longitudinal axis.

Adoral zone occupies 28–35% of cell length in vivo,

Body 160–205 × 40–55 ␮m in vivo, length to width ratio approximately 3.5–4:1 in living cells, 3.7:1 in stained cells. 25–30% in protargol-stained cells, composed of 35–40

Body non-contractile but flexible, usually slender to ellip- membranelles (Figs. 2A, F, H, 3 A, H). Bases of largest ␮ soidal in shape, anterior end more or less narrowed, posterior membranelles about 18 m long (Figs. 2A, 3 A, B). Dis- broadly rounded; left margin slightly convex, right margin tal portion of adoral zone extends dorsally and slightly slightly concave in normal cells (Figs. 2A, 3 A–C). Dorsoven- toward the right side, with a DE-value of 0.12 in the trally flattened about 1.5:1 (Fig. 3F). Cytoplasm colourless holotype specimen (Figs. 2F, H, 3 H; for explanation of DE-value, see Berger 2006, p. 18). Endoral and paroral rel- containing numerous refringent globules measuring 5–10 ␮m atively short, latter extends slightly further anteriorly and in diameter, colorless globules (about 1–5 ␮m across) and posteriorly than the former (Figs. 2F, H, 3 H). Frontal

ingested green algae (about 5–25 ␮m across) in mid-body ␮ and posterior region (Figs. 2A, 3 A–C, E). Cortical gran- cirri approximately 15 m long in vivo, other somatic cirri

10–15 ␮m long. Frontal cirri inconspicuous, form oblique

ules yellowish, rod-shaped, about 2 × 1 ␮m in vivo, arranged in short irregular rows on ventral side and in sparse clus- pseudorow close behind distal portion of adoral zone, buccal ters on dorsal side (Figs. 2B, C, 3 G). Contractile vacuole cirrus located at level of anterior end of endoral, parabuc- cal (= III/2) cirrus behind rightmost frontal cirrus (= III/3). about 18 ␮m across, located slightly anterior of mid-body near left cell margin, with anterior and posterior collecting Two frontoterminal cirri near anterior end of midventral canals, pulsating at intervals of 10 s (Figs. 2D, E, 3 C, D). complex. Midventral complex composed of 14–25 pairs of L. Chen, J. Dong, W. Wu et al. / European Journal of Protistology 72 (2020) 125646 5

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 (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 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 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 ␮m (A–C); 15 ␮m (H, J–K). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) cirri arranged in a zig-zag pattern, extending about 90% Morphology of American population of down length of body (Figs. 2F, H, 3 H, I). Single left Holosticha pullaster (Müller, 1773) Foissner and right marginal row composed of 35–48 and 33–46 et al., 1991 (Figs. 4A–F, 5 A–G; Table 1) cirri, respectively; marginal rows overlap at posterior end

of cell (Fig. 2H). Four to six transverse cirri, each pro-

Body 70–80 × 30–40 ␮m in vivo, 53–81 × 22–34 ␮min trudes slightly beyond posterior cell margin. One or two protargol-preparations; long elliptical to broadly fusiform, (due to lack of ontogenetic data, we cannot be precise) pre- widest at about mid-body, anterior body portion usually transverse cirri situated right of transverse cirri (Figs. 2H, more or less curved leftwards (Figs. 4A, B, 5 A, B). Pel- 3 I). Four bipolar dorsal kineties, comprising 17–28, licle flexible. Cytoplasm colourless, usually with numerous 17–25, 17–26, and 22–31 dikinetids, respectively (Figs. 2I, refringent globules ca. 5 ␮m in diameter and food vacuoles 3 K). (Figs. 4A, B, 5 A, B). Cortical granules lacking. Contrac- tile vacuole situated approximately 70% down body length, 6 L. Chen, J. Dong, W. Wu et al. / European Journal of Protistology 72 (2020) 125646

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

Characteristica 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 Body width 40 70 53.0 53.5 10.7 20.1 8 22 34 27.4 29.0 4.0 14.5 20 Adoral zone, length 49 62 55.5 56.0 5.0 9.0 8 22 30 26.1 27.0 2.1 8.2 19 Adoral membranelles, total number 35 40 36.6 36.5 1.6 4.4 8 21 24 21.9 22.0 1.1 5.0 20 Membranelles in AZM1, number – – – – – – – 6 10 7.5 8.0 0.9 12.0 20 Membranelles in AZM2, number – – – – – – – 13 16 14.4 15.0 0.9 6.3 20 Buccal cirri, number 1 1 1.0 1.0 0 0 10 1 1 1.0 1.0 0 0 20 Parabuccal cirri, number 1 1 1.0 1.0 0 0 8 1 1 1.0 1.0 0 0 20 Frontal cirri, number 3 3 3.0 3.0 0 0 10 3 3 3.0 3.0 0 0 20 Frontoterminal cirri, number 2 2 2.0 2.0 0 0 10 2 2 2.0 2.0 0 0 20 Midventral pairs, number 14 25 19.5 19.0 4.1 20.9 8 6 8 7.0 7.0 0.5 6.8 19 Midventral cirri, number 29 51 40.0 39.0 8.1 20.4 8 13 17 15.0 15.0 1.0 6.3 19 Left marginal cirri, number 35 48 40.7 40.0 4.8 11.7 7 9 16 11.3 11.5 1.6 13.8 19 Right marginal cirri, number 33 46 40.0 41.0 4.8 12.0 8 10 15 12.4 13.0 1.2 9.4 20 Pretransverse ventral cirri, number 2 2 2.0 2.0 0 0 8 2 2 2.0 2.0 0 0 20 Transvers cirri, number 5 5 5.0 5.0 0 0 8 8 10 8.9 9.0 1.0 10.9 20 Dorsal kineties, number 4 4 4.0 4.0 0 0 8 4 4 4.0 4.0 0 0 20 Dorsal kinety 1, number of bristles 17 28 21.6 20.5 3.8 17.5 8 10 15 12.7 13.0 1.0 7.8 15 Dorsal kinety 2, number of bristles 17 25 21.0 20.5 2.6 12.5 8 12 16 14.1 14.5 1.0 7.0 14 Dorsal kinety 3, number of bristles 17 26 21.6 22.5 3.3 15.2 8 11 16 13.8 14.0 1.6 11.2 11 Dorsal kinety 4, number of bristles 22 31 25.1 25.0 2.9 11.3 8 9 15 12.2 12.0 1.7 14.2 13 Macronuclear nodules, number 12 22 15.6 15.0 3.0 19.3 8 2 2 2.0 2.0 0 0 20

a All data are based on protargol-prepared specimens. Measurements in ␮m. 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; Min, minimum; n, sample number; SD, standard deviation. “–” data unavailable.

approximately 12 ␮m in diameter (Figs. 4C, D, 5 B, D). imens, bipartite with conspicuous gap between distal and Invariably two macronuclear nodules located right of body proximal part which comprise 6–10 (on average 8) and 13–16 midline (Figs. 4B, F, 5 E–G). Locomotion by slowly crawling (on average 14) membranelles, respectively (Figs. 4E, 5 E, on substrate or on bottom of Petri dish, occasionally jerking F). Distal end of adoral zone extends down right-ventral side back and forth (twitching movement). of cell with DE-value of 0.37 (Fig. 4E). Paroral and endoral Somatic ciliature as shown in Fig. 4E, F. Adoral zone almost equal in length, slightly curved leftward and optically occupies about 40% of body length in protargol-stained spec- intersect in mid-region (Figs. 4E, 5 E, F). L. Chen, J. Dong, W. Wu et al. / European Journal of Protistology 72 (2020) 125646 7

Fig. 4. A–F. Morphology of Holosticha pullaster from life (A–D) and after staining with protargol (E, 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 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; 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, transverse cirri; 1–4, dorsal kineties.

Scale bars = 30 ␮m.

Cilia of apical membranelles and ventral cirri roughly Stomatogenesis

10–12 ␮m long in vivo, those of transverse cirri about 14 ␮m long. Constantly three slightly enlarged frontal cirri and The very first stage in the dedifferentiation of the parental one buccal cirrus distinctly ahead of undulating membranes adoral zone of membranelles was not observed. During the (Figs. 4E, 5 E, F). One parabuccal cirrus in frontal portion middle stage, new membranelles begin to organize in a poste- and two relatively fine frontoterminal cirri positioned behind riad direction formed from the oral primordium in the opisthe rightmost frontal cirrus (= III/3). Midventral complex ends (Figs. 5H, 6 A). In the middle-late stage, the anterior end about at level of anteriormost transverse cirri, composed of of the newly built adoral zone for the opisthe bends to the 6–8 (on average 7) pairs of cirri arranged in a typical zigzag right and the differentiation of membranelles is completed pattern. Usually 8–10 (on average 9) transverse cirri, although (Figs. 5J, L, 6 C, E, G). The parental adoral zone is retained in one divider 11 were present in opisthe (Fig. 6G), well completely intact by the proter. The undulating membranes developed, arranged in a J-shaped pseudorow (Figs. 4E, 5 E, anlage (anlage I) appears independently in both proter and F). Posterior ends of marginal rows separated by transverse opisthe since there is no connection with the oral primordium. cirri. Left marginal row with 9–16 cirri, anterior portion (three The leftmost frontal cirrus is generated from the anterior end cirri) distinctly curved rightwards. Right marginal row with of anlage I in each daughter (Figs. 5H, 6 A). Each undulating 10–15 cirri, adjacent to midventral complex (Figs. 4E, 5 E, membrane anlage splits longitudinally to form two streaks F). Four more or less bipolar dorsal kineties comprising in from which the paroral and endoral originate (Figs. 5J, L, 6 total about 53 dikinetids (Figs. 4F, 5 G; Table 1). C, E, G).

Somatic ciliature Notes on morphogenesis of Holosticha pullaster (Müller, 1773) Foissner et al., 1991 (Figs. 5H–M, In middle-stage dividers, two axial groups of frontal- 6 A–H) ventral-transverse cirral anlagen (FVT-anlagen) are formed, each consisting of nine or 10 short diagonal streaks (Figs. 5H, Only middle and middle-late stages of binary fission were 6 A). The posterior field, which will form the cirri of the opis- found. the, lies mostly to the right of the parental midventral complex 8 L. Chen, J. Dong, W. Wu et al. / European Journal of Protistology 72 (2020) 125646

Fig. 5. A–M. Photomicrographs of Holosticha pullaster in vivo (A–D) and after protargol staining (E–M). (A) Ventral view of a representative cell. (B–D) Ventral views of different individuals 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 cirrus. (G) Dorsal view showing dorsal kineties (1–4). (H, I) Ventral and dorsal views of an early-middle 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 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 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, 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 ␮m. at about the same level as the oral primordium; the anterior The formation of the dorsal kineties follows the Holosticha field will form the cirri of the proter (Figs. 5H, 6 A). It is pattern, i.e. the four dorsal kinety anlagen are formed beside unknown whether each group remains united where it crosses the old kineties in both dividers. The anlage of dorsal kinety the midventral row in earlier stages of division. In addition, 2 develops de novo to the right and left of parental dor- we cannot confirm whether the cirri of the parental midventral sal kinety 2 in the proter and opisthe, respectively (double complex contribute to the formation of the FVT-anlagen. arrowheads in Fig. 6). Thus, four dorsal kinety anlagen are In the next stage, the FVT cirri differentiate and begin to formed in total. No caudal cirri develop at the end of the dorsal migrate to their final positions. The frontoterminal cirri are kineties. the two anteriormost cirri of the last FVT cirral streak which subsequently move anteriad (Figs. 5H, J, L, 6 C, E, G). Streak Development of macronuclear apparatus I becomes the leftmost frontal cirrus, streak II provides the middle frontal and the buccal cirrus, streak III produces the The macronuclear apparatus develops in the usual way, i.e., rightmost frontal and parabuccal cirrus, streaks IV to n-2 during the middle-late stage, the two macronuclear nodules each develops a midventral cirral pair and a transverse cirrus, fuse to a single mass; the division of the micronucleus was streak n-1 provides a midventral cirral pair, a pretransverse not observed (Figs. 5I, K, M, 6 B, D, F, H). ventral and a transverse cirrus, and the last streak (streak n) develops the two frontoterminal cirri, a pretransverse ventral Deposition of voucher slides of Holosticha cirrus and one transverse cirrus (Figs. 5H, J, L, 6 C, E, G). Finally, in the opisthe, new marginal cirral anlagen develop pullaster within the parental structures on each side whereas in the pro- ter they develop beside the parental structures, ahead of the Eight protargol slides have been deposited as voucher parental row and intrakinetally on the left side. All these anla- slides (registration numbers CLY2015011102/A–H) in the gen move further apart to replace the old structures, which Laboratory of Microbiota, College of Life Science, North- west Normal University, Lanzhou, China. are now being resorbed (Figs. 5H, J, L, 6 C, E, G). L. Chen, J. Dong, W. Wu et al. / European Journal of Protistology 72 (2020) 125646 9

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 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 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 ␮m. SSU rDNA sequence data and phylogenetic Topologies of the SSU rDNA tree inferred from ML analyses (Fig. 7) and BI methods are nearly congruent, therefore only the ML tree is shown here (Fig. 7). The genus Holosticha is The SSU rDNA sequences of Holosticha pullaster (Amer- monophyletic (ML/BI, 100/1.00) and is the sister group to ican population) and Anteholosticha songi nov. spec. have the genus Uncinata. The new population of H. pullaster been deposited in GenBank. The length, GC content and (MK713373) clusters with two populations of H. diade- GenBank accession number of these sequences are: 1650 bp, mata (KF306396 and DQ059583) and H. heterofoissneri 45.82% and MK713373 for Holosticha pullaster, and 1648 (DQ09582 and KP717082) with low support (less than bp, 45.57% and MK713372 for Anteholosticha songi nov. 50/0.50). spec. 10 L. Chen, J. Dong, W. Wu et al. / European Journal of Protistology 72 (2020) 125646

Fig. 7. Maximum likelihood (ML) tree inferred from the SSU rDNA sequences showing the 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 circles. The scale bar corresponds to 0.02 expected substitutions per site. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Anteholosticha songi nov. spec. clusters tightly with two than 30 macronuclear nodules, four dorsal kineties, and the Pseudourostyla species (P. cristatoides JN88767 and P. midventral complex extends more or less to the posterior end nova KJ645976) with high support values (ML/BI, 99/1.00). of the cell. Thus it can be easily separated from its congeners This clade is the sister group to Anteholosticha monilata (Table 2). (HM568416) (ML/BI, 96/1.00). In terms of body shape and size, somatic ciliature, posses- sion of cortical granules, and habitat, two congeners, namely A. australis (Blatterer and Foissner, 1988) Berger, 2003 and Discussion A. sigmoidea (Foissner, 1982) Berger, 2003, should be com- pared with A. songi nov. spec. in detail. Anteholosticha songi nov. spec With reference to the general infraciliature, Anteholosticha australis resembles A. songi nov. spec. However, the former Morphological comparison with related taxa can be distinguished from the latter by having fewer adoral Anteholosticha is a speciose genus with more than 40 nom- membranelles (27–33 vs. 35–40) and midventral pairs (7–11 inal species (Berger 2003, 2006, 2008; Chen et al. 2018; Fan vs. 14–25), a shorter adoral zone relative to the body length et al. 2014, 2016; Jung et al. 2016; Kumar et al. 2010; Li in vivo (27% vs. 28–35%) and the appearance of the left et al. 2007, 2008, 2011; Park et al. 2012, 2013; Xu et al. marginal row which is almost confluent posteriorly with the 2011). However, it is not a monophyletic group and lacks right marginal in A. australis (vs. the posterior part of the left morphological apomorphies (Berger 2003, 2006; Fanetal. marginal row is bent to the right side of the body in A. songi 2014; Li et al. 2009; Lv et al. 2015; Lyu et al. 2018c; Zhao nov. spec.) (for revision, see Berger 2006).

et al. 2015). Anteholosticha songi nov. spec. differs from A. sigmoidea

Anteholosticha songi nov. spec. matches the diagnosis of by its larger body size in vivo (160–205 × 40–55 ␮m vs. the genus Anteholosticha (Berger 2003, 2006, 2008). The key 90–130 × 20–30 ␮m) and having more adoral membranelles diagnostic features of A. songi nov. spec. are that it has fewer L. Chen, J. Dong, W. Wu et al. / European Journal of Protistology 72 (2020) 125646 11

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 (␮m) 160–205 139–190 90–130 Body, width in vivo (␮m) 40–55 30–40 20–30 Length:width ratioin vivo 3.5–4:1 4:1a -

Body, length (protargol, ␮m) 168–223 105–163 50–98

Body, width (protargol, ␮m) 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 yellowish Colourless Position of contractile vacuole 40% 46%a 44%a Collecting canals present present absent Ratio of adoral zone of membranelles in vivo 28–35% 27% 25–33% Adoral zone of membranelles, length (␮m) 49–62 30–41 - 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 Dikinetids in dorsal kinety 1, number 17–28 16a 9–16a Dikinetids in dorsal kinety 2, number 17–25 20a 14–15a Dikinetids in dorsal kinety 3, number 17–26 16a 15–18a Dikinetids in dorsal kinety 4, number 22–31 20a 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), Blatterer and Foissner (1988)Foissner (1982), Foissner (1984)

a Data from drawings. (35–40 vs. 16–28), midventral pairs (14–25 vs. 10) and Holosticha pullaster (Müller, 1773) Foissner macronuclear nodules (12–22 vs. 5–12) (for revision, see et al., 1991 Berger 2006). Patterns of morphogenesis have long been used to infer Identification evolutionary relationships among ciliates in general and Currently there are eight valid species of Holosticha urostylids in particular (Berger 2006; Foissner 1996). Even Wrzesniowski,´ 1877: H. gibba (Müller, 1786) Wrzesniowski,´ though morphogenetic data of Anteholosticha are only avail- 1877, H. diademata (Rees, 1884) Kahl, 1932, H. pullaster able for ten species, i.e., A. monilata, A. multistilata, A. (Müller, 1773) Foissner et al., 1991, H. foissneri Petz et al., manca, A. marimonilata, A. multicirrata, A. heterocirrata, 1995, H. spindleri Petz et al., 1995, H. heterofoissneri Hu and A. pulchra, A. paramanca, A. randani, and A. intermedia Song, 2001, H. hamulata Lei et al., 2005, and H. muuiensis (Berger 2006, 2008; Chen et al. 2018; Fan et al. 2014, 2016; Kim et al., 2017. Holosticha pullaster can be easily sepa- Jung et al. 2016; Kumar et al. 2010; Li et al. 2007, 2008, rated from its congeners by the pattern of its infraciliature and 2011; Park et al. 2012, 2013; Xu et al. 2011), members of this the position of its contractile vacuole (Berger 2003, 2006). genus are known to be highly diverse in terms of their modes The identity of the American population of H. pullaster is of ontogenesis. For example, the parental adoral zone can be therefore not in doubt (Fig. 4A–F, 5 A–D; Table 1). inherited intact by the proter or be completely resorbed; the undulating membranes can be inherited intact by the proter Comparison with other populations of Holosticha or the anlage can dedifferentiate; and the FVT-anlagen can be pullaster generated in one of two ways, i.e. from the primary primor- Holosticha pullaster was originally discovered by Müller dia or form separately (Berger 2006, 2008; Chen et al. 2018). (1773) in an infusion of plant material and was subsequently Therefore, as morphogenetic data become available for more redescribed by numerous researchers (for details, see Berger nominal species of Anteholosticha, a taxonomic revision of 2006). The most detailed redescriptions were provided by the genus will likely become necessary. Wang and Nie (1932), Foissner et al. (1991) and Petz et al. 12 L. Chen, J. Dong, W. Wu et al. / European Journal of Protistology 72 (2020) 125646

(1995). The American population of H. pullaster closely Hu and Song 2001; Kim et al. 2017; Li et al. 2009; Lv et al.

resembles these descriptions although it shows slight differ- 2015). Phylogenetic relationships within Anteholosticha and

ences in terms of body size in vivo (70–80 × 30–40 ␮m vs. Holosticha inferred from SSU rDNA sequence data (Fig. 7)

50–70 × 20–26 ␮m) and the numbers of adoral membranelles are consistent with previous studies and show that Holosticha (21–24, on average 22 vs. 10–21, on average 19), midventral is monophyletic whereas Anteholosticha is polyphyletic (Gao cirral pairs (6–8, on average 7 vs. 10–11, on average 10), et al. 2010; Kim et al. 2017; Luo et al. 2015; Lv et al. 2015; transvers cirri (8–10, on average 9 vs. 6–8, on average 7), Park et al. 2013; Shao et al. 2011; Xu et al. 2011; Yi et al. left (9–16, on average 11 vs. 9–12, on average 9) and right 2010; Zhao et al. 2015). (10–15, on average 12 vs. 9–11, on average 10) marginal In the present study, all populations of the genus Holosticha cirri. These differences are likely due to their widely sepa- form a clade, albeit with low support (Fig. 7). An environ- rated habitats and habitat types, i.e., freshwater habitats in mental sequence of an indeterminate species of Holosticha Denmark, Germany, France, Romania, and Switzerland, a (AY462947) clusters with members of the Oxytrichidae littoral area in Hungary, and marine habitats in Antarctica rather than with Holosticha, suggesting that its taxonomic (Wedell Sea) and China (Bay of Amoy) (Foissner et al. 1991; identity is doubtful. It is noteworthy that three popula- Gellért and Tamás 1959; Müller 1773, 1786; Petz et al. 1995; tions of H. heterofoissneri were scattered within the genus Sterki 1878; Vuxanovici 1963; Wang and Nie 1932). Holosticha. However, morphological information is only available for one of these (KP717082) and the identity of the other two remains uncertain. In addition, the tight cluster Morphogenesis formed by two populations of Holosticha cf. heterofoissneri (ML/BI, 95/0.98) is distinctly separated from the three popu- Hemberger (1982) reported the morphogenetic process of lations of H. heterofoissneri, suggesting that “Holosticha cf. a European population of H. pullaster, which is basically sim- heterofoissneri” might be a distinct species (Luo et al. 2015). ilar to that of the American population reported here. Some The diagnostic characters that distinguish Anteholosticha early stages are lacking in our study, therefore it remains from Holosticha are not supported by SSU rDNA phylo- unclear whether the formation of FVT-anlagen is by way of genies (Luo et al. 2015; Lv et al. 2015; Park et al. 2013; primary primordia, i.e. common anlagen for both dividers. Shao et al. 2011; Xu et al. 2011; Zhao et al. 2015). The To date, primary primordia have only been recognized for H. present SSU rDNA tree shows that Anteholosticha is non- heterofoissneri (Hu and Song 2001). Here we report that the monophyletic since A. songi nov. spec. groups with two formation of the frontoterminal cirri in H. pullaster is ple- species of Pseudourostyla (P. cristatoides and P. nova), and siomorphic, i.e. they are formed from the last streak of the other Anteholosticha species are scattered among different FVT-anlage (streak n). This clearly differs from Hemberger’s branches of the urostylid assemblage (Fig. 7). This is con- illustration (Berger 2006, Fig. 28e), so we consider Hem- sistent with previous findings and supports the assertion that berger’s observation to be a misinterpretation. The present Anteholosticha is a heterogeneous collection of species that study reveals that the anlage for the left marginal row of lacks morphological synapomorphies and should probably be the proter originates de novo, a feature that is also known divided into separate genera (Berger 2003, 2006; Gao et al. for H. heterofoissneri and H. diademata (Berger 2006; Hu 2010; Li et al. 2009; Lv et al. 2015; Kim et al. 2017). 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 Author contributions for Holosticha whereby each of the four dorsal kinety anla- gen is formed beside a parental dorsal kinety. In the present Y.N designed and supervised the research study. L.C, J.D study, the anlage of dorsal kinety 2 develops de novo to the and Y.Z wrote the manuscript. W.W and Y.X collected sam- right and left of parental dorsal kinety 2 in the proter and ples and performed staining. A.W revised and improved the opisthe, respectively. This information on the dorsomarginal manuscript. kinety largely substantiates Hemberger’s original observa- tions, which were once thought to be questionable due to their insufficiently detailed description (Berger 2006; Hemberger Acknowledgements 1982). This work was supported by the National Natural Sci- Phylogenetic analyses ence Foundation of China (project numbers: 41761056 to Y. Ning, 31702009 to J. Li), the Project for Enhancing the The genera Anteholosticha and Holosticha were estab- Research Capability of Young Teachers and undergraduate lished with the following combination of characters: three students in Northwest Normal University (NWNU-LKQN- distinct frontal cirri, a zig-zag midventral complex composed 16-11, NWNU-LKQN-18-9 and NWNU2019KT159) and only of cirral pairs, transverse cirri, single left and right the Project for Enhancing the Innovation Capability of Higher marginal rows and without caudal cirri (Berger 2003, 2006; School in Gansu province (2019A-001). Our thanks are L. Chen, J. Dong, W. Wu et al. / European Journal of Protistology 72 (2020) 125646 13 also given to Helmut Berger and anonymous reviewers for Namib desert. Part I: text and line drawings. Part II: photographs. improving this manuscript. Denisia 5, 1–1459. Foissner, W., Blatterer, H., Berger, H., Kohmann, F., 1–478 1991. Taxonomische und ökologische Revision der Ciliaten des Sapro- biensystems. 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