Two Urosoma species (Ciliophora, Hypotrichia): A multidisciplinary approach provides new insights into their ultrastructure and systematics

Journal Pre-proof Two Urosoma species (Ciliophora, Hypotrichia): a multidisciplinary approach provides new insights into their ultrastructure and systematics Jingyi Dong, Lifang Li, Xinpeng Fan, Honggang Ma, Alan Warren

PII:

S0932-4739(19)30098-7

DOI:

https://doi.org/10.1016/j.ejop.2019.125661

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EJOP 125661

To appear in:

European Journal of Protistology

Received Date:

18 June 2018

Revised Date:

18 November 2019

Accepted Date:

27 November 2019

Please cite this article as: Dong J, Li L, Fan X, Ma H, Warren A, Two Urosoma species (Ciliophora, Hypotrichia): a multidisciplinary approach provides new insights into their ultrastructure and systematics, European Journal of Protistology (2019), doi: https://doi.org/10.1016/j.ejop.2019.125661

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European Journal of Protistology

Two Urosoma species (Ciliophora, Hypotrichia): a multidisciplinary approach provides new insights into their ultrastructure and systematics Jingyi Donga, b, *, Lifang Lic, *, Xinpeng Fanb, **, Honggang Maa, **, Alan Warrend a

Institute of Evolution and Marine Biodiversity and Key Laboratory of Mariculture, Ministry

of Education, Ocean University of China, Qingdao 266003, China School of Life Sciences, East China Normal University, Shanghai 200241, China

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Marine College, Shandong University, Weihai 264209, China

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

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b

**Corresponding author

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Xinpeng Fan, e-mail: [email protected]

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*both authors contributed equally

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Honggang Ma, e-mail: [email protected]

Abstract

The general morphology and ultrastructure of two soil hypotrichous ciliates, Urosoma

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emarginata and U. salmastra, were investigated using light microscopy, scanning electron microscopy and transmission electron microscopy. Phylogenetic analyses, based on the newly sequenced small subunit ribosomal (SSU) rRNA genes, were conducted on three U. emarginata

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populations and one U. salmastra population. Our findings support for the validity of Perilemmaphora Berger, 2008, a rankless taxon comprising spirotrich ciliates having a

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perilemma. The cortical granules of both species are extrusomes representing a new type of mucocyst in U. emarginata and possibly a new type of pigmentocyst in U. salmastra. Additionally, the lithosomes were revealed as subglobose structures composed of a low electron-dense, homogeneous inner part and an electron-dense outer part. The ultrastructural features of the cortical granules, together with ontogenetic and molecular phylogenetic data, suggest that the genus Urosoma might need to be divided. It is posited that ultrastructural features of hypotrichous ciliates in general may have important taxonomic value warranting further investigation. 1

Keywords: Extrusome; Perilemma; Perilemmaphora; Spirotrichea; Ultrastructure; 18S rRNA gene

Introduction The hypotrichs are the most highly differentiated ciliate group, containing numerous taxa, many of which have unresolved phylogenetic relationships (Berger 1999, 2001; Borror 1972; Foissner 1982; Hewitt et al. 2003; Kahl 1932; Kumar et al. 2015; Li et al. 2018; Luo et al. 2018; Park K. et al. 2013; Park M.H. et al. 2017; Singh and Kamra 2015). In recent decades, a

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large number of new hypotrich species from a wide variety of habitats across the world have been described and many little-known species have been re-described (e.g., Bharti et al. 2015; Bourland 2015; Dragesco and Dragesco-Kernéis 1986; Foissner 1994, 1998, 2016; Foissner et al. 2002; Heber et al. 2014; Kumar and Foissner 2016; Lu et al. 2018; Luo et al. 2017;

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Méndez-Sánchez et al. 2018; Paiva et al. 2016; Valbonesi and Luporini 1990; Warren et al. 2002). However, systematic ambiguities persist, requiring the application of more detailed

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studies of their morphology, ontogenesis, and molecular phylogeny (Gao et al. 2017; Park et al. 2013; Schmidt et al. 2007; Shazib et al. 2016; Wang C. et al. 2017). Furthermore,

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taxonomic and phylogenetic conflicts between the morphological, ontogenetic and molecular evidence require reconciliation. These problems will undoubtedly require the accumulation of

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even larger amounts of molecular data (including whole genomes, transcriptomes and metatranscriptomes) as well as more detailed morphological and ontogenetic studies (Chen X. et

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al. 2016; Gentekaki et al. 2017; Hewitt et al. 2003; Huang et al. 2014; Wylezich et al. 2010; Xu et al. 2018; Yan et al. 2018; Zhang T. et al. 2018; Zhao et al. 2018). Most importantly, the

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molecular, morphological and ontogenetic data should not be considered in isolation but rather must be considered, together with ecological data, in an integrative approach (Clamp and Lynn 2017).

Like the ciliary pattern, many ultrastructural features are taxonomically informative for

ciliates (Lynn 2008). In hypotrichs, it has been demonstrated that features such as the extrusomes (cortical granules), the arrangment of microtubules associated with basal bodies, and features of the resting cyst have taxonomic value (Bakowska and Jerka-Dziadosz 1978, 1980; Bencatova et al. 2016; Farmer 1980; Foissner and Foissner 1987; Grim 1967, 1972; 2

Grimes 1973; Grimes and Adler 1976; Gutierrez et al. 1983; Li Q. et al. 2016; Tang et al. 2016; Torres et al. 1986; Wirnsberger and Hausmann 1988; Zhang X. et al. 2012, 2014). Ultrastructural information is, however, available for comparatively few hypotrich taxa, e.g., Pseudokeronopsis carnea, Paraurostyla weissei, Pseudourostyla nova; Gastrostyla steinii, Euplotes eurystomus, Engelmanniella mobilis, Euplotidium itoi, Uronychia transfuga, Stylonychia mytilus, and Oxytricha fallax (Grim 1967; Grim 1972; Grimes 1973; JerkaDziadosz and Frankel 1969; Jerka-Dziadosz 1982; Jerka-Dziadosz and Wienicka 1992; Lenzi and Rosati 1993; Morelli et al. 1996; Sui et al. 2001; Wirnsberger and Hausmann 1988; Wirnsberger-Aescht and Foissner 1989; Zhou et al. 2011).

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Urosoma Kowalewski, 1882 is a dorsomarginalian genus comprising 10 species that share a Gonostomum-like adoral zone. Furthermore, the parabuccal cirrus (III/2) is distinctly displaced anteriad (Berger 1999, 2008). In the present study, four Urosoma populations (three of U. emarginata and one of U. salmastra) were investigated morphologically and by molecular sequencing. In addition, the ultrastructure of Urosoma was studied by transmission electron

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microscopy for the first time. Several ultrastructural characters were revealed that might be applicable to the systematics of this other hypotrichous ciliates. The implications of these

Material and Methods

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Sampling and cultivation

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and systematics (Clamp and Lynn 2017).

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findings are discussed in the context of an integrative approach to investigate ciliate biodiversity

The Urosoma emarginata Lanzhou population was collected on 24 February 2015 from surface soil of a dry riverbed in Yintan Wetland Park (36°05'19"N; 109°42'55"E), about 13 km

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west of the city of Lanzhou, Gansu Province, China. The U. emarginata Wuhan population was collected on 4 May 2017 from surface soil in a Pinus massoniana forest in Ma'an Mountain

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Forest Park (30°30'48"N; 114°26'39"E), about 25 km southeast of the city of Wuhan, Hubei Province, China. The U. emarginata Saudi Arabia population was collected from soil at a farmland (26°22'01"N; 44°46'03"E), about 12 km northwest of Zulfi city (Az Zulfi), Riyadh Province, Saudi Arabia. All samples were collected from the upper 10 cm layer of soil. The non-flooded Petri dish method was applied to stimulate ciliates to excyst (Foissner 1987; Foissner et al. 2002). Urosoma salmastra was collected on 14 November 2015 from the surface of a marsh wetland, which is usually flooded but intermittently desiccates (water salinity 10 PSU), on Chongming Island (31°38'10"N; 121°33'05"E), Shanghai, China. 3

Several cells from each sample were isolated using micropipettes and cultivated at 26 ºC in Petri dishes using Nongfu Spring mineral water (U. emarginata) or brackish water prepared with Golden Trump sea salt (salinity 10 PSU; U. salmastra). Wheat grains were added to enrich bacterial food organisms.

Light microscopy Isolated cells were observed in vivo using bright field and differential interference contrast microscopy. The protargol method according to Wilbert (1975) was used to reveal the infraciliature. The protargol powder was prepared according to Pan et al. (2013). Measurements

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of protargol-stained specimens were performed with an ocular micrometer.

Scanning and transmission electron microscopy

Individuals for scanning electron microscopy (SEM) were prepared mainly according to the method reported by Gu and Ni (1993). Cells of U. emarginata (in about 1.4 ml culture

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medium) were fixed in a mixture containing 200 μl 1% OsO4, 600 μl saturated solution of HgCl2 and 600 μl 2.5% glutaraldehyde. Cells of U. salmastra (in about 1.4 ml culture medium) were

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fixed in a mixture containing 200 μl 1% OsO4 and 1200 μl saturated solution of HgCl2. The fixed cells were dehydrated in a graded series of ethanol, dried in a critical point dryer (Leica

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EM CPD300), and coated with gold by a sputter coater (Leica EM ACE600). Cells were observed using a scanning electron microscope (Hitachi S-4800) at an accelerating voltage of 10 kV.

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For transmission electron microscopy (TEM), specimens were treated according to the procedure in Gu et al. (2002). The ciliates (in about 700 μl culture medium) were fixed in a mixture containing 600 μl 2.5% glutaraldehyde and 100 μl 2% OsO4, post-fixed in 2% OsO4 in

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0.2 M cacodylate buffer and dehydrated in a graded acetone series. The specimens were embedded in Eponate 12 resin. Ultrathin sections (about 70 nm) were stained with uranyl

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acetate and lead citrate, and observed with a Hitachi HT7700 transmission electron microscope at an accelerating voltage of 100 kV.

Voucher material Two protargol slides of Urosoma emarginata Lanzhou population (DJY2015022402-01, 02), three protargol slides of U. emarginata Wuhan population (ZTY2017062301-01, -02, -03) and two protargol slides of U. salmastra (DJY2015111406-01, -02) have been deposited in the collection of the Laboratory of Protozoology, Ocean University of China (OUC), China. One 4

protargol slide (S007) of U. emarginata Saudi Arabia population has been deposited in the collection of the Laboratory of Protozoology, East China Normal University, China.

DNA extraction, PCR amplification, and sequencing Genomic DNA extraction was performed according to the following method: several cells of each population were isolated from the raw culture, washed five times with 0.22 μm filtered culture water to remove potential contamination and transferred into a 1.5 ml microcentrifuge tube with a minimum volume of water. The total genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) according to the

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methods described by Gao et al. (2016). Q5 Hot Start High-Fidelity DNA Polymerase (Cat. #M0493 L, New England Biolabs, USA) was used to amplify the small subunit (SSU) rRNA gene with primers 18S-F (5'-AACCTGGTTGATCCTGCCAGT-3') or 82-F (5'-

GAAACTGCGAATGGCTC-3') and 18S-R (5'-TGATCCTTCTGCAGGTTCACCTAC-3')

designed by Elwood et al. (1985) and Medlin et al. (1988). Conditions for polymerase chain

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reaction (PCR) amplification were according to Huang et al. (2016).

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Phylogenetic analyses

The SSU rRNA gene sequences of U. emarginata and U. salmastra were aligned with 41

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other hypotrich sequences obtained from the GenBank database using the online program GUIDANCE with default parameters (Penn et al. 2010). The alignments comprised a matrix of 1726 characters. Both ends of the alignments were trimmed and ambiguous columns were

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removed based on confidence scores <0.93 calculated by GUIDANCE. Maximum likelihood (ML) analyses were conducted with RAxML-HPC2 on XSEDE (8.2.10) (Stamatakis et al. 2008) on the CIPRES Science Gateway (http://www.phylo.org/sub sections/portal). The program

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MrModeltest v.2.0 (Nylander 2004) selected GTR + Ι + Γ as the best model of nucleotide substitution with Akaike Information Criterion (AIC), which was then used for Bayesian

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inference (BI) analysis. The BI analysis was performed with MrBayes on XSEDE 3.2.6, with two sets of four chains for 10,000,000 generations (Ronquist and Huelsenbeck 2003). The first 25% of sampled trees were discarded as burn-in prior to constructing the majority rule consensus tree from the remaining trees. MEGA 6.0 was used to visualize tree topologies (Talavera and Castresana 2007; Tamura et al. 2013).

Topology testing 5

To test the monophyly of the genus Urosoma, constrained ML trees enforcing the monophyly of all Urosoma populations were created in the RAxML framework (Stamatakis et al. 2008) and compared with the unconstrained (i.e. best scoring) ML tree topology. The approximately unbiased (AU) test was carried out using CONSEL ver. 0.1k (Shimodaira 2002, 2008; Shimodaira and Hasegawa 2001).

ZooBank LSID The genus Urosoma Kowalewskiego, 1882 has been registered in ZooBank (registration number: urn:lsid:zoobank.org:act:B832597B-4CB7-42A6-B2F8-3FCE3022A73A) including

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the two species: Urosoma emarginata (Stokes, 1885) Berger, 1999 (registration number: urn:lsid:zoobank.org:act:9DB46584-99F1-4B50-8FCE-86651B4DDF04) and Urosoma salmastra (Dragesco and Dragesco-Kernéis, 1986) Berger, 1999 (registration number:

urn:lsid:zoobank.org:act:67494C41-5E90-4399-BEA1-3AD849BE9D4E). The ZooBank

registration number of the present work is: urn:lsid:zoobank.org:pub:AC93FB11-EE04-43EC-

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B9C7-6F85D12B35FF.

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Terminology

Terminology is according to Berger (1999, 2008) and Foissner and Al-Rasheid (2006).

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Classification is according to Berger (1999, 2008). The terminology regarding ultrastructure is mainly according to Lynn (2008). Some infrequently used terms are as follows: Epiplasm: the fibrillar or filamentous pellicular layer directly underlying the alveoli and/or

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the plasma membrane.

Perilemma: additional outermost “unit membrane-like” structure, covering the pellicle. Lateral membranellar cilia: the cilia that derive from the rightmost adoral membranellar

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kinetosomes, project rightward parallel with the cell surface across the oral cavity, and may be used in prey selection and feeding.

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When used with reference to extrusomes, the verbs ‘eject’ and ‘extrude’ are used

interchangeably.

Results Urosoma emarginata (Stokes, 1885) Berger, 1999 (Figs 1A−P, 2A−K, 3A−D, 4A−I, 5A−J; Table 1) General morphology of Lanzhou population (Figs 1A−P, 2A−K, 11C−F; Table 1) 6

Body 150−185 μm × 25−50 μm in vivo (n = 5) (cells shrink markedly after SEM preparation), elongated, anterior end rounded, posterior end tapered (Fig. 1A, H, I). Cytoplasm colourless. Three to five (usually four) lithosomes longitudinally arranged left of ventral meridian and evenly spaced, spherical measuring up to 7 μm in diameter. Large vacuoles, each containing a single lithosome, often seen during live observation of cells compressed by coverslip (Fig. 1A, B). These vacuoles differ from food vacuoles because they remain stationary (i.e. do not undergo cyclosis) and their contents, i.e., lithosomes, are never digested. At low magnification, lithosomes usually ring-like in appearance, some with additional smaller inner “ring” (Fig. 1A, B). At high magnification, focusing continuously from dorsal to ventral side,

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inner and outer parts of lithosomes refract light differently (Fig. 1C−E). Lithosomes are not recognizable in protargol preparations (Fig. 11C–F). No distinct cortical granules observed in vivo, but numerous clusters of granules arranged in longitudinally oriented rows are seen in protargol and SEM preparations (Figs. 1F, 2A−K; see “cortical structure of the Lanzhou population” for detailed description). Contractile vacuole pore slightly above equatorial region

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of cell immediately left of dorsal kinety 2, about 5 μm long with discrete lip-like margin during diastole, elliptical during systole (Fig. 1I, M, N). Cytoproct slit-like, about 10 μm long, in

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posterior quarter of cell, immediately left of dorsal kinety 2 (Fig. 1I, O, P). Swims moderately rapidly while rotating about main cell axis or jerking hastily to and fro on substrate.

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Two ellipsoidal macronuclear nodules usually located in mid-body region left of cell midline, about 23−43 μm × 12−28 μm in size (Fig. 11C–F). One to seven (usually two) micronuclei, adjacent to macronuclear nodules, ovoid to ellipsoid, about 4 × 3 μm in size (Fig.

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11C–F).

Adoral zone about 30% of body length; membranellar cilia highly differentiated: (1) cilia of rows 1 to 3 with acicular ends (Fig. 1G); (2) row 4 usually consists of one acicular lateral

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membranellar cilium (located rightmost) facing buccal cavity margin, and two minute cilia with bluntly rounded ends (Fig. 1G, J, K); (3) rightmost kinetosome of row 2 sometimes bears one

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lateral membranellar cilium, otherwise barren of cilia (loss of cilia during preparation cannot be excluded) (Fig. 1K); (4) rightmost kinetosomes of rows 1 and 3 usually without cilia (Fig. 1K). Anteriormost membranelle bearing lateral membranellar cilium is located at level of anterior end of paroral membrane (Fig. 1G). Paroral membrane inserted in slot on buccal lip, cilia of paroral membrane about 3 μm long in SEM preparations (Fig. 1G, J). Three slightly enlarged frontal cirri about 20 μm long; single buccal cirrus located right of paroral membrane; frontoventral-transverse ciliature comprises 18 cirri (Figs. 1H, 11C, D). Approximately 90% of individuals have a conspicuous cortical groove in midline on ventral 7

side, extending from anterior postoral ventral cirrus (IV/2) to anterior pretransverse ventral cirrus (V/2) (Fig. 1H). One left and one right marginal row consisting of about 34 and 38 cirri on average, respectively; gap between posterior ends of right marginal row, which ends subterminally, and left marginal row, which extends around posterior end of cell (Figs 1A, H, I, 11C−F). Usually five transverse cirri arranged in a hook shaped-row, each cirrus with about 12 basal bodies, 1−3 of which are barren of cilia (Fig. 1L). Two pretransverse ventral cirri. Invariably four dorsal kineties, three bipolar and one dorsomarginal which is posteriorly shortened. Three caudal cirri (Figs. 1I, 11E, F).

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General morphology of populations from Saudi Arabia and Wuhan (Fig. 11A, B, G-N; Table 1)

Body size in vivo 70−103 μm × 15−20 μm in Wuhan population, 132−195 μm×20−24 μm in Saudi Arabia population. Body flexible but not contractile, anterior end broadly rounded, posterior end pointed. Single contractile vacuole. Cortical granules not observed in vivo,

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however numerous ellipsoidal to cylindrical granules observed in specimens after protargol staining and by SEM. Adoral zone composed of 28–33 membranelles in Saudi Arabia

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population, 25–31 membranelles in Wuhan population. Three enlarged frontal cirri, single buccal cirrus and four frontoventral cirri located right of buccal field. Usually four transverse

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cirri in a hook-shaped row in posterior region of cell, with two pretransverse ventral cirri close to them. Right marginal row comprises 41–57 cirri in Saudi Arabia population, 24–34 cirri in Wuhan population; left marginal row comprises 44–47 cirri in Saudi Arabia population, 23–36

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cirri in Wuhan population. Three dorsal kineties and one dorsomarginal kinety. Three caudal cirri, one at posterior end of each of dorsal kineties 1–3. For morphometric characterization of

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each population, see Table 1.

Cortical structure of the Lanzhou population (Figs 2A−K, 3A−D, 4A−I, 5A−J)

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Pellicle. The cell is covered by up to six unit membranes. In non-ciliated parts of the cell,

2−6 membranes are loosely arranged, the innermost and outermost membranes are about 100 nm apart (Fig. 3A). It is difficult to distinguish between the plasma membrane and perilemma. The inner unit membrane is considered to be the plasma membrane (Fig. 3C, D). In ciliated parts of the cell, cirri are covered by the plasma membrane and perilemma (Fig. 3B). Vesicular structures, bounded by inner and outer unit membranes and located below the plasma membrane, are considered to be cortical alveoli (Fig. 3C, D). An epiplasm-like structure with electron-dense strands is located beneath the cortical 8

alveoli and is closely associated with the underlying unit membrane (Fig. 3C, D). We cannot confirm that this structure is the epiplasm or a product of depolymerization of a microtubule layer as no sub-pellicular microtubules were seen. Cortical granules. Cortical granules were not observed in vivo, but numerous clusters of argyrophilic granules arranged in longitudinally oriented rows are apparent in protargol preparations (Fig. 1F). In SEM preparations, ellipsoidal to cylindrical structures 2.0 μm × 0.5 μm, i.e., with the same size and arrangement as the cortical granules seen in protargol preparations, can be seen extruding from the cell surface: on the dorsal side these are located in longitudinal rows between or near the dorsal kineties (Fig. 2D, I); on the ventral side they are

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located near the bases of the adoral membranelles and the marginal, ventral and transverse cirri, and are also distributed irregularly in the glabrous area between the marginal cirral rows (Fig. 2A−C, E). Residual "pits" can be observed after ejection (Fig. 2G, I). The surface of ejected and ejecting granules is decorated by spiraling ribs (Fig. 2F−H, J). Material of the central core protrudes from apical openings in the granules (Fig. 2F, J, K). In TEM preparations, the

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granules are invariably clustered near mitochondria. They are encased by electron-dense cytoplasm, occasionally together with paraglycogen particles (Fig. 4A−C, E). Each granule is

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enclosed by a unit membrane, is narrowly rounded distally and broadly rounded proximally (Fig. 4G−I). The granules comprise two parts: the central core which consists of electron-dense

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material, and the peripheral coat which is composed of more finely granular and less electrondense material (Fig. 4D, G−I). The core in some granules is more diffuse, possibly reflecting various stages of ejection (Fig. 4H, I, cf. Fig. 2F, J). Many short, electron-dense, rod-like

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fragments, possibly cytoplasmic structures ejected together with granule contents, surround ejected granules (Fig. 4F). The enveloping membrane of the granule appears to remain in the

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cortex following ejection (Fig. 4F, H, I). Cytoplasm and nuclear apparatus of Lanzhou population (Fig. 5A−J)

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The cytoplasm appears as low electron-dense cytosol, i.e., the “background”, and numerous

scattered electron-dense “islands”. Several short (about 0.2 μm long), stout, rod-like particles are scattered in the cytosol (Fig. 5b). Each “island” is about 5 μm across, surrounded by a membrane, and contains electron-dense cytosol, mitochondria, cortical granules, endoplasmic reticulum, and paraglycogen particles (Figs. 4E, 5a). Mitochondria are usually present in those “islands” close to the pellicle (Fig. 5A, H). The Golgi apparatus and transport vesicles are occasionally also observed in these “islands” (Fig. 5I). Lithosomes usually consist of two parts: a homogenous, low electron-dense center and an 9

electron-dense outer layer of variable thickness, occasionally with embedded paraglycogen particles (Fig. 5A−E, H). In longitudinal sections of the cell only a single center was observed in each lithosome (Fig. 5A−C). In transverse sections of the cell, however, a second, smaller center was observed in some lithosomes (Fig. 5D, E, H). The two elliptical macronuclear nodules each contain several electron-dense globular structures, most likely nucleoli, and scattered granular material, most likely chromatin (Fig. 5F). Occasionally multilamellate structures are seen attached to the macronuclear membrane (Fig. 5G). The micronuclei, usually adjacent to a macronuclear nodule, contain homogenous, electron-dense chromatin (Fig. 5F; Table 1). Numerous food vacuoles containing bacteria were

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also observed (Fig. 5J).

Urosoma salmastra (Dragesco and Dragesco-Kernéis, 1986) Berger, 1999 (Figs 6A−K, 7A−G, 8A−H, 9A−D; Table 1) General morphology (Fig. 6A−K; Table 1)

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Body 100−135 μm × 20−30 μm in vivo, elongate, anterior and posterior ends rounded, flexible but not contractile (Fig. 6A, E, F). Cytoplasm colourless. Posterior end usually packed

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with numerous irregular crystals rendering this part of the cell dark at low magnification with bright field illumination (Fig. 6A). Cortical granules spherical, arranged in short irregular rows

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(Fig. 6A−G; for detailed description, see “Cortical granules” in “Fine structure of cortex” below). Cytoproct located subterminally slightly left of dorsal kinety 2 (i.e. at same location as in U. emarginata), about 4 μm long, slit-like during diastole, elliptical during systole (Fig. 6F,

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H). Locomotion by moderately rapid swimming or jerking hastily to and fro on substrate. Two ellipsoidal macronuclear nodules about 9−25 μm × 4−11 μm in size, usually located along, or slightly left of, cell midline (Fig. 6B). Two micronuclei, about 2−5 μm × 1−3 μm in

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size, adjacent to macronuclear nodules (Fig. 6B). Adoral zone about 30% of body length, with typical Gonostomum-pattern. Paroral

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membrane comprises about 14 cilia arranged in a single row, located in anterior portion of slot on buccal lip; buccal lip extends to posterior end of adoral zone (Fig. 6D). Intermembranellar ridges of adoral zone inconspicuous (Fig. 6G). Membranellar cilia differentiated as follows: (1) cilia generally acicular (Fig. 6D, G); (2) rightmost kinetosomes of row 1 sometimes unciliated (Fig. 6J, K); (3) rightmost kinetosomes of rows 2 and 3 unciliated (Fig. 6J, K); (4) row 4 of each membranelle always composed of two minute cilia and one rightmost lateral membranellar cilium (Fig. 6D, J, K). One buccal cirrus; three enlarged frontal cirri about 12 μm long; frontoventral cirrus III/2 10

located anterior of level of cirrus VI/4; three postoral ventral cirri located beneath buccal vertex; usually four transverse cirri and one pretransverse ventral cirrus; one left and one right marginal row consisting of about 20−32 and 25−35 cirri, respectively (Fig. 6B, E). Four dorsal kineties and three caudal cirri (Fig. 6F). Fine structure of cortex (Figs. 7A−G, 8A−H, 9A−D) Pellicle. The cell surface is covered by a perilemma, a plasma membrane and cortical alveoli (Fig. 8A−C). The perilemma, composed of one or two unit membranes, lies near the plasma membrane and covers the entire cell surface including the cirri. The prominent cortical

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alveoli appear flattened (Fig. 8B, C). A longitudinal layer of microtubules lies beneath the inner alveolar membrane (Fig. 8B, C). One cytoplasmic membrane is sometimes observed beneath the microtubular layer and, in some parts of the cell, fuses with the inner alveolar membrane enclosing several microtubules (Fig. 8D); elsewhere it extends to the cell interior forming cytoplasmic vesicles (Fig. 8A, F).

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Cortical granules. The cortical granules are <0.5 μm in diameter in vivo, spherical, colorless, and arranged in short longitudinally oriented rows (Fig. 6C). In SEM preparations,

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numerous spherical, rough-surfaced, ejected granules, sometimes in short rows and with size similar to those seen in vivo, are seen on the dorsal and ventral surfaces (Fig. 7A−C, E−G). In

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TEM preparations, some ultrathin sections showed 3−5 granules, similar in arrangement and size to those observed by light microscopy (<0.5 μm in diameter), usually located in rows beneath the pellicle (Fig. 8G). The cortical granules appear as spherical, membrane-bound

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vesicles of varying electron density (Fig. 8G). Cortical granules eject through the pellicle, sometimes leaving partially collapsed vesicles (Fig. 8H). Cytoplasmic organelles and nuclear apparatus. The cytoplasm is a translucent colloidal

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matrix containing numerous large vesicles, granules and food vacuoles. Large, closely arranged, irregular, membrane-bound vesicles (about 3 μm in diameter), occupy most of the cytoplasm.

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These vesicles contain mitochondria, paraglycogen particles, cytoplasm, and, sometimes, small granules (Fig. 9C, D). Cytoplasmic granules are of two kinds: (1) small (about 0.1 μm diameter), spherical, electron-dense, scattered mainly beneath the pellicle; (2) large (0.2−0.7 μm diameter), of lower electron density, and distributed between the large vesicles (Fig. 9A). Food vacuoles are approximately 5 μm in diameter and contain large amounts of food debris, possibly of bacterial origin (Fig. 9C). Mitochondria are about 3 μm long, ellipsoidal with tubular cristae, usually contained in large vesicles, and mainly distributed under the cortex (Fig. 9B). Macronuclear nodules are composed of numerous irregular granular chromatin bodies that 11

are more or less homogenous and several nucleoli that contain scattered, punctate, electrondense particles (Fig. 9F). Micronuclei were not seen.

SSU rRNA gene sequences and phylogenetic analyses (Fig. 10) The accession numbers, lengths and GC content of the four newly sequenced SSU rRNA genes are as follows: U. salmastra Shanghai population (MH393884, 1729 bp, 45.58%), U. emarginata Lanzhou population (MH393885, 1727 bp, 45.17%), U. emarginata Wuhan population (MH393887, 1651 bp, 45.55%), and U. emarginata Saudi Arabia population (MH393886, 1727 bp, 45.28%).

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The topologies of the SSU rRNA gene trees based on two different methods of analyses (ML and BI) were similar, therefore only the ML tree is presented here (Fig. 10).

In both analyses, the sequences of the genus Urosoma separated into two large assemblages. In one assemblage, the two new Chinese populations of U. emarginata (Lanzhou and Wuhan) nested in a strongly supported clade (ML/BI, 95/1.00) with other two Chinese populations, i.e.,

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Yuzhong (MF289776) and Xi’an (MF289777). The U. emarginata Saudi Arabia population is sister to the latter clade with moderate support (ML/BI, 74/0.96). The three populations of U.

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salmastra, that is, Shanghai (present study), Zhanjiang (KF951419), and Weinan (MF289778), clustered together with full support (ML/BI, 100/1.00) in clade that is sister to U. emarginata

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but with low support. The other assemblage included U. karinae sinense Sangke population (KF951418), Urosoma sp. Fujian population (KY922826) and several other hypotrichs including Urosomoida subtropica KP280064, Monomicrocaryon elegans AM412767, and

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Hemiurosoma terricola qingdaoensis KM222091.

In order to assess the monophyly of morphology-based taxa, the Approximately Unbiased (AU) test was performed to compare the constrained tree (i.e. Urosoma is monophyletic) with

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the best ML tree (Table 4). At the 5% significance level, the hypothesis that Urosoma is

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monophyletic was rejected (P = 0.005 <0.05).

Discussion

Comparison with other populations of Urosoma emarginata and Urosoma salmastra (Table 2) Although the Lanzhou, Wuhan and Saudi Arabia populations of Urosoma emarginata closely resemble other populations in most key characters, several differences are present: (1) the average numbers of right and left marginal cirri is significantly higher in the Saudi Arabia population compared to other populations, although ranges overlap among populations 12

(Foissner 1982, 1983; Kahl 1932; Stokes 1885; Wang et al. 2017b); (2) long contractile vacuole collecting canals were observed only in Austria population 1 (Foissner 1982); (3) cortical granules are indiscernible in vivo in all populations except for Austria population 1 which has mitochondrion-like cortical granules (Foissner 1982, 1983; Kahl 1932; Stokes 1885; Wang et al. 2017); (4) lithosomes were observed in Yuzhong, Xi’an, Lanzhou, Saudi Arabia, and Wuhan populations, but not mentioned for any other populations (Foissner 1982, 1983; Kahl 1932; Stokes 1885; Wang et al. 2017). We consider all these differences as environment-, population-, or observer-dependent. Based on the reported data of Urosoma salmastra, the Shanghai population is consistent

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with the previous descriptions (Dragesco and Dragesco-Kernéis 1986; Shao et al. 2014; Wang J. et al. 2017). The morphometrics are all within the expected range of variation, so we believe that our identification is beyond doubt.

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Possible fixation problem of Urosoma emarginata during TEM preparation

The difficulty in obtaining optimum fixation for hypotrichs has been well documented

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(Shigenaka et al. 1973). We fixed U. emarginata several times with different fixatives. In poorly fixed specimens (e.g., fixed using 200 μl 2.5% glutaraldehyde and 200 μl 2% OsO4, and

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post-fixed in 1% OsO4), unit membranes were not well preserved and the perilemma was not observed (Supplementary Figure 1). The perilemma was observed in the sections from our bestfixed specimens in which other unit membranes (e.g. those of the mitochondria and the plasma

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membrane) were also well-preserved. These findings support the objective existence of the perilemma, although the spaces between its constituent membranes might be artifacts. Extrusomes were observed to have an identical morphology in every specimen observed,

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whether fixed well or not (Supplementary Figure 1) and thus their description is reliable. Although the epiplasm is present in some ciliates (Aufderheide 1983; Hausmann 1979;

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Hausmann and Hausmann 1981; Mainwaring 1972; Peck 1977; Williams and Luft 1968; Willams et al. 1979, 1981), it has never previously been reported in hypotrichs (Jerka-Dziadosz 1982; Grim 1972; Grimes 1972; Wirnsberger-Aescht and Foissner 1989; Zhang X. et al. 2014). Furthermore, the subpellicular layer of microtubules is widely reported in other ciliates (Aufderheide 1983; Calvo et al. 1986; Gliddon 1966; Grimes 1972; Paramá et al. 2006; Puytorac et al. 1976; Saxena et al. 1978). Consequently, the presence of an epiplasm-like structure beneath the pellicle, and the absence of a subpellicular layer of microtubules, in U. emarginata are both unusual. Therefore, it cannot be excluded that the epiplasm-like structure 13

in our specimens derives from microtubules that had depolymerized due to the failure of our fixative to preserve them.

Perilemma (Table 3) The perilemma is a characteristic feature of species of the subclass Choreotrichia (e.g., Laval-Peuto 1975; Wasik and Mikolajczyk 1992). A perilemma has also been reported in the subclass Oligotrichia, such as Strombidium viride (Agatha and Struder-Kypke 2007; Greuet et al. 1986; Rogerson et al. 1989). In these taxa, the perilemma covers the whole cell and numerous layers may be found in the cytopharynx (Bardele 1981). Montagnes (1991) pointed out that the

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presence or absence of a perilemma may have phylogenetic importance. A perilemma has also been reported in hypotrichous ciliates, including Urostyla grandis, Uroleptus caudatus, Engelmanniella mobilis and Epiclintes felis (Bardele 1981; Carey and Tatchell 1983; Wirnsberger-Aescht and Foissner 1989). While the perilemma covers the cell completely in some species (e.g., Engelmanniella mobilis), in others (e.g., Epiclintes felis) it appears to be

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confined to the oral and perioral areas (Carey and Tatchell 1983; Wirnsberger-Aescht and Foissner 1989). In our study, the perilemma invariably covered the whole cell surface of the

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two Urosoma species. More importantly, the presence of the perilemma in our populations of Urosoma strongly supports the validity of Perilemmaphora Berger, 2008, a rankless taxon

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unifying the oligotrichs (now the subclasses Choreotrichia and Oligotrichia) and hypotrichs (now the subclass Hypotrichia) based on the presence of a perilemma (Berger 2008).

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Extrusomes (Table 3)

The cortical granules of U. emarginata and U. salmastra are membrane-bound structures that can extrude their contents as shown in SEM and TEM preparations. Thus, they are

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extrusomes according to the definition given by Hausmann (1978). Knowledge of extrusome ultrastructure in hypotrichs is limited. To date, only four

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extrusome types have been well described: (1) trichocyst-like extrusomes (e.g. in Pseudourostyla cristata, P. nova and Anteholosticha monilata) which have a distinctive cap and central shaft (Grim and Manganaro 1985; Gu et al. 2002; Jerka-Dziadosz 1970; Oberschmidleitner and Aescht 1996; Suganuma 1973; Tesarova and Foissner 1999; Zhang J. et al. 2011; Zhang X. et al. 2012; Zhou et al. 2011); (2) pigmentocysts (e.g. in Pseudokeronopsis carnea and Thigmokeronopsis jahodai) which contain homogenous pigment and sometimes also have a release channel connected with pellicular membranes (Wicklow 1981; Wirnsberger and Hausmann 1988); (3) “typical” mucocysts (e.g. in Urostyla grandis and Stylonychia mytilus) 14

which contain a tip and body parts (Görtz 1982a; Zhang J. et al. 2007); and (4) cup-shaped extrusomes (in Architricha indica and Oxytricha granulifera), which are possibly a special kind of mucocyst that ejects mucosubstances onto the cell surface (Tang et al. 2016; Zhang X. et al. 2014). It is worth mentioning that some extrusomes described as mucocysts (e.g., in Paraurostyla weissei and Diaxonella pseudorubra) might be pigmentocysts because they are spherical, membrane-bounded structures containing homogeneous material in TEM preparations and are colored in vivo, i.e., yellow-greenish in P. weissei and red in D. pseudorubra (Jerka-Dziadosz 1982; Sun et al. 2014; Wirnsberger et al. 1985). The extrusomes of Urosoma emarginata have heterogeneous contents bounded by a

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membrane, similar to the typical mucocysts and cup-shaped extrusomes (Cai et al. 2017; Görtz 1982a; Zhang J. et al. 2007; Zhang X. et al. 2014). However, unlike either typical mucocysts or cup-shaped extrusomes, the extrusomes in U. emarginata have an electron-dense core that is ejected during extrusion, surrounded by less electron-dense material. Thus, the extrusomes of U. emarginata might represent a new type of mucocyst. Considering their spherical structure,

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the extrusomes of U. salmastra are possibly pigmentocysts, similar to those of Blepharisma japonicum (Harumoto et al. 1998). However, the contents of the U. salmastra extrusomes did

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not show the very high electron-density typical of pigmentocysts in other hypotrichs (JerkaDziadosz 1982; Sun et al. 2014; Wicklow 1981; Wirnsberger and Hausmann 1988). Further

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TEM-based investigations are needed to better characterize these structures. Extrusomes are widely distributed in ciliates and have phylogenetic importance (Rosati and Modeo 2003). Examples include the intensively studied trichocysts of peniculids (Bannister

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1972; Eisler and Peck 1998; Wessenberg and Antipa 1970; Yusa 1963) and cortical ampules which are unique to the genus Euplotes (Dallai and Luporini 1981; Gliddon 1966; Görtz 1982b; Hammond 1937; Roth 1957). Trichocyst-like extrusomes have also been described in some

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genera of the order Urostylida (Zhang J. et al. 2011; Zhang X. et al. 2012; Zhou et al. 2011). Thus, the presence of mucocyst-like extrusomes in Urosoma emarginata and pigmentocyst-

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like extrusomes in U. salmastra suggests that the genus Urosoma may need to be divided.

Ultrastructural features of lithosomes (Table 3) Although terminology varies, what we refer to as “lithosomes” in the present study were

first recorded by Stein (1859) in Tachysoma pellionellum and appeared “ring-like” in vivo. They have been reported in Urosoma macrostyla, Apogastrostyla rigescens, Oxytricha lithofera, and several species of Amphisiella, using different terms, e.g., “lithosomes” in O. lithofera and Birojimia litoralis, “refringent globules” in T. pellionellum, and “ring-like structures” in U. 15

macrostyla, Apogastrostyla rigescens and Amphisiella species (Berger 2004; Chen et al. 2017; Foissner 2016; Hu et al. 2004; Li L. et al. 2016). A lithosome usually appears as a “ring” in vivo in Urosoma emarginata, which is consistent with the reports mentioned above. However, this ring-like arrangement might be an artifact based on a two-dimensional view due to compression of the specimen by the coverslip during live observation. Based on our observations of U. emarginata cells without a coverslip, we found that the lithosome is a more or less globular rather than ring-like. Our ultrastructural results indicate that lithosomes are regular structures with a low electron-dense, homogeneous nodule within an electron-dense envelope (Fig. 5B−E). In other

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words, lithosomes are egg-like (i.e., with a “yolk-like” center and an “albumin-like” coat) rather than ring-like, the different refractive power of the central nodule and outer envelope causing the “ring-like” appearance in light microscopy. Another type of lithosome found in Engelmanniella mobilis and Pseudokeronopsis carnea, differs from the “egg-like” lithosome

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in U. emarginata by having multiple lamellae (Wirnsberger-Aescht and Foissner 1989;

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Wirnsberger and Hausmann 1988).

Taxonomic assignment of Urosoma species

As revealed by previous studies (Shao et al. 2014; Wang J. et al. 2017), the genus Urosoma

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is not monophyletic based on SSU rRNA gene sequence data. Moreover, the results of the AU test (Table 4) indicated that the monophyly of Urosoma was rejected (P = 0.005 <0.05). Wang

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J. et al. (2017). This suggests that Urosoma species can be divided into two groups based on a combination of morphological and morphogenetic data: group I (U. salmastra and U. gigantea) without a tail and with cirrus III/2 joining in the formation of the frontoventral transverse

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anlagen (FVT-anlagen); group II (U. emarginata and U. macrostyla) with a tail and cirrus III/2 not joining in the formation of FVT-anlagen. In the present study, it was revealed that U.

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emarginata and U. salmastra have different types of extrusomes. Thus, the new ultrastructural evidence supports the separation of the two groups (Wang J. et al. 2017). However, since only three of the ten nominal species were included in the phylogenetic analyses and the data sets for each species are incomplete (either lacking ultrastructural or morphogenetic information), integrative studies of more Urosoma species are needed in order to verify this separation. Hypotrichous ciliates are a highly differentiated and diverse ciliate group. Previous studies presumed that some characters commonly used in taxonomy may have evolved convergently during evolution, e.g., Cyrtohymena-undulating membrane pattern has evolved (very likely) 16

convergently, once in the stylonychines, e.g., Rigidohymena, and again in the non-stylonychine oxytrichids, e.g., Cyrtohymena (Berger 2011; Foissner et al. 2004; Yang et al. 2015). This causes confusion when we try to elucidate the relationships of higher taxa. As in the case of Urosoma, molecular phylogenetic analyses suggest that many other genera, e.g., Oxytricha and Urosomoida, are non-monophyletic (Shao et al. 2014; Wang J. et al. 2016, 2017). Several studies have attempted to provide more robust interpretations of their relationships by using multi-gene approaches (ITS, LSU/SSU-rRNA, α-tubulin genes), but have not completely resolved these problems (Huang et al. 2016). In the present study, distinct differences between congeners indicate that differentiation among hypotrichous ciliates at the ultrastructural level

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might be more complicated than previously thought and may be taxonomically significant. Inclusion of ultrastructure as part of an integrative approach is needed in order to improve resolution of the systematics of hypotrichous ciliates.

Author contributions

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HG Ma and XP Fan conceptualized the project; JY Dong carried out the research, LF Li performed phylogenetic analysis; JY Dong, LF Li, XP Fan and A Warren wrote the manuscript.

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Acknowledgements

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All authors approved the final version of the manuscript.

This work was supported by the National Nature Science Foundation of China (project numbers: 31572223 to XP Fan; 31772431 to LF Li; 31430077 to WB Song). We thank Dr. Bing

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Ni (ECNU), for his helpful guidance in SEM and TEM, Mr. Tengyue Zhang (OUC), for his protargol-stained specimens of U. emarginata Wuhan population, and Dr William Bourland, Boise State University, USA, for his help in improving the manuscript. Many thanks are also

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due to three anonymous reviewers and the AE for their kind advices on this work.

17

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Figure Legends.

Fig. 1A−P Urosoma emarginata Lanzhou population from life (A−E), after protargol preparation (F) and in SEM (G−P). (A) Ventral view of a slightly compressed individual, arrows

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show lithosomes with single inner part, double-arrowheads mark lithosomes with double inner parts, arrowheads show large vacuoles surrounding lithosomes. (B) Four lithosomes (arrows)

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around one of which a large vacuole can be observed (arrowhead). (C−E) Different focal planes of a representative lithosome, from dorsal (C) to ventral (E) side. (F) Cortical granules (arrows).

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(G) Proximal portion of oral apparatus showing lateral membranellar cilia (arrows), membranelles of adoral zone (asterisks), and paroral membrane (PM), minute cilia (arrowheads) and acicular ends of cilia (double-arrowheads). (H) Ventral view, arrow shows long conspicuous groove, arrowhead marks species-specific caudal emargination, doublearrowheads indicate ejected contents of cortical granules (extrusomes). (I) Dorsal view, arrow shows contractile vacuole pore, arrowhead shows cytoproct immediately left of dorsal kinety 2, asterisk marks extraneous debris. (J, K) Adoral membranelles and paroral membrane (PM); numbers (1−4) refer to the four ciliary rows that comprise each membranelle; arrows show 30

lateral membranellar cilia located in row 4, and sometimes also in row 2; double-arrowheads indicate minute cilia in row 4, arrowheads mark barren kinetosomes situated in rows 1 and 3. (L) Transverse cirri, arrows mark kinetosomes without cilia. (M, N) Contractile vacuole excretory pore, in diastole (M), in systole (N). (O, P) Cytoproct closed (O) and open (P). PM, paroral membrane; 1−4, rows 1−4 of adoral membranelles (row 4 is shortest row). Scale bars =

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50 μm (A, H, I); 10 μm (G, K); 5 μm (J, O); 2 μm (L−N, P).

Fig. 2A−K Scanning electron micrographs of cortical granules (extrusomes) of Urosoma

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emarginata Lanzhou population. (A, B, E) Ventral view showing extrusomes in anterior portion (A, arrows), within marginal cirri row (B), and left of adoral membranelles (E). (C, F) Apical

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views of ejecting extrusomes. (D) Dorsal view showing a large number of ejecting extrusomes located in tail of cell. (G, I) Dorsal view showing the residual "pits" (arrows) after extrusomes

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have been ejected. (H) Lateral view of a fractured cell showing an extrusome in process of ejecting. (J, K) Lateral (J) and apical (K) view of ejecting extrusomes showing spiraling ribs on surface and central ejecta at tip. Scale bars = 10 μm (A−C, D, I); 5 μm (E, G); 2 μm (F); 0.5 μm (H, J, K).

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Fig. 3A−D Transmission electron micrographs of cortex of Urosoma emarginata Lanzhou

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population. (A, B) Cortex of unciliated area (A) and near a cirrus (B); arrow in (A) shows the innermost unit membrane which might be the plasma membrane; arrow in (B) shows the plasma

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membrane, double-arrowhead in (B) marks the perilemma. (C, D) Perilemma (PL), plasma membrane (arrow), alveoli (arrowhead), and epiplasm-like structure (E) with its underlying membrane (double-arrowheads). PL, perilemma. E, epiplasm-like structure. Scale bars = 1 μm

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(A); 0.5 μm (B, C); 0.3 μm (D).

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Fig. 4A−I Transmission electron micrographs of extrusomes of Urosoma emarginata Lanzhou population. (A−C) Single or multiple extrusomes inset in “islands” of dense cytoplasm together

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with mitochondria. (D) Cross-section of extrusome, showing irregular core part (arrows). (E) Single extrusome grouped with a mitochondrion and a paraglycogen particle (asterisk). (F) Ejected extrusomes, arrowheads show rod-like fragments, possibly cytoplasmic structures

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ejected together with granules. (G−I) Longitudinal section of extrusomes, showing pyknotic (I) and more diffuse (H) electron-dense core surrounded by the less electron-dense coat; arrowhead in (I) shows ejection of electron-dense core; arrows indicate membrane of extrusome which

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remains in place after ejection. Scale bars = 0.5 μm.

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Fig. 5A−J Transmission electron micrographs of cytoplasmic structures of Urosoma

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emarginata Lanzhou population. (A, H) Longitudinal (A) and transverse (H) section of cell, to show lithosomes (asterisks), food vacuoles (arrowhead), cytoplasmic “islands” (double-

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arrowheads) which sometimes contain paraglycogen (arrows); inset (a) shows two electrondense “islands” with packed mitochondria (Mt) and electron-dense cytosol; inset (b) shows the rod-like particles (arrows) scattered in low electron-dense cytosol “background”. (B−E) Cross-

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sections of different lithosomes, arrowheads mark glycogen granules or paraglycogen particles, arrows show low electron-dense spherical structure of lithosomes, double-arrowheads show

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electron-dense material of lithosomes. (F) Macronucleus (Ma) and micronucleus (Mi) showing chromatin bodies (arrow) and nucleoli (arrowheads). (G) Multilamellar membrane structures attached to macronuclear nodule. (I) Golgi apparatus, arrows show Golgi vesicles, arrowheads

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show ribosomes. (J) Food vacuoles (arrows) containing bacteria. Ma, macronuclear nodule; Mi, micronuclei; Mt, mitochondria. Scale bars = 20 μm (A, H); 1 μm (a, b); 2 μm (B−E, J); 0.5 μm (F, G, I).

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Fig. 6A−K Light micrographs (A−C) and scanning electron micrographs (D−K) of Urosoma salmastra Shanghai population. (A, B) Ventral view of a representative individual from life (A) and after protargol staining (B). (C) Colourless cortical granules (extrusomes) from life, which

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are often arranged in clusters below the cell surface. (D) Buccal region, showing lateral membranellar cilia (arrows), and buccal lip (arrowhead) with paroral membrane. (E, F) Ventral (E) and dorsal (F) view of representative individuals, showing the cytoproct (arrow in F). (G)

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Small intermembranellar ridges (arrows) between adoral membranelles. (H) Cytoproct when open. (I) Transverse cirri, showing barren kinetosomes (arrowhead). (J, K) Differentiation of

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membranellar cilia in adoral zone; numbers (1−4) refer to the four ciliary rows that comprise each membranelle. Arrows show lateral membranellar cilia in row 4, double-arrowheads mark minute cilia in row 4; arrowheads in (J) show barren kinetosomes in rows 2 and 3, sometimes also in row 1. Ma, macronuclear nodule; Mi, micronuclei; 1−4, rows 1−4 of adoral membranelles (row 4 is the shortest row). Scale bars = 50 μm (A); 30 μm (B);10 μm (D); 40 μm (E, F); 5 μm (G); 2 μm (H−J); 1 μm (K).

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Fig. 7A−G Scanning electron micrographs of ejecting cortical granules (extrusomes) of Urosoma salmastra. (A, E) Ventral view showing arrangement of ejected extrusomes in frontal region (A) and unciliated region (E). (B, F) Dorsal view, showing arrangement of ejected

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extrusomes on dorsal region. (C, G) Ejected extrusomes arranged in short rows. (D) Apical

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view of ejecting extrusome. Scale bars = 10 μm (A, E); 5 μm (B, F); 1 μm (C, G); 0.5 μm (D).

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Fig. 8A−H Transmission electron micrographs of cortex and extrusomes of Urosoma salmastra. (A) Pellicle near dorsal kinety unit, arrowheads show the membrane underneath microtubular

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layer which envelops cytoplasm. (B, C) Pellicle, perilemma (PL) and alveoli (arrow), doublearrowhead points to subpellicular microtubule, arrowheads mark membrane underneath

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subpellicular microtubular layer. (D) Subpellicular microtubule layer; membrane underneath subpellicular microtubule layer sometimes fuses with membrane of alveoli (arrow) enclosing several microtubules; double-arrowhead marks microtubule. (E) Perilemma (arrowheads)

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covering each cilium. (F) Subpellicular microtubule layer and underlying membrane (arrowhead) enclosing the cytoplasm. (G) Extrusomes under pellicle. Arrows mark spherical structure which is possibly pigment. (H) Showing extrusome membrane fused with pellicle and

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ejected contents, possibly pigment (arrow). PL, perilemma. Scale bars = 1 μm (A, F); 0.5 μm

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(B, C, G, H); 0.3 μm (D).

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Fig. 9A−D Transmission electron micrographs of extrusomes and cytoplasmic structures of

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Urosoma salmastra. (A) Granules beneath pellicle. Small granules (arrows) with high electrondensity and large granules (arrowheads) with low electron-density. (B) Large vesicles (doublearrowhead) encasing mitochondria (Mt) near pellicle, arrow and arrowhead indicate small and

ur

large granules, respectively. (C) Food vacuoles (FV) containing bacteria, large vesicles (arrow) and large granules (arrowhead) in cytoplasm. (D) Macronucleus and large vesicles containing

Jo

paraglycogen particles (double-arrowhead); arrow marks nucleoli, arrowheads mark chromatin bodies. FV, food vacuoles; Ma, Macronucleus; Mt, mitochondria. Scale bar = 0.5 μm (A); 2 μm (B−D).

38

ro of -p re

Fig. 10 Maximum likelihood (ML) tree inferred from the SSU rRNA gene sequences showing

lP

representative hypotrich taxa and species newly sequenced (in red). Bootstrap values above 50 for the Maximum likelihood or/and Bayesian inference are given at the individual nodes. Scale

Jo

ur

na

bar = one substitution per 100 nucleotide positions.

39

ro of -p

re

Fig. 11 A–N Urosoma emarginata Lanzhou population (C–F), Wuhan population (A, G–J) and Saudi Arabia population (B, K–N) from life (A, B) and after protargol staining (C–N). (A, B) Ventral views, arrows show lithosomes. (C, D, G, H, K, L) Ventral views of representative

lP

individuals, to show ventral ciliature. (E, F, I, J, M, N) Dorsal views of representative individuals to show dorsal kineties and nuclear apparatus. CC, caudal cirri; FC, frontal cirri;

na

FVC, frontoventral cirri; LMR, left marginal row; Ma, macronuclear nodules; Mi, micronuclei; PVC, postoral ventral cirri; PTVC, pretransverseventral cirri; RMR, right marginal row; TC,

Jo

ur

transverse cirri;1–4, dorsal kineties. Scale bars = 50 μm.

40

Table 1. Morphometric characterization of Urosoma emarginata Lanzhou population (Lan), U. emarginata Wuhan population (Wuh), U. emarginata Saudi Arabia population (Sau) and Urosoma salmastra (sal).

Length of adoral zone/Length of body of body Length of paroral

Length of endoral

Anterior body end to anterior end of paroral membrane Anterior body end to endoral membrane

ur

Number of adoral membranelles

na

Anterior body end to buccal cirrus

Jo

Number of buccal cirri

Number of frontal cirri

Number of frontoventral cirri

Number of postoral ventral cirri Number of pretransverse ventral cirri

Mean 175.3 173.7 233.9 90.9 39.2 35.7 54.5 24.7 4.5 5.0 4.4 3.7 48.7 41.3 53.5 24.5 0.3 0.3 0.2 0.3 16.4 14.3 17.4 5.2 15.3 7.1 12.1 9.7 26.1 23.4 30.8 12.4 23.7 20.6 30.1 14.2 25.0 1.0 29.4 13.2 31.7 27.5 29.8 26.1 1.0 1.0 1.0 1.0 3.0 3.0 3.0 3.0 4.0 4.0 4.0 4.0 3.0 2.6 3.0 3.1 1.9 1.6

41

M 181 185 236 88 37 33 54 23 4.0 5.0 4.3 4.0 49 42 53 23 0.3 0.2 0.2 0.3 17 13 19 5 15 6 12 10 26 24 30. 11 23 20 30 14 26 1 29 12 32 27 30 25 1 1 1 1 3 3 3 3 4 4 4 4 3 3 3 3 2 2

SD 18. 3 36. 9 17. 2 18. 3 6.4 7.9 9.5 5.1 0.6 1.1 0.7 0.5 4.8 4.4 3.1 6.0 0.3 0.1 0 0.6 2.5 4.4 3.9 1.5 3.6 2.9 3.4 1.5 4.0 3.9 4.0 3.2 4.0 3.5 3.8 3.5 3.3 0 4.3 2.8 3.0 1.6 1.7 2.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0.7 0 0.5 0.2 0.5

CV 10.4 21.3 7.4 20.1 16.3 22.0 17.4 20.5 13.5 21.7 0.1 12.6 9.9 10.7 5.8 24.5 11.2 26.3 0.1 20.8 15.2 30.6 22.6 29.0 23.3 41.0 28.0 15.2 15.4 16.5 13.0 26.1 16.9 17.1 12.6 24.5 13.1 0 14.8 21.4 9.5 5.8 5.6 9.9 0 0 0 0 0 0 0 0 0 0 0 0 0 24.9 0 16.0 12.1 30.3

n 22 15 8 20 22 15 8 20 22 14 8 20 22 15 8 20 22 14 8 20 22 14 8 19 20 14 8 19 22 14 8 19 21 14 8 19 22 15 8 20 22 15 8 20 22 15 8 20 22 15 8 20 22 15 8 20 15 13 8 16 18 14

ro of

Length of adoral zone

Max 196 212 264 130 56 53 72 35 6.1 6.3 5.4 4.4 57 46 60 33 0.3 0.4 0.3 0.4 21 28 21 9 25 13 16 12 35 28 39 20 32 26 36 21 32 1 37 18 38 31 33 32 1 1 1 1 3 3 3 3 4 4 4 4 3 3 3 5 2 2

-p

Length of body / Width of body

Min 143 90 205 71 31 26 38 17 3.3 2.7 3.3 2.8 40 30 50 11 0.2 0.2 0.2 0.1 11 10 10 4 10 3 6 6 20 16 27 8 18 15 25 8 19 1 22 9 26 25 28 22 1 1 1 1 3 3 3 3 4 4 4 4 3 1 3 3 1 1

re

Width of body

Population Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh

lP

Character Length of body

Number of caudal cirri

Number of macronuclear nodules Length of anterior macronuclear nodule Width of anterior macronuclear nodule Number of micronuclei

Length of micronucleus

na

Width of micronucleus

2 1 5 4 4 4 38 32 50 29 34 34 44 28 4 3 4 4 3 3 3 3 2 2 2 2 32 22 25 14 14 8 11 7 2 3 4 2 4 3 5 3 3 2 3 2

0.4 0.5 0.4 0.9 0.4 0.8 5.3 2.6 5.6 2.0 5.6 4.3 4.2 2.8 0.5 0 0 0 0.4 0 0. 0.3 0.2 0.3 0.5 0 5.5 8.8 7.5 4.2 4.7 3.0 2.7 1.7 1.5 1.4 1.1 0 1.0 0.5 1.0 1.0 1.0 0.5 0.6 0.7

20.4 36.1 8.7 20.9 9.8 22.0 13.5 8.4 11.3 7.0 16.1 13.2 9.9 10.5 12.1 0 0.0 0 12.9 0 0.0 9.0 12.1 12.9 21.4 0 17.5 35.5 30.0 28.2 30.8 33.39 27.1 % 23.7 54.2 45.1 29.1 0 23.2 15.0 21.8 33.3 28.5 21.1 20.5 33.9

All data are based on protargol-stained specimens, measurements in μm. Abbreviations: Min,

ur

minimum; Max, maximum; Mean, arithmetic mean; M, median; SD, standard deviation; CV,

Jo

coefficient of variation in %; n, sample size.

42

7 18 19 14 7 20 21 15 8 20 21 14 8 20 12 13 6 15 6 6 5 13 18 14 8 20 10 14 8 20 10 14 8 20 19 13 8 17 19 13 8 17 19 13 8 17

ro of

Number of dorsal kineties

1.9 1.3 4.8 4.1 3.9 3.5 39.3 31.1 49.5 28.6 34.5 32.3 42.6 26.8 3.8 3.0 4.0 4.0 3.2 3.0 3.0 3.1 1.9 2.1 2.5 2.0 31.5 24.9 24.9 14.8 15.3 8.9 10.1 7.1 2.7 3.0 3.9 2.0 4.2 3.4 4.8 3.0 3.3 2.5 3.1 1.9

-p

Number of left marginal cirri

2 2 5 5 4 4 50 34 57 35 45 36 47 32 4 3 4 4 4 3 3 4 2 3 3 2 43 48 34 25 28 18 15 11 7 6 5 2 6 4 6 5 6 3 4 3

re

Number of right marginal cirri

1 1 4 2 3 2 31 24 41 25 23 23 44 20 3 3 4 4 3 3 3 3 1 2 2 2 23 17 13 9 12 4 7 4 1 1 2 2 2 3 3 2 2 2 2 1

lP

Number of transverse cirri

Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal Lan Wuh Sau sal

f

Body width in vivo

Body length

Body width

No. of adoral membr anelles

No. of cirri in LMR/RMR

No. Ma

No. PTVC

No. TC

U. emarginata America pop.

125−145

−

−

−

−

−/−

−

−

U. emarginata Germany pop.

120−150

−

−

−

−

−/−

−

−

U. emarginata Austria pop.1a

150−190

40−60

73−95

20−25

24−27

28−32/32−36

2

U. emarginata Austria pop.2a

−

−

83−140

22−31

26−34

27−40/31−43

U. emarginata Yuzhong pop.

139−165

25−33

96−202

30−69

26−32

U. emarginata Xi’an pop.

102−130

23−25

107−146

31−43

U. emarginata Lanzhou pop.

150−185

25−50

143−196

U. emarginata Wuhan pop.

70−103

15−20

U. emarginata Saudi. pop.

132−195 −

U. salmastra Benin pop. U. salmastra Zhanjiang pop.

Cortical granules (extrusomes) in EM

Lithosomes

Habitat

Population

Data source

−

−

−

−

−

USA

Stokes (1885)

−

−

−

−

Soil

Germany

Kahl (1932)

1−3

4−5

1.5−2μm, yellowish

−

−

Soil

Austria

2

2

4−5

Absent

−

−

Soil

Austria

26−33/31−36

2

1−2

4−5

Absent

−

Present

Soil

Yuzhong

Wang J. et al. (2017b)

25−30

29−35/36−49

2

1−2

4−5

Absent

−

Present

Soil

Xi’an

Wang J. et al. (2017b)

31−56

26−38

23−45/31−50

1−2

1−2

4−5

−

Prolate ellipsoidal

Present

Soil

Lanzhou

Original

90−212

26−53

25−31

23−36/24−34

2−3

1−2

2−5

−

Prolate ellipsoidal

Present

Soil

Wuhan

Original

20−24

205−264

38−72

28−33

44−47/41−57

2−3

1−2

3−4

−

Prolate ellipsoidal

Present

Soil

Saudi Arabia

Original

−

48−101

15−30

21−26

16−25/22−30

2

1

3−5

−

−

Absent

Salt marsh

Benin

Dragesco and Dragesco-

30−50

84−142

22−59

27−43

21−31/19−39

2

1−2

3−4

Colourless, spherical, arranged in short

Spherical, arranged in short

Present

Intertida l belt

Zhanjiang

110−150

e-

Pr

na l

Jo ur

Species

Cortical granules in vivo

pr

Body length in vivo

oo

Table 2 Morphological comparison of all reported populations of U. emarginata and U. salmastra.

43

Foissner (1982） Foissner (1983）

kerneis （1986）

Shao et al. (2014）

30−40

100−160

27−50

22−27

21−31/27−36

2

1−2

4

U. salmastra Shanghai pop.

100−135

20−30

71−114

17−33

22−32

20−32/26−35

2

1−2

2−4

Colourless, spherical, arranged in short

Spherical, arranged in short

Absent

Saline soil

Weinan

Wang J. et al. (2017b)

Colourless, spherical, arranged in short

Spherical, arranged in short

Absent

Soils

Shanghai

Original

f

110−160

oo

U. salmastra Weinan pop.

Jo ur

na l

Pr

e-

pr

All data are based on protargol−stained specimens, measurements in μm. −: data not available. *: data from specimens in vivo. a: these two populations of Foissner (1982, 1983) were described as Urosoma macrostyla, U. emarginata was re-activated by Berger (1999). Abbreviations: AZM: adoral zone of membranelles; EM: electron microscopy; LMC: left marginal cirri; Ma: macronuclear nodules; pop.: population; PTVC: pretransverse ventral cirri; RMC: right marginal cirri; TC: transverse cirri.

44

Table 3 Ultrastructural comparison of reported species of Hypotricha. Species

Perilemma

Extrusomes

Lithosomes

Data source

Engelmanniella

Multilamellate

? (original designation

Multiple

Wirnsberger-Aescht

"subpellicular granules")

lamellae

et al. (1989)

“Typical” mucocysts

−

Bardele (1981);

mobilis Urostyla grandis

Single

Zhang J. et al. (2007) Stylonychia mytilus

Single

“Typical” mucocysts

−

Görtz (1982a)

Uroleptus caudatus

Single

−

−

Bardele (1981)

Epiclintes felis

Multilamellate

−

−

Carey and Tatchell

and/or single Pseudourostyla

(1983)

−

Trichocyst-like extrusomes

−

cristata

Suganuma (1973); Grim and Manganaro

ro of

(1985); Zhang J. et al. (2011)

Pseudourostyla

−

Trichocyst-like extrusomes

−

Zhou et al. (2011)

−

Trichocyst-like extrusomes

−

Zhang X et al. (2012)

Absent

Pigmentocysts

nova Anteholosticha Pseudokeronopsis carnea −

Pigmentocysts (originally

re

Thigmokeronopsis

-p

monilata

designated as "vesicles")

Diaxonella

Pigmentocysts (originally

pseudorubra Paraurostyla

lamellae

Hausmann (1988)

−

Wicklow (1981)

−

Sun et al. (2014)

−

Pigmentocysts (originally

−

Jerka-Dziadosz (1982)

designated as "mucocysts")

− −

Oxytricha Pseudoamphisiella

Jo

−

Zhang X. et al. (2014)

Cup-shaped extrusomes

−

Tang et al. (2016)

−

Mucocyst

−

Cai et al. (2017)

Multilamellate

Mucocyst-like extrusomes

Bipartition

Original

1−2

Pigmentocyst-like

−

Original

ur

granulifera

Cup-shaped extrusomes

na

Architricha indica

Urosoma

Wirnsberger and

designated as "mucocysts")

weissei

lacazei

lP

jahodai

Multiple

emarginata

Urosoma salmastra

extrusomes

−: data not available. ?: the subpellicular granules were neither described as extrusomes nor observed to extrude.

45

Table 4. Approximately Unbiased (AU) test results for tree comparison considering different topological scenarios.

Topology constraints

Unconstrained Monophyly of Urosoma

-Ln likelihood

AU value (p)

6602.61405 genus 6639.13164

0.995 0.005

Jo

ur

na

lP

re

-p

ro of

p < 0.05 refutes monophyly; p > 0.05 does not refute the possibility of monophyly.

46