IJSEM Papers in Press. Published June 13, 2014 as doi:10.1099/ijs.0.062281-0
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International Journal of Systematic and Evolutionary Microbiology
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Morphology and phylogenesis of two oxytrichid soil ciliates from China, Oxytricha
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paragranulifera n. sp. and Oxytricha granulifera Foissner and Adam, 1983 (Protista,
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Ciliophora, Hypotrichia)
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Chen SHAO1, Zhao LV,1 Ying PAN,2 Khaled A. S. AL-RASHEID,3 and Zhenzhen YI4
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1
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of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China
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2
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University of China, Qingdao 266003, China
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3
Zoology Department, King Saud University, Riyadh 11451, Saudi Arabia
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4
Laboratory of Protozoology, Key Laboratory of Ecology and Environmental Science in
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Guangdong Higher Education, College of Life Science, South China Normal University,
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Guangzhou 510631, China
The Key Laboratory of Biomedical Information Engineering, Ministry of Education, School
Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean
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Correspondence:
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Chen Shao
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Email: [email protected]
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Telephone number: +86 29 8266 8463 Ext 423
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Running title: C. Shao et al. Description of two hypotrichous ciliates
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Contents category: New Taxa—Eukaryotic Micro-organisms
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Accession number of the SSU rRNA gene sequence of Oxytricha paragranulifera n. sp. is
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KJ081200
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Accession number of the SSU rRNA gene sequence of O. granulifera is KJ081199
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1
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The morphology and infraciliature of two hypotrichous ciliates, Oxytricha paragranulifera n.
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sp. and Oxytricha granulifera Foissner and Adam, 1983, collected respectively from the
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surface of sandy soil in the Huguang mangrove forest, Zhanjiang, China, and the surface of
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soil in a forest beside Ziwu Road, Xian, northwest China. Oxytricha paragranulifera n. sp. is
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characterized by an elongate body with slightly tapered anterior end, two macronuclear
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nodules and two micronuclei, paroral and endoral in Stylonychia-pattern, colourless cortical
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granules distributed in clusters or irregular short rows, adoral zone occupying 37% of body
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length, marginal rows almost confluent posteriorly, six dorsal kineties and three caudal cirri,
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caudal cirri and dorsal bristles almost indistinguishable when viewed in vivo. The well-known
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Oxytricha granulifera Foissner and Adam, 1983 was also redescribed and can be separated
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from the new species by having cortical granules arranged along dorsal kineties and marginal
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rows on both sides (vs. grouped in clusters as well as in short irregular rows), paroral and
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endoral in Oxytricha-pattern (vs. in Stylonychia-pattern), macronuclear nodules obviously
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detached (vs. adjacent) and a non-saline terrestrial habitat (vs. saline terrestrial). The
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separation of these two taxa is also firmly supported by the molecular data which shows a
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significant discrepancy between the two in their SSU rRNA gene sequences (similarity
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97.1%). Phylogenetic analyses based on SSU rRNA gene sequence data suggest a close
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relationship within the Oxytrichidae assemblage between Oxytricha paragranulifera n. sp.
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and Oxytricha granulifera.
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2
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INTRODUCTION
58 59
There is consensus among most taxonomists (Song, 1990, 2001; Song & Wilbert, 1997a, b,
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2002; Berger, 1999; Foissner, 1999; Song & Warren, 1999; Foissner et al., 2002, 2008;
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Küppers et al., 2011; Chen et al., 2013b; Lv et al., 2013; Shao et al., 2013a, b; Shi et al., 2002;
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Singh & Kamra, 2013; Singh et al., 2013) that the following characters are important for
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species separation in oxytrichids: (1) body size, shape and colour; (2) characteristics of
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cortical granules; (3) whether dorsal cilia and caudal cirri are indistinguishable or
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distinguishable from marginal cirri; (4) relative length of buccal apparatus and DE-value; (5)
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characteristics of macronuclear nodules and micronuclei; (6) the pattern of the buccal and
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somatic ciliature; (7) morphometric data relating to the ciliature; and (8) habitat.
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Oxytricha is one of the oldest genera (Berger, 1999). Its systematics, however, have
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traditionally been highly confused, not least because a properly defined type species was not
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fixed until relatively recently (Berger, 1999). The status of Oxytricha, along with three other
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oxytrichid genera with flexible bodies and 18 frontoventral transverse cirri, was recently
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reviewed by Shao et al. (2012), who defined it as follows: flexible 18-cirri Oxytrichidae with
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undulating membranes in the Oxytricha pattern and adoral zone in the shape of a question
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mark; frontoventral cirri in a V-shaped pattern; one left and one right marginal row; five or six
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dorsal kineties in the Oxytricha-pattern, that is, one or two dorsomarginal kineties and simple
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kinety three; fragmentation present; caudal cirri on dorsal kineties 1, 2 and 4. The genus
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Oxytricha currently includes ca. 48 morphospecies, several of which have overlapping
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species-level characters with respect to their living morphology, infraciliature, morphometric
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data and habitat.
80 81
In November 2010 and April 2012, two oxytrichid ciliates were isolated from the surface of
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sandy soil in the Huguang town mangrove forest, Zhanjiang, southern China, and the surface
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of soil in a forest beside Ziwu Road, Xian, northwest China, respectively. Subsequent
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observations demonstrated them to be members of the genus Oxytricha. In the present paper,
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their morphology is described and their SSU rRNA genes sequenced.
86 87 88
METHODS
89 90
Sampling and cultivation (Fig. 1). Oxytricha paragranulifera n. sp. was collected from the
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surface of sandy soil in the Huguang town mangrove forest, Zhanjiang (21°06 N; 110°18 E),
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China on November 25, 2010 when the soil temperature was 24 °C, salinity 25.5 and pH 7.3.
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Oxytricha granulifera was collected from the surface of soil in the centre of Zhuque National
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3
′
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Forest Park, Xi’an (33°55 N; 108°32 E), China on April 30, 2012 when the soil temperature
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was 28 °C, salinity 3 and pH 8. Ciliates were stimulated to excyst and emerge from the soil
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samples by employing the non-flooded Petri dish method described by Foissner (1987) and
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updated in Foissner et al. (2002). Isolated specimens were maintained as non-clonal cultures
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in Petri dishes at room temperature (20 °C) using boiled freshwater with rice grains to enrich
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bacterial food organisms.
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′
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Morphology. Living cells were observed with bright field and differential interference
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contrast microscopy (Chen et al., 2013a). The protargol silver staining method according to
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Wilbert (1975) was used to reveal the infraciliature (Pan et al., 2013). Measurements of the
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stained specimens were carried out with an ocular micrometer (Paiva et al., 2012). Drawings
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of stained specimens were performed at 1,250× magnification with the aid of a camera lucida
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(Foissner, 2012).
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Terminology. General terminology is mainly according to Berger (1999); for explanation of
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terms specific for hypotrichs (e.g., pseudorow, mixed row, DE-value), see Berger & Foissner
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(1997), Berger (1999, 2006, 2008, 2011), Foissner & Stoeck (2011) and Foissner &
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Al-Rasheid (2006). For the designation of the frontoventral-transverse cirri, the numbering
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system by Wallengren (1900) is used (details see Berger 1999: 16). The term 18-cirri
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hypotrich means a hypotrich with 18 frontal-ventral-transverse cirri (e.g., Berger, 2008: 27).
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DNA extraction, PCR amplification and sequencing. One or more cells were isolated from
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the ciliate cultures, and then washed three times with sterilized fresh/saline water (0.22 m
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filtered). These cells were then transferred to a 1.5 ml microfuge tube with a minimum
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volume of water. Genomic DNA of Oxytricha paragranulifera n. sp. and O. granulifera were
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extracted using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the
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manufacturer’s instructions (Gao et al., 2013; Huang et al., 2014). The PCR amplification of
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the SSU rRNA gene was performed according to Li et al. (2013) and Yi et al. (2012), with
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primers 18s-F (5’-AAC CTG GTT GAT CCT GCC AGT-3’) and 18s-R (5’-TGA TCC TTC
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TGC AGG TTC ACC TAC-3’) (Medlin et al., 1988).
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Phylogenetic analyses. Using the online program Muscle 3.7 (Edgar, 2004), the SSU rRNA
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gene sequences of Oxytricha paragranulifera n. sp. and O. granulifera were aligned with 51
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hypotrichid sequences obtained from the GenBank database. Three urostylid species were
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selected as the outgroup taxa. These sequences were edited manually using Bioedit 7.0.0 in
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order to remove ambiguous gaps (Hall, 1999). The program MrModeltest v.2.0 (Nylander,
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2004) selected the GTR + I (= 0.6678) + G (= 0.5202) as the best model with the AIC
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criterion, which was then used for Bayesian (BI) and Maximum Likelihood (ML) analyses. BI
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analysis was performed with MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003). ML trees were
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constructed using RaxML-HPC2 (Stamatakis et al., 2008) at the CIPRES website
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(http://www.phylo.org/). MEGA 4.0 (Tamura et al., 2007) was used to visualize tree
μ
4
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topologies.
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Topology testing. A constraint tree in which the genus Oxytricha was a monophyletic clade
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was defined and loaded into PAUP 4.0 (Calendini & Martin, 2005), using ML criterion and
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heuristic search with the tree bisection-reconnection (TBR) and 10 random sequence addition
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replicates. The site-wise likelihoods were calculated in PAUP 4.0 under the GTR + I + G
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model with parameters as suggested by MrModeltest for all trees. Then the constraint tree was
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compared to unconstrained best and 100 random ML topologies using the Approximately
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Unbiased (AU) test (Shimodaira, 2002) as implemented in the CONSEL software package
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(Shimodaira & Hasegawa, 2001; Huang et al., 2012; Huang et al., 2014).
140 141
RESULTS
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Oxytricha paragranulifera n. sp. (Table 1, Figs 2a-e and 3a-p)
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Diagnosis. Body elongate with slightly tapered anterior end, about 80–110 m × 35–50 µm in
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size. Two macronuclear nodules and two micronuclei. Cortical granules colourless, densely
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grouped in clusters or irregular short rows. One contractile vacuole located at about mid-body
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near left margin. Adoral zone occupies 37% of body length in vivo and is composed of ca. 27
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membranelles on average. Paroral and endoral in the Stylonychia-pattern. Invariably 18
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frontoventral transverse cirri. Marginal rows almost confluent posteriorly. Six dorsal kineties
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and three caudal cirri. Caudal cirri and dorsal bristles almost indistinguishable when viewed
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in vivo.
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Etymology. The species name paragranulifera (pa.ra.gra.nu.li'fe.ra. Gr. pref. para- beside,
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along; N.L. fem. adj. granulifera little seed bearing) referring to the species Oxytricha
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granulifera; N.L. fem. adj. paragranulifera similar to Oxytrichia granulifera.
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Type locality. Ciliates were collected in November 2010 from the surface of sandy soil in
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Huguang town mangrove forest, Zhanjiang (21°07 N; 110°14 E), Guangdong Province, China,
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when the soil temperature was 24 °C, pH 7.3, and salinity 25.5‰.
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Type specimens deposited. The slide (No. PY10112501A) containing the holotype specimen
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(Fig. 2d, e) and a paratype slide (No. PY10112501B) with protargol-impregnated specimens
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are deposited in the Laboratory of Protozoology, OUC, China.
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Description (Table 1, Figs 2a-e and 3a-p). Body about 80–110 m × 35–50 m in vivo,
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non-contractile and flexible, usually slender oval to elliptical with anterior end usually narrow,
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posterior broadly rounded; right margin slightly convex, left margin strongly convex (Figs 2a
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and 3a–c, e). Dorsoventrally flattened (about 3:1), ventral side flat, dorsal side convex in
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middle portion. Cytoplasm colourless to greyish, containing numerous shining globules and
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crystals. Cells frequently with many lipid droplets (ca 2–4 µm across) and food vacuoles (ca
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2–5 µm across). Cortical granules colourless, round, about 1 µm across, densely grouped in
μ
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′
μ
5
μ
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clusters and irregular short rows on dorsal side, while slightly sparsely arranged in irregular
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short rows on ventral side (Figs 2b and 3i). Contractile vacuole about 15 µm across, located at
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the mid-body, near left body margin, contracting at intervals of about 1 min (Figs 2a and 3d).
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Invariably two macronuclear nodules, usually arranged closely in line at about the mid-body
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and slightly left of the midline; individual nodules ellipsoidal (length:width ratio about 2:1),
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with small to moderately large nucleoli. Usually one micronucleus attached to each
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macronuclear nodule (Figs 2a, e and 3a, l).
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Locomotion by continuous rapid crawling on bottom of Petri dish and surface of water. When
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suspended, cells often swim continuously in circles.
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Infraciliature as shown in Figs 2c–e and 3m–p. Adoral zone occupies 37% of cell length (Fig.
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2d) and is composed of 25–29 membranelles. Distal portion of adoral zone extends slightly
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posteriorly onto right side of cell, that is, a DE-value 0.23 in the holotype specimen (Fig. 2d;
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for explanation of DE-value, see Berger 2006: 18). Paroral and endoral in parallel optically
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(Figs 2c and 3m). Frontoventral-transverse ciliature comprising 18 cirri: three slightly
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enlarged frontal cirri with cilia about 15 µm long; single buccal cirrus near anterior end of
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undulating membranes; frontoventral cirri arranged in a short, mixed, asymmetric, V-shaped
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row; cirrus III/2 slightly ahead of the level of cirrus VI/3; cirrus III/2 closer to the remaining
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frontoventral cirri than to the paroral and endoral; three postoral ventral cirri located
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intermediately behind the vertex and distinctly separated from the two pre-transverse ventral
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cirri; postoral ventral cirrus IV/2 arranged more anteriorly than V/4; five transverse cirri in a
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hook-shaped row, with cilia about 20 µm long; two pretransverse ventral cirri, cirrus VI/2
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located at about the same level as the leftmost transverse cirrus, cirrus V/2 closer to cirrus
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VI/2 than cirrus V/3; distance between cirri V/3 and V/4 shorter than that between cirri V/3
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and V/2 (Figs 2c, d and 3g, j, n, o). The posterior ends of marginal rows are almost confluent;
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left row with 18–25 cirri, right row with 18–25 cirri; cilia of marginal cirri about 10 µm long
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(Figs 2d, e and 3f).
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Six dorsal kineties; leftmost two (dorsal kineties 1 and 2) bipolar, each comprising about 20
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pairs of basal bodies; third and fifth dorsal kineties start at about the anterior end of cell and
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terminate at 4/5 and 2/5 of cell length respectively; fourth dorsal kinety commences near the
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equatorial level of cell and stretches to the posterior end; rightmost dorsal kinety (dorsal
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kinety 6) composed of two or three dikinetids (Figs 2e and 3p). Dorsal cilia about 3 µm long
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in vivo. Three caudal cirri, thin, located at posterior body margin, narrowly separated, one
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each on dorsal kineties 1, 2, and 4 (Figs 2e and 3h); cilia of caudal cirri about 12 µm long in
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vivo and thus almost indistinguishable from marginal cirri (Fig. 2a and 3a, j).
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Oxytricha granulifera Foissner and Adam, 1983 (Table 1, Fig 4a-h)
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Description. Body about 90–130 m × 30–50 m in vivo, length:width ratio approximately μ
μ
6
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3:1 in vivo, 1.8:1 on average in protargol preparations. Body flexible but not contractile,
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usually slender oval to elliptical with anterior end usually narrow, posterior broadly rounded
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(Fig. 4a, b). Dorsoventrally flattened (about 3:1), ventral side flat, dorsal side convex in
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middle portion; right cell margin slightly convex to slightly concave, left margin strongly
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convex, usually widest in front of mid-body. Cytoplasm colourless to greyish, containing
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numerous shining globules and crystals (Fig. 4c). Cells with many lipid droplets (ca 4–5 µm
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across) and food vacuoles (ca 5–10 µm across). Cortical granules colourless, round, about 1
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µm across, arranged along dorsal kineties and marginal rows on both sides (Fig. 4d).
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Contractile vacuole located at mid-body, near left body margin, contracting at intervals of
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about 10s (Fig. 4b, c). Invariably, two macronuclear nodules, usually arranged at anterior and
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posterior 1/3, as well as slightly left of the midline (Fig. 4f). Two micronuclei, one each
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beside the two macronuclear nodules (Fig. 4f). Locomotion by slow to moderately fast
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crawling on substrate, but usually motionless.
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Infraciliature as shown in Fig. 4e-h. Adoral zone occupies 39% of cell length on average in
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protargol preparations, composed of 28–38 adoral membranelles. Distal portion of adoral
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zone extends slightly posteriorly on to the right side of cell, that is, DE-value about 0.30 (Fig.
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4e; for explanation of DE-value, see Berger 2006: 18). Paroral and endoral intersect each
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other optically behind the buccal cirrus (Fig. 4e). Three slightly enlarged frontal cirri with
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cilia about 15 µm long; single buccal cirrus near anterior end of paroral and endoral;
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frontoventral cirri arranged in a short, mixed, asymmetric, V-shaped row; cirrus III/2 slightly
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ahead of level of cirrus VI/3; cirrus III/2 closer to remaining frontoventral cirri than to paroral
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and endoral (Fig. 4e) Three postoral ventral cirri located intermediately behind vertex and
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distinctly separated from the two pre-transverse ventral cirri; postoral ventral cirrus IV/2
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arranged more anteriorly than V/4. Five transverse cirri in a hook-shaped row, with cilia
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about 20 µm long; two pretransverse ventral cirri, cirrus VI/2 located near rightmost
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transverse cirrus, cirrus V/2 closer to cirrus VI/2 than cirrus V/3; distance between cirri V/3
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and V/4 less than that between cirri V/3 and V/2 (Fig. 4g). Posterior ends of marginal rows
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are almost confluent; left row with 23–31 cirri, right row with 26–32 cirri; cilia of marginal
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cirri about 10 µm long (Fig. 4g).
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Six dorsal kineties; leftmost two (dorsal kineties 1 and 2) bipolar, each comprising about 20
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pairs of basal bodies; third and fifth dorsal kineties start at about the anterior end of cell and
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terminate at 4/5 and 2/5 of cell length respectively; fourth dorsal kinety commences near the
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equatorial level of cell and stretches to the posterior end; rightmost dorsal kinety (dorsal
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kinety 6) composed of two or three dikinetids (Fig. 4h). Dorsal cilia about 3 µm long in vivo.
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Three caudal cirri, thin, located at the posterior body margin, slightly separated, one each on
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dorsal kineties 1, 2 and 4.
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Phylogenetic analyses (Fig. 5)
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The SSU rRNA gene sequence of Oxytricha paragranulifera n. sp. and O. granulifera were
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deposited into GenBank, with accession numbers KJ081200 and KJ081199, respectively. The
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SSU rDNA gene sequence of our Oxytricha granulifera is 1620 bp long and has a GC content
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of 45.68%, and that of O. granulifera (GenBank accession number AF164122) is 1774 bp
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long and has a GC content of 45.55%. The similarity between them is 99.7% indicating that
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they are conspecific. The topologies of the ML and BI trees were similar, therefore only the
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ML tree is shown (Fig. 5). In both analyses, Oxytricha paragranulifera n. sp. and Oxytricha
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granulifera fall into the Oxytrichinae assemblage. However, the Oxytricha is not a
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monophyletic group: fourteen species fall into nine clades. And the monophyly of the genus
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Oxytricha is rejected by the AU test (P = 0.003). Oxytricha granulifera clustered with
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Oxytricha sp. 1 MD-2012 with high support values (ML/BI, 83/0.93), and O. paragranulifera
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n. sp. was sister to Onychodromopsis flexilis with low and high support values (ML/BI,
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77/0.96).
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DISCUSSION
258 259
Oxytricha paragranulifera n. sp. (Table 1, Figs 2a-e and 3a-p)
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Comparison with congeners. In terms of its slender oval to elliptical body shape and slightly
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tapered anterior end, two macronuclear nodules and indistinguishable dorsal cilia and caudal
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cirri, 23 congeners resemble Oxytricha paragranulifera n. sp. Thus, we compare Oxytricha
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paragranulifera n. sp with each of these.
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Oxytricha granulifera Foissner and Adam, 1983 has a very similar body size and body shape
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to O. paragranulifera n. sp. but can be distinguished from the latter by having cortical
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granules arranged along the dorsal kineties and marginal rows on both sides (vs. grouped in
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clusters as well as in short irregular rows). Furthermore, the paroral and endoral intersect each
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other optically (vs. in parallel optically), five (in Austrian population) or six (in Chinese
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population) dorsal kineties (vs. six), the macronuclear nodules are obviously detached (vs.
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adjacent) and the habitat is non-saline terrestrial (vs. saline terrestrial) (Berger, 1999). The
271
separation of these two taxa is also firmly supported by the molecular data which shows a
272
significant discrepancy between the two in their SSU rRNA gene sequences (similarity
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97.1%).
274
Considering the somatic ciliature, body shape, and the appearance of the undulating
275
membranes, O. chlorelligera Kahl, 1932 is similar to O. paragranulifera n. sp., although the
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former can be recognized by the presence of symbiotic algae (vs. absent) (Berger, 1999).
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Oxytricha fallax Stein, 1859 can be separated from O. paragranulifera n. sp. by its obviously
278
detached (vs. adjacent) macronuclear nodules, the Oxytricha-pattern (vs. Stylonychia-pattern) 8
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of its paroral and endoral and the absence (vs. presence) of cortical granules (Berger, 1999).
280
Compared with the well-known O. granulosa Schmitz, 1986, O. paragranulifera n. sp. has a
281
smaller body (80–110 µm vs. 155–280 µm), colourless cortical granules which are grouped in
282
clusters as well as in short irregular rows (vs. yellow-green in colour and arranged in 14–18
283
longitudinal rows), fewer membranelles (25–29 vs. 44–50), more dorsal kineties (six vs. four
284
or five), paroral and endoral in the Stylonychia-pattern (vs. in the Oxytricha-pattern) and two
285
(vs. one) micronuclei (Berger, 1999).
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Oxytricha hymenostoma Stokes, 1887 differs from O. paragranulifera n. sp. by having a
287
broadly rounded (vs. slightly tapered) anterior end, slightly separated (vs. well separated)
288
postoral and pretransverse cirri, dorsal kinety 4 starting almost at the anterior end (vs.
289
commencing at the mid-body) and obviously detached (vs. adjacent) macronuclear nodules
290
(Berger, 1999).
291
Compared with O. paragranulifera n. sp., O. longicirrata Kahl, 1932 has its paroral and
292
endoral in the Oxytricha-pattern (vs. in the Stylonychia-pattern), a lack of cortical granules (vs.
293
their presence), obviously detached (vs. adjacent) macronuclear nodules and distinctly
294
separated (vs. confluent) marginal rows posteriorly (Berger, 1999).
295
Considering the somatic ciliature, body shape, and the appearance of the undulating
296
membranes, O. paragranulifera n. sp. is similar to O. aeruginosa Wrzesniowskiego, 1866,
297
although the former can be recognized by its colourless (vs. russet and black) cortical
298
granules, smaller body size (80–110 µm vs. 120–165 µm) and adjacent (vs. obviously
299
detached) macronuclear nodules (Berger, 1999).
300
Oxytricha paragranulifera n. sp. differs from O. longissima Dragesco and Njine, 1971 in
301
having a smaller body (80–110 µm vs. 250 µm), transverse cirri slightly (vs. distinctly)
302
displaced anteriorly, fewer right and left marginal cirri (18–25 and 18–25 vs. 40–54 and
303
37–42) and adjacent (vs. obviously detached) macronuclear nodules (Berger, 1999).
304
In terms of its somatic ciliature and body shape, O. paragranulifera n. sp. is most similar to O.
305
multiseta Dragesco, 1966. The former, however, can be recognized by the number of
306
transverse cirri (consistently five vs. six or seven) and adjacent (vs. obviously detached)
307
macronuclear nodules (Berger, 1999).
308
Oxytricha quadricirrata Blatterer and Foissner, 1988 can be separated from O.
309
paragranulifera n. sp. by its distinctly separated (vs. confluent) marginal rows posteriorly, its
310
cortical granules being arranged along the dorsal kineties and marginal rows (vs. in clusters
311
and in short irregular rows) and fewer membranelles (19–21 vs. 25–29), transverse cirri (four
312
vs. five), right marginal cirri (14–17 vs. 18–25) and left marginal cirri (13–18 vs. 18–25)
313
(Berger, 1999).
314
Oxytricha variabilis Grolière, 1975 differs from O. paragranulifera n. sp. in the number of
315
micronuclei (four vs. two), obviously detached (vs. adjacent) macronuclear nodules and in 9
316
having five (vs. three) postoral ventral cirri and five (vs. six) dorsal kineties (Berger, 1999).
317
Oxytricha auripunctata Blatterer and Foissner, 1988 can be separated from O.
318
paragranulifera n. sp. in having one to five (vs. invariably two) micronuclei; four to five (vs.
319
three) caudal cirri, distinctly separated (vs. confluent) marginal rows posteriorly;
320
orange-yellow (vs. colourless) cortical granules; paroral and endoral arranged in the
321
Oxytricha-pattern (vs. in the Stylonychia-pattern) and obviously detached (vs. adjacent)
322
macronuclear nodules (Berger, 1999).
323
Oxytricha arabica Foissner et al., 2008 can be separated from O. paragranulifera n. sp. in
324
having five (vs. six) dorsal kineties and an absence of cortical granules (vs. their presence)
325
(Foissner et al., 2008).
326
Oxytricha lanceolata Shibuya, 1930 has a similar body size and shape to O. paragranulifera
327
n. sp. but can be separated from the latter by having an absence of cortical granules (vs. their
328
presence), four (vs. six) dorsal kineties and obviously detached (vs. adjacent) macronuclear
329
nodules (Berger, 1999).
330
Oxytricha matritensis Ramirez-Montesinos and Perez-Silva, 1966 differs from O.
331
paragranulifera n. sp. in the number of right marginal cirri (about 17 (data from drawing) vs.
332
18–25) and left marginal cirri (about 15 (data from drawing) vs. 18–25), the probable absence
333
of caudal cirri (vs. their presence) and obviously detached (vs. adjacent) macronuclear
334
nodules (Berger, 1999).
335
Oxytricha proximata Shibuya, 1930 differs from O. paragranulifera n. sp. in its obviously
336
detached (vs. adjacent) macronuclear nodules, four (vs. five) transverse cirri and in the
337
rectangular arrangement (vs. a V-shaped arrangement) of its frontoventral cirri (Berger, 1999).
338
Oxytricha longigranulosa Berger and Foissner, 1989 resembles O. paragranulifera n. sp. in
339
its dorsal ciliature, however, the former can be distinguished by the arrangement of cortical
340
granules (in short lines vs. in clusters and short irregular lines), body shape (elliptical vs.
341
mostly slender oval) and in the distinctly separated (vs. confluent) marginal rows posteriorly
342
(Berger, 1999).
343
Compared to O. paragranulifera n. sp., O. pseudofusiformis Dragesco and Dragesco-Kernéis,
344
1986 has a smaller body size (35–46 µm after protargol impregnation vs. 80–110 µm in vivo),
345
fewer cirri in the frontal area (five to seven vs. eight), membranelles (12–18 vs. 25–29), right
346
marginal (7–11 vs. 18–25) and left marginal cirri (seven to nine vs. 18–25) as well as only one
347
(vs. two) micronucleus and transverse cirri that are distinctly (vs. slightly) displaced
348
anteriorly (Berger, 1999).
349
Oxytricha tenella Song and Wilbert, 1989 can be separated from O. paragranulifera n. sp. in
350
having a smaller body (50–70 µm vs. 80–110 µm); cortical granules arranged irregularly (vs.
351
in clusters and short irregular lines); paroral and endoral arranged in the Oxytricha-pattern (vs.
352
in the Stylonychia-pattern), as well as postoral and pretransverse cirri slightly separated (vs. 10
353
well separated) (Berger, 1999).
354
Oxytricha rubripuncta Berger and Foissner, 1987 differs from O. paragranulifera n. sp. in
355
having two to four (vs. two) micronuclei, cirrus III/2 located between cirri IV/3 and VI/3 (vs.
356
between cirri VI/3 and VI/4), paroral and endoral arranged in the Oxytricha-pattern (vs. in the
357
Stylonychia-pattern), reddish (vs. colourless) cortical granules, obviously detached (vs.
358
adjacent) macronuclear nodules and dorsal kinety 5 bipolar (vs. terminating at the mid-body)
359
(Berger, 1999).
360
Oxytricha durhamiensis Berger, 1999 can be separated from O. paragranulifera n. sp. by
361
having cortical granules in linear groups of two to five parallel to the dorsal kineties (vs. in
362
clusters and short irregular lines) and cirrus III/2 located between cirri IV/3 and VI/3 (vs.
363
between cirri VI/3 and VI/4) (Berger, 1999).
364
Oxytricha oxymarina Berger, 1999 can be distinguished from O. paragranulifera n. sp. in
365
having three bipolar dorsal kineties (vs. six dorsal kineties, four of which are not bipolar),
366
distinctly separated (vs. confluent) marginal rows posteriorly, and an adoral zone that is rather
367
long relative to the body length (51% vs. 37% after protargol impregnation) (Berger, 1999).
368
Compared to O. paragranulifera n. sp., Oxytricha alfredi Berger, 1999 has one (vs. two)
369
micronucleus and a rather long posterior-most marginal (or caudal?) cirri (vs.
370
indistinguishable) (Berger, 1999).
371
Compared with O. paragranulifera n. sp., O. acidotolerans Weisse et al., 2013 has its paroral
372
and endoral in the Oxytricha-pattern (vs. in the Stylonychia-pattern), a lack of cortical
373
granules (vs. their presence), distinctly separated (vs. confluent) marginal rows posteriorly
374
and dorsal ciliature (dorsal kineties 1 and 2 shortened anteriorly and each comprising about
375
10 pairs of basal bodies (vs. bipolar and each comprising about 20 pairs of basal bodies) and
376
third dorsal kinety terminating at midline (vs. 4/5) of cell length) (Weisse et al., 2013).
377 378 379
Oxytricha granulifera Foissner and Adam, 1983 (Table 1, Fig. 4a-h)
380
Comparison with Austrian populations. According to the original report (Foissner and
381
Adam, 1983), our population closely resembles the Austrian population of Oxytricha
382
granulifera, the main difference being the number of dorsal kineties (five in Australian
383
population vs. six in the present population. We believe that this difference is intraspecific and
384
therefore not significant for species level separation. Since all other key characters, i.e. body
385
size, shape, macronuclear and micronuclear features, arrangement of cirri, morphometric data
386
(Table 1, Foissner and Adam 1983) and biotope are all consistent with the original description.
387
The identification of the Xi’an population is therefore not in doubt. Further, the similarity
388
between our O. granulifera and O. granulifera (AF164122) is 99.7%, indicating that they are
389
conspecific. 11
390 391
Phylogenetic analyses.
392
In the present SSU rRNA gene trees, Oxytricha granulifera was sister to O. sp. 1 MD-2012,
393
and O. paragranulifera n. sp. clustered with Onychodromopsis flexilis, O. ottowi 1 MD-2012,
394
O. ottowi 2 MD-2012, O. longigranulosa and Urosomoida lanceolata. Each of these species
395
has an 18 frontal-ventral-transverse cirral pattern, dorsomarginal kineties (information in
396
respect to Onychodromopsis unknown) and a flexible body, and has been assigned to the
397
Oxytrichinae based on both morphology and molecular information (Berger, 1999). The
398
fourteen oxytrichids species are distributed among nine clades, and AU test also rejects the
399
monophyly of the genus Oxytricha. This is consistent with the results of many previous
400
studies (for example, Hu et al., 2011; Shao et al., 2011) . The non-monophyly of Oxytricha
401
indicates that the 18-cirri pattern might not be a apomorphy of this genus. Additionally, O.
402
paragranulifera n. sp. was not sister to the two O. granulifera sequences, which demonstrated
403
that O. granulifera and O. paragranulifera n. sp. were different species.
404 405
ACKNOWLEDGEMENTS
406
This work was supported by the Natural Science Foundation of China (Project numbers:
407
31372148, 31172041), the Fundamental Research Funds for the Central Universities and
408
Funded by Research Group Project No. RGP-VPP-083, King Saud University Deanship of
409
Scientific Research.
410 411 412
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413 414 415 416 417 418 419 420 421 422 423 424 425
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563
Figures and Legends
564 565
Fig. 1. The sample sites and surrounding areas. (a) Map showing the locations of forest beside
566
Ziwu Road, Xi’an, China and Huguang Mangrove Forest, Zhanjiang, China. (b) Showing the
567
location at the Forest beside Ziwu Road where the sample containing Oxytricha granulifera
568
was collected (blue square). (c, d) Showing the location at the Huguang Mangrove Forest
569
where the sample containing O. paragranulifera n. sp. was collected.
570 571 572
Fig. 2. Morphology of Oxytricha paragranulifera n. sp. (a) Ventral view of a representative
573
individual. (b) Distribution of cortical granules on dorsal side. (c) Ventral view of anterior
574
portion, to show the paroral and endoral, and frontoventral cirri. (d, e) Ventral (d) and dorsal
575
(e) view of the holotype, to show the general infraciliature. AZM, adoral zone of
576
membranelles; BC, buccal cirri; CC, caudal cirri; E, endoral; FC, frontal cirri; FVC,
577
frontoventral cirri; LMR, left marginal row; Ma, macronuclear nodules; Mi, micronuclei; P,
578
paroral; PTVC, pretransverse ventral cirri; PVC, postoral ventral cirri; RMR, right marginal
579
row; TC, transverse cirri. 1–6, dorsal kineties. Bars, 35 m (a, d, e); 15 m (b). μ
μ
580 581 582
Fig. 3. Photomicrographs of Oxytricha paragranulifera n. sp. in vivo (a–l) and after protargol
583
impregnation (m–p). (a) Ventral view of a typical cell. (b–e) Ventral views of different
584
individuals, arrow in (d) points to the contractile vacuole. (f) Ventral view, to show the
585
left/right marginal rows (arrows). (g) Ventral view of the anterior portion, showing the frontal
586
cirri (arrowheads). (h, i, k) Dorsal views, to show the arrangement of the small cortical
587
granules and dorsal cilia (arrowheads). (j) Ventral view of the cell end, arrow indicates the
588
five strong transverse cirri. (l) Dorsal view, to show the macronuclear nodules. (m) Ventral
589
view of the anterior portion, showing the frontal, buccal (arrow) and frontoventral (circle)
590
cirri. (n) Ventral view of the middle portion, arrowheads indicate the postoral ventral cirri. (o)
591
Ventral view of the posterior portion, to show the transverse cirri, caudal cirri (arrows) and
592
the posterior ends of the right and left marginal rows. (p) Dorsal view, to show the dorsal
593
kineties. FC, frontal cirri; Ma, Macronuclear nodules; TC, transverse cirri; 2, 3, 5, 6, dorsal
594
kineties. Bars, 35 m (a–e); 15 m (i, m). μ
μ
595 596 597
Fig. 4. Photomicrographs of Oxytricha granulifera from life (a–d) and after protargol
598
impregnation (e–h). (a) Ventral view of a typical cell. (b) Ventral view of a different
599
individual. (c) Ventral view, to show the contractile vacuole and crystals (arrows). (d) Dorsal 17
600
view, to show the arrangement of the cortical granules (arrows). (e) Ventral view of the
601
anterior portion, showing the frontal, buccal (arrowhead) and frontoventral (arrows) cirri. (f)
602
Ventral view, to show the postoral ventral cirri (arrows), macronuclear nodules and
603
micronuclei. (g) Ventral view of the cell end, to show the left (arrowhead) and right
604
(double-arrowhead) marginal, pretransverse and transverse cirri. (h) Dorsal view, to show the
605
dorsal kinetids (arrows). AZM, adoral zone of membranelles; CV, contractile vacuole; FC,
606
frontal cirri; Ma, macronuclear nodules; Mi, micronuclei; PTVC, pretransverse ventral cirri;
607
TC, transverse cirri. Bars, 50 m (a, b); 20 m (d-h). μ
μ
608 609 610
Fig. 5. A maximum-likelihood (ML) phylogenetic tree inferred from the SSU rRNA gene
611
sequences of 53 hypotrichs showing the position of Oxytricha paragranulifera n. sp. and
612
Oxytricha granulifera. The new sequences from the present study are indicated by bold type.
613
Numbers near nodes are nonparametric bootstrap values for ML and posterior probability
614
values for Bayesian inference (BI). “*” refers to a disagreement in the topology of the BI tree.
615
All branches are drawn to scale. The scale bar corresponds to 0.01 expected substitution per
616
site.
18
617
Table 1. Characterization of Oxytricha paragranulifera n. sp. (upper line) and Oxytricha
618
granulifera Foissner and Adam, 1983 (lower line). Character a
HT
Min
Max
Mean
SD
CV
n
Body length
101
71
106
93.4
9.4
10.1
20
90
127
105.5
8.4
7.9
20
39
57
49.4
5.4
11.0
20
45
75
57.7
6.6
11.5
20
28
43
34.6
3.4
9.9
20
35
45
40.7
3.1
7.6
20
25
29
26.7
1.0
3.7
20
28
38
30.7
2.2
7.2
20
1
1
1.0
0
0
20
1
1
1.0
0
0
20
3
3
3.0
0
0
20
3
3
3.0
0
0
20
4
4
4.0
0
0
20
4
4
4.0
0
0
20
3
3
3.0
0
0
20
3
3
3.0
0
0
20
2
2
2.0
0
0
20
2
2
2.0
0
0
20
5
5
5.0
0
0
20
5
5
5.0
0
0
20
18
25
21.5
2.1
9.6
20
23
31
27.9
2.1
7.4
20
18
25
21.6
2.0
9.2
20
26
32
29.1
1.6
5.5
20
3
3
3.0
0
0
20
3
4
3.2
0.4
11.9
20
6
6
6.0
0
0
20
6
6
6.0
0
0
20
15
23
-
-
-
3
-
-
-
-
-
-
16
23
-
-
-
3
Body width
Adoral zone, length
Adoral membranelles, No.
Buccal cirri, No.
Frontal cirri, No.
Frontoventral cirri, No.
Postoral ventral cirri, No.
Pretransverse ventral cirri,
40
42
28
1
3
4
3
2
No. Transverse cirri, No.
Cirri in left marginal row,
5
22
No. Cirri in right marginal row,
21
No. Caudal cirri, No.
Dorsal kineties, No.
Dorsal kinety 1, bristles no.
Dorsal kinety 2, bristles no.
3
6
21
21
19
Dorsal kinety 3, bristles no.
Dorsal kinety 4, bristles no.
Dorsal kinety 5, bristles no.
Dorsal kinety 6, bristles no.
Bristles in total no.
Macronuclear nodules, No.
Micronuclei, No.
Macronuclear nodule, length
Macronuclear nodule, width
14
6
7
2
71
2
2
22
16
-
-
-
-
-
-
13
17
-
-
-
3
-
-
-
-
-
-
6
7
-
-
-
4
-
-
-
-
-
-
6
7
-
-
-
4
-
-
-
-
-
-
2
2
-
-
-
3
-
-
-
-
-
-
60
77
-
-
-
3
-
-
-
-
-
-
2
2
2.0
0
0
20
2
2
2.0
0
0
20
1
2
1.9
0.4
19.8
20
2
5
3.4
0.9
27.9
20
14
26
20.5
3.6
17.8
20
10
14
11.7
1.5
12.4
20
9
16
10.9
1.5
14.1
20
6
8
6.8
0.7
10.2
20
619
a
620
unavailable. Abbreviations: CV, coefficient of variation in %; HT, holotype; Max, maximum;
621
Mean, arithmetic mean; Min, minimum; n, number of cells measured; SD, standard deviation.
All data are based on protargol-impregnated specimens. Measurements in
622
20
m. - data
μ
1
International Journal of Systematic and Evolutionary Microbiology
2 3
Morphology and phylogenesis of two oxytrichid soil ciliates from China, Oxytricha
4
paragranulifera n. sp. and Oxytricha granulifera Foissner and Adam, 1983 (Protista,
5
Ciliophora, Hypotrichia)
6 7 8
Chen SHAO1, Zhao LV,1 Ying PAN,2 Khaled A. S. AL-RASHEID,3 and Zhenzhen YI4
9 10
1
11
of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China
12
2
13
University of China, Qingdao 266003, China
14
3
Zoology Department, King Saud University, Riyadh 11451, Saudi Arabia
15
4
Laboratory of Protozoology, Key Laboratory of Ecology and Environmental Science in
16
Guangdong Higher Education, College of Life Science, South China Normal University,
17
Guangzhou 510631, China
The Key Laboratory of Biomedical Information Engineering, Ministry of Education, School
Laboratory of Protozoology, Institute of Evolution and Marine Biodiversity, Ocean
18 19 20
Correspondence:
21
Chen Shao
22
Email: [email protected]
23
Telephone number: +86 29 8266 8463 Ext 423
24 25 26
Running title: C. Shao et al. Description of two hypotrichous ciliates
27
Contents category: New Taxa—Eukaryotic Micro-organisms
28 29 30 31
Accession number of the SSU rRNA gene sequence of Oxytricha paragranulifera n. sp. is
32
KJ081200
33
Accession number of the SSU rRNA gene sequence of O. granulifera is KJ081199
34 35
1
36
The morphology and infraciliature of two hypotrichous ciliates, Oxytricha paragranulifera n.
37
sp. and Oxytricha granulifera Foissner and Adam, 1983, collected respectively from the
38
surface of sandy soil in the Huguang mangrove forest, Zhanjiang, China, and the surface of
39
soil in a forest beside Ziwu Road, Xian, northwest China. Oxytricha paragranulifera n. sp. is
40
characterized by an elongate body with slightly tapered anterior end, two macronuclear
41
nodules and two micronuclei, paroral and endoral in Stylonychia-pattern, colourless cortical
42
granules distributed in clusters or irregular short rows, adoral zone occupying 37% of body
43
length, marginal rows almost confluent posteriorly, six dorsal kineties and three caudal cirri,
44
caudal cirri and dorsal bristles almost indistinguishable when viewed in vivo. The well-known
45
Oxytricha granulifera Foissner and Adam, 1983 was also redescribed and can be separated
46
from the new species by having cortical granules arranged along dorsal kineties and marginal
47
rows on both sides (vs. grouped in clusters as well as in short irregular rows), paroral and
48
endoral in Oxytricha-pattern (vs. in Stylonychia-pattern), macronuclear nodules obviously
49
detached (vs. adjacent) and a non-saline terrestrial habitat (vs. saline terrestrial). The
50
separation of these two taxa is also firmly supported by the molecular data which shows a
51
significant discrepancy between the two in their SSU rRNA gene sequences (similarity
52
97.1%). Phylogenetic analyses based on SSU rRNA gene sequence data suggest a close
53
relationship within the Oxytrichidae assemblage between Oxytricha paragranulifera n. sp.
54
and Oxytricha granulifera.
55 56
2
57
INTRODUCTION
58 59
There is consensus among most taxonomists (Song, 1990, 2001; Song & Wilbert, 1997a, b,
60
2002; Berger, 1999; Foissner, 1999; Song & Warren, 1999; Foissner et al., 2002, 2008;
61
Küppers et al., 2011; Chen et al., 2013b; Lv et al., 2013; Shao et al., 2013a, b; Shi et al., 2002;
62
Singh & Kamra, 2013; Singh et al., 2013) that the following characters are important for
63
species separation in oxytrichids: (1) body size, shape and colour; (2) characteristics of
64
cortical granules; (3) whether dorsal cilia and caudal cirri are indistinguishable or
65
distinguishable from marginal cirri; (4) relative length of buccal apparatus and DE-value; (5)
66
characteristics of macronuclear nodules and micronuclei; (6) the pattern of the buccal and
67
somatic ciliature; (7) morphometric data relating to the ciliature; and (8) habitat.
68
Oxytricha is one of the oldest genera (Berger, 1999). Its systematics, however, have
69
traditionally been highly confused, not least because a properly defined type species was not
70
fixed until relatively recently (Berger, 1999). The status of Oxytricha, along with three other
71
oxytrichid genera with flexible bodies and 18 frontoventral transverse cirri, was recently
72
reviewed by Shao et al. (2012), who defined it as follows: flexible 18-cirri Oxytrichidae with
73
undulating membranes in the Oxytricha pattern and adoral zone in the shape of a question
74
mark; frontoventral cirri in a V-shaped pattern; one left and one right marginal row; five or six
75
dorsal kineties in the Oxytricha-pattern, that is, one or two dorsomarginal kineties and simple
76
kinety three; fragmentation present; caudal cirri on dorsal kineties 1, 2 and 4. The genus
77
Oxytricha currently includes ca. 48 morphospecies, several of which have overlapping
78
species-level characters with respect to their living morphology, infraciliature, morphometric
79
data and habitat.
80 81
In November 2010 and April 2012, two oxytrichid ciliates were isolated from the surface of
82
sandy soil in the Huguang town mangrove forest, Zhanjiang, southern China, and the surface
83
of soil in a forest beside Ziwu Road, Xian, northwest China, respectively. Subsequent
84
observations demonstrated them to be members of the genus Oxytricha. In the present paper,
85
their morphology is described and their SSU rRNA genes sequenced.
86 87 88
METHODS
89 90
Sampling and cultivation (Fig. 1). Oxytricha paragranulifera n. sp. was collected from the
91
surface of sandy soil in the Huguang town mangrove forest, Zhanjiang (21°06 N; 110°18 E),
92
China on November 25, 2010 when the soil temperature was 24 °C, salinity 25.5 and pH 7.3.
93
Oxytricha granulifera was collected from the surface of soil in the centre of Zhuque National
′
3
′
94
Forest Park, Xi’an (33°55 N; 108°32 E), China on April 30, 2012 when the soil temperature
95
was 28 °C, salinity 3 and pH 8. Ciliates were stimulated to excyst and emerge from the soil
96
samples by employing the non-flooded Petri dish method described by Foissner (1987) and
97
updated in Foissner et al. (2002). Isolated specimens were maintained as non-clonal cultures
98
in Petri dishes at room temperature (20 °C) using boiled freshwater with rice grains to enrich
99
bacterial food organisms.
′
′
100
Morphology. Living cells were observed with bright field and differential interference
101
contrast microscopy (Chen et al., 2013a). The protargol silver staining method according to
102
Wilbert (1975) was used to reveal the infraciliature (Pan et al., 2013). Measurements of the
103
stained specimens were carried out with an ocular micrometer (Paiva et al., 2012). Drawings
104
of stained specimens were performed at 1,250× magnification with the aid of a camera lucida
105
(Foissner, 2012).
106
Terminology. General terminology is mainly according to Berger (1999); for explanation of
107
terms specific for hypotrichs (e.g., pseudorow, mixed row, DE-value), see Berger & Foissner
108
(1997), Berger (1999, 2006, 2008, 2011), Foissner & Stoeck (2011) and Foissner &
109
Al-Rasheid (2006). For the designation of the frontoventral-transverse cirri, the numbering
110
system by Wallengren (1900) is used (details see Berger 1999: 16). The term 18-cirri
111
hypotrich means a hypotrich with 18 frontal-ventral-transverse cirri (e.g., Berger, 2008: 27).
112
DNA extraction, PCR amplification and sequencing. One or more cells were isolated from
113
the ciliate cultures, and then washed three times with sterilized fresh/saline water (0.22 m
114
filtered). These cells were then transferred to a 1.5 ml microfuge tube with a minimum
115
volume of water. Genomic DNA of Oxytricha paragranulifera n. sp. and O. granulifera were
116
extracted using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the
117
manufacturer’s instructions (Gao et al., 2013; Huang et al., 2014). The PCR amplification of
118
the SSU rRNA gene was performed according to Li et al. (2013) and Yi et al. (2012), with
119
primers 18s-F (5’-AAC CTG GTT GAT CCT GCC AGT-3’) and 18s-R (5’-TGA TCC TTC
120
TGC AGG TTC ACC TAC-3’) (Medlin et al., 1988).
121
Phylogenetic analyses. Using the online program Muscle 3.7 (Edgar, 2004), the SSU rRNA
122
gene sequences of Oxytricha paragranulifera n. sp. and O. granulifera were aligned with 51
123
hypotrichid sequences obtained from the GenBank database. Three urostylid species were
124
selected as the outgroup taxa. These sequences were edited manually using Bioedit 7.0.0 in
125
order to remove ambiguous gaps (Hall, 1999). The program MrModeltest v.2.0 (Nylander,
126
2004) selected the GTR + I (= 0.6678) + G (= 0.5202) as the best model with the AIC
127
criterion, which was then used for Bayesian (BI) and Maximum Likelihood (ML) analyses. BI
128
analysis was performed with MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003). ML trees were
129
constructed using RaxML-HPC2 (Stamatakis et al., 2008) at the CIPRES website
130
(http://www.phylo.org/). MEGA 4.0 (Tamura et al., 2007) was used to visualize tree
μ
4
131
topologies.
132
Topology testing. A constraint tree in which the genus Oxytricha was a monophyletic clade
133
was defined and loaded into PAUP 4.0 (Calendini & Martin, 2005), using ML criterion and
134
heuristic search with the tree bisection-reconnection (TBR) and 10 random sequence addition
135
replicates. The site-wise likelihoods were calculated in PAUP 4.0 under the GTR + I + G
136
model with parameters as suggested by MrModeltest for all trees. Then the constraint tree was
137
compared to unconstrained best and 100 random ML topologies using the Approximately
138
Unbiased (AU) test (Shimodaira, 2002) as implemented in the CONSEL software package
139
(Shimodaira & Hasegawa, 2001; Huang et al., 2012; Huang et al., 2014).
140 141
RESULTS
142 143
Oxytricha paragranulifera n. sp. (Table 1, Figs 2a-e and 3a-p)
144
Diagnosis. Body elongate with slightly tapered anterior end, about 80–110 m × 35–50 µm in
145
size. Two macronuclear nodules and two micronuclei. Cortical granules colourless, densely
146
grouped in clusters or irregular short rows. One contractile vacuole located at about mid-body
147
near left margin. Adoral zone occupies 37% of body length in vivo and is composed of ca. 27
148
membranelles on average. Paroral and endoral in the Stylonychia-pattern. Invariably 18
149
frontoventral transverse cirri. Marginal rows almost confluent posteriorly. Six dorsal kineties
150
and three caudal cirri. Caudal cirri and dorsal bristles almost indistinguishable when viewed
151
in vivo.
152
Etymology. The species name paragranulifera (pa.ra.gra.nu.li'fe.ra. Gr. pref. para- beside,
153
along; N.L. fem. adj. granulifera little seed bearing) referring to the species Oxytricha
154
granulifera; N.L. fem. adj. paragranulifera similar to Oxytrichia granulifera.
155
Type locality. Ciliates were collected in November 2010 from the surface of sandy soil in
156
Huguang town mangrove forest, Zhanjiang (21°07 N; 110°14 E), Guangdong Province, China,
157
when the soil temperature was 24 °C, pH 7.3, and salinity 25.5‰.
158
Type specimens deposited. The slide (No. PY10112501A) containing the holotype specimen
159
(Fig. 2d, e) and a paratype slide (No. PY10112501B) with protargol-impregnated specimens
160
are deposited in the Laboratory of Protozoology, OUC, China.
161
Description (Table 1, Figs 2a-e and 3a-p). Body about 80–110 m × 35–50 m in vivo,
162
non-contractile and flexible, usually slender oval to elliptical with anterior end usually narrow,
163
posterior broadly rounded; right margin slightly convex, left margin strongly convex (Figs 2a
164
and 3a–c, e). Dorsoventrally flattened (about 3:1), ventral side flat, dorsal side convex in
165
middle portion. Cytoplasm colourless to greyish, containing numerous shining globules and
166
crystals. Cells frequently with many lipid droplets (ca 2–4 µm across) and food vacuoles (ca
167
2–5 µm across). Cortical granules colourless, round, about 1 µm across, densely grouped in
μ
′
′
μ
5
μ
168
clusters and irregular short rows on dorsal side, while slightly sparsely arranged in irregular
169
short rows on ventral side (Figs 2b and 3i). Contractile vacuole about 15 µm across, located at
170
the mid-body, near left body margin, contracting at intervals of about 1 min (Figs 2a and 3d).
171
Invariably two macronuclear nodules, usually arranged closely in line at about the mid-body
172
and slightly left of the midline; individual nodules ellipsoidal (length:width ratio about 2:1),
173
with small to moderately large nucleoli. Usually one micronucleus attached to each
174
macronuclear nodule (Figs 2a, e and 3a, l).
175
Locomotion by continuous rapid crawling on bottom of Petri dish and surface of water. When
176
suspended, cells often swim continuously in circles.
177
Infraciliature as shown in Figs 2c–e and 3m–p. Adoral zone occupies 37% of cell length (Fig.
178
2d) and is composed of 25–29 membranelles. Distal portion of adoral zone extends slightly
179
posteriorly onto right side of cell, that is, a DE-value 0.23 in the holotype specimen (Fig. 2d;
180
for explanation of DE-value, see Berger 2006: 18). Paroral and endoral in parallel optically
181
(Figs 2c and 3m). Frontoventral-transverse ciliature comprising 18 cirri: three slightly
182
enlarged frontal cirri with cilia about 15 µm long; single buccal cirrus near anterior end of
183
undulating membranes; frontoventral cirri arranged in a short, mixed, asymmetric, V-shaped
184
row; cirrus III/2 slightly ahead of the level of cirrus VI/3; cirrus III/2 closer to the remaining
185
frontoventral cirri than to the paroral and endoral; three postoral ventral cirri located
186
intermediately behind the vertex and distinctly separated from the two pre-transverse ventral
187
cirri; postoral ventral cirrus IV/2 arranged more anteriorly than V/4; five transverse cirri in a
188
hook-shaped row, with cilia about 20 µm long; two pretransverse ventral cirri, cirrus VI/2
189
located at about the same level as the leftmost transverse cirrus, cirrus V/2 closer to cirrus
190
VI/2 than cirrus V/3; distance between cirri V/3 and V/4 shorter than that between cirri V/3
191
and V/2 (Figs 2c, d and 3g, j, n, o). The posterior ends of marginal rows are almost confluent;
192
left row with 18–25 cirri, right row with 18–25 cirri; cilia of marginal cirri about 10 µm long
193
(Figs 2d, e and 3f).
194
Six dorsal kineties; leftmost two (dorsal kineties 1 and 2) bipolar, each comprising about 20
195
pairs of basal bodies; third and fifth dorsal kineties start at about the anterior end of cell and
196
terminate at 4/5 and 2/5 of cell length respectively; fourth dorsal kinety commences near the
197
equatorial level of cell and stretches to the posterior end; rightmost dorsal kinety (dorsal
198
kinety 6) composed of two or three dikinetids (Figs 2e and 3p). Dorsal cilia about 3 µm long
199
in vivo. Three caudal cirri, thin, located at posterior body margin, narrowly separated, one
200
each on dorsal kineties 1, 2, and 4 (Figs 2e and 3h); cilia of caudal cirri about 12 µm long in
201
vivo and thus almost indistinguishable from marginal cirri (Fig. 2a and 3a, j).
202 203
Oxytricha granulifera Foissner and Adam, 1983 (Table 1, Fig 4a-h)
204
Description. Body about 90–130 m × 30–50 m in vivo, length:width ratio approximately μ
μ
6
205
3:1 in vivo, 1.8:1 on average in protargol preparations. Body flexible but not contractile,
206
usually slender oval to elliptical with anterior end usually narrow, posterior broadly rounded
207
(Fig. 4a, b). Dorsoventrally flattened (about 3:1), ventral side flat, dorsal side convex in
208
middle portion; right cell margin slightly convex to slightly concave, left margin strongly
209
convex, usually widest in front of mid-body. Cytoplasm colourless to greyish, containing
210
numerous shining globules and crystals (Fig. 4c). Cells with many lipid droplets (ca 4–5 µm
211
across) and food vacuoles (ca 5–10 µm across). Cortical granules colourless, round, about 1
212
µm across, arranged along dorsal kineties and marginal rows on both sides (Fig. 4d).
213
Contractile vacuole located at mid-body, near left body margin, contracting at intervals of
214
about 10s (Fig. 4b, c). Invariably, two macronuclear nodules, usually arranged at anterior and
215
posterior 1/3, as well as slightly left of the midline (Fig. 4f). Two micronuclei, one each
216
beside the two macronuclear nodules (Fig. 4f). Locomotion by slow to moderately fast
217
crawling on substrate, but usually motionless.
218
Infraciliature as shown in Fig. 4e-h. Adoral zone occupies 39% of cell length on average in
219
protargol preparations, composed of 28–38 adoral membranelles. Distal portion of adoral
220
zone extends slightly posteriorly on to the right side of cell, that is, DE-value about 0.30 (Fig.
221
4e; for explanation of DE-value, see Berger 2006: 18). Paroral and endoral intersect each
222
other optically behind the buccal cirrus (Fig. 4e). Three slightly enlarged frontal cirri with
223
cilia about 15 µm long; single buccal cirrus near anterior end of paroral and endoral;
224
frontoventral cirri arranged in a short, mixed, asymmetric, V-shaped row; cirrus III/2 slightly
225
ahead of level of cirrus VI/3; cirrus III/2 closer to remaining frontoventral cirri than to paroral
226
and endoral (Fig. 4e) Three postoral ventral cirri located intermediately behind vertex and
227
distinctly separated from the two pre-transverse ventral cirri; postoral ventral cirrus IV/2
228
arranged more anteriorly than V/4. Five transverse cirri in a hook-shaped row, with cilia
229
about 20 µm long; two pretransverse ventral cirri, cirrus VI/2 located near rightmost
230
transverse cirrus, cirrus V/2 closer to cirrus VI/2 than cirrus V/3; distance between cirri V/3
231
and V/4 less than that between cirri V/3 and V/2 (Fig. 4g). Posterior ends of marginal rows
232
are almost confluent; left row with 23–31 cirri, right row with 26–32 cirri; cilia of marginal
233
cirri about 10 µm long (Fig. 4g).
234
Six dorsal kineties; leftmost two (dorsal kineties 1 and 2) bipolar, each comprising about 20
235
pairs of basal bodies; third and fifth dorsal kineties start at about the anterior end of cell and
236
terminate at 4/5 and 2/5 of cell length respectively; fourth dorsal kinety commences near the
237
equatorial level of cell and stretches to the posterior end; rightmost dorsal kinety (dorsal
238
kinety 6) composed of two or three dikinetids (Fig. 4h). Dorsal cilia about 3 µm long in vivo.
239
Three caudal cirri, thin, located at the posterior body margin, slightly separated, one each on
240
dorsal kineties 1, 2 and 4.
241 7
242
Phylogenetic analyses (Fig. 5)
243
The SSU rRNA gene sequence of Oxytricha paragranulifera n. sp. and O. granulifera were
244
deposited into GenBank, with accession numbers KJ081200 and KJ081199, respectively. The
245
SSU rDNA gene sequence of our Oxytricha granulifera is 1620 bp long and has a GC content
246
of 45.68%, and that of O. granulifera (GenBank accession number AF164122) is 1774 bp
247
long and has a GC content of 45.55%. The similarity between them is 99.7% indicating that
248
they are conspecific. The topologies of the ML and BI trees were similar, therefore only the
249
ML tree is shown (Fig. 5). In both analyses, Oxytricha paragranulifera n. sp. and Oxytricha
250
granulifera fall into the Oxytrichinae assemblage. However, the Oxytricha is not a
251
monophyletic group: fourteen species fall into nine clades. And the monophyly of the genus
252
Oxytricha is rejected by the AU test (P = 0.003). Oxytricha granulifera clustered with
253
Oxytricha sp. 1 MD-2012 with high support values (ML/BI, 83/0.93), and O. paragranulifera
254
n. sp. was sister to Onychodromopsis flexilis with low and high support values (ML/BI,
255
77/0.96).
256 257
DISCUSSION
258 259
Oxytricha paragranulifera n. sp. (Table 1, Figs 2a-e and 3a-p)
260
Comparison with congeners. In terms of its slender oval to elliptical body shape and slightly
261
tapered anterior end, two macronuclear nodules and indistinguishable dorsal cilia and caudal
262
cirri, 23 congeners resemble Oxytricha paragranulifera n. sp. Thus, we compare Oxytricha
263
paragranulifera n. sp with each of these.
264
Oxytricha granulifera Foissner and Adam, 1983 has a very similar body size and body shape
265
to O. paragranulifera n. sp. but can be distinguished from the latter by having cortical
266
granules arranged along the dorsal kineties and marginal rows on both sides (vs. grouped in
267
clusters as well as in short irregular rows). Furthermore, the paroral and endoral intersect each
268
other optically (vs. in parallel optically), five (in Austrian population) or six (in Chinese
269
population) dorsal kineties (vs. six), the macronuclear nodules are obviously detached (vs.
270
adjacent) and the habitat is non-saline terrestrial (vs. saline terrestrial) (Berger, 1999). The
271
separation of these two taxa is also firmly supported by the molecular data which shows a
272
significant discrepancy between the two in their SSU rRNA gene sequences (similarity
273
97.1%).
274
Considering the somatic ciliature, body shape, and the appearance of the undulating
275
membranes, O. chlorelligera Kahl, 1932 is similar to O. paragranulifera n. sp., although the
276
former can be recognized by the presence of symbiotic algae (vs. absent) (Berger, 1999).
277
Oxytricha fallax Stein, 1859 can be separated from O. paragranulifera n. sp. by its obviously
278
detached (vs. adjacent) macronuclear nodules, the Oxytricha-pattern (vs. Stylonychia-pattern) 8
279
of its paroral and endoral and the absence (vs. presence) of cortical granules (Berger, 1999).
280
Compared with the well-known O. granulosa Schmitz, 1986, O. paragranulifera n. sp. has a
281
smaller body (80–110 µm vs. 155–280 µm), colourless cortical granules which are grouped in
282
clusters as well as in short irregular rows (vs. yellow-green in colour and arranged in 14–18
283
longitudinal rows), fewer membranelles (25–29 vs. 44–50), more dorsal kineties (six vs. four
284
or five), paroral and endoral in the Stylonychia-pattern (vs. in the Oxytricha-pattern) and two
285
(vs. one) micronuclei (Berger, 1999).
286
Oxytricha hymenostoma Stokes, 1887 differs from O. paragranulifera n. sp. by having a
287
broadly rounded (vs. slightly tapered) anterior end, slightly separated (vs. well separated)
288
postoral and pretransverse cirri, dorsal kinety 4 starting almost at the anterior end (vs.
289
commencing at the mid-body) and obviously detached (vs. adjacent) macronuclear nodules
290
(Berger, 1999).
291
Compared with O. paragranulifera n. sp., O. longicirrata Kahl, 1932 has its paroral and
292
endoral in the Oxytricha-pattern (vs. in the Stylonychia-pattern), a lack of cortical granules (vs.
293
their presence), obviously detached (vs. adjacent) macronuclear nodules and distinctly
294
separated (vs. confluent) marginal rows posteriorly (Berger, 1999).
295
Considering the somatic ciliature, body shape, and the appearance of the undulating
296
membranes, O. paragranulifera n. sp. is similar to O. aeruginosa Wrzesniowskiego, 1866,
297
although the former can be recognized by its colourless (vs. russet and black) cortical
298
granules, smaller body size (80–110 µm vs. 120–165 µm) and adjacent (vs. obviously
299
detached) macronuclear nodules (Berger, 1999).
300
Oxytricha paragranulifera n. sp. differs from O. longissima Dragesco and Njine, 1971 in
301
having a smaller body (80–110 µm vs. 250 µm), transverse cirri slightly (vs. distinctly)
302
displaced anteriorly, fewer right and left marginal cirri (18–25 and 18–25 vs. 40–54 and
303
37–42) and adjacent (vs. obviously detached) macronuclear nodules (Berger, 1999).
304
In terms of its somatic ciliature and body shape, O. paragranulifera n. sp. is most similar to O.
305
multiseta Dragesco, 1966. The former, however, can be recognized by the number of
306
transverse cirri (consistently five vs. six or seven) and adjacent (vs. obviously detached)
307
macronuclear nodules (Berger, 1999).
308
Oxytricha quadricirrata Blatterer and Foissner, 1988 can be separated from O.
309
paragranulifera n. sp. by its distinctly separated (vs. confluent) marginal rows posteriorly, its
310
cortical granules being arranged along the dorsal kineties and marginal rows (vs. in clusters
311
and in short irregular rows) and fewer membranelles (19–21 vs. 25–29), transverse cirri (four
312
vs. five), right marginal cirri (14–17 vs. 18–25) and left marginal cirri (13–18 vs. 18–25)
313
(Berger, 1999).
314
Oxytricha variabilis Grolière, 1975 differs from O. paragranulifera n. sp. in the number of
315
micronuclei (four vs. two), obviously detached (vs. adjacent) macronuclear nodules and in 9
316
having five (vs. three) postoral ventral cirri and five (vs. six) dorsal kineties (Berger, 1999).
317
Oxytricha auripunctata Blatterer and Foissner, 1988 can be separated from O.
318
paragranulifera n. sp. in having one to five (vs. invariably two) micronuclei; four to five (vs.
319
three) caudal cirri, distinctly separated (vs. confluent) marginal rows posteriorly;
320
orange-yellow (vs. colourless) cortical granules; paroral and endoral arranged in the
321
Oxytricha-pattern (vs. in the Stylonychia-pattern) and obviously detached (vs. adjacent)
322
macronuclear nodules (Berger, 1999).
323
Oxytricha arabica Foissner et al., 2008 can be separated from O. paragranulifera n. sp. in
324
having five (vs. six) dorsal kineties and an absence of cortical granules (vs. their presence)
325
(Foissner et al., 2008).
326
Oxytricha lanceolata Shibuya, 1930 has a similar body size and shape to O. paragranulifera
327
n. sp. but can be separated from the latter by having an absence of cortical granules (vs. their
328
presence), four (vs. six) dorsal kineties and obviously detached (vs. adjacent) macronuclear
329
nodules (Berger, 1999).
330
Oxytricha matritensis Ramirez-Montesinos and Perez-Silva, 1966 differs from O.
331
paragranulifera n. sp. in the number of right marginal cirri (about 17 (data from drawing) vs.
332
18–25) and left marginal cirri (about 15 (data from drawing) vs. 18–25), the probable absence
333
of caudal cirri (vs. their presence) and obviously detached (vs. adjacent) macronuclear
334
nodules (Berger, 1999).
335
Oxytricha proximata Shibuya, 1930 differs from O. paragranulifera n. sp. in its obviously
336
detached (vs. adjacent) macronuclear nodules, four (vs. five) transverse cirri and in the
337
rectangular arrangement (vs. a V-shaped arrangement) of its frontoventral cirri (Berger, 1999).
338
Oxytricha longigranulosa Berger and Foissner, 1989 resembles O. paragranulifera n. sp. in
339
its dorsal ciliature, however, the former can be distinguished by the arrangement of cortical
340
granules (in short lines vs. in clusters and short irregular lines), body shape (elliptical vs.
341
mostly slender oval) and in the distinctly separated (vs. confluent) marginal rows posteriorly
342
(Berger, 1999).
343
Compared to O. paragranulifera n. sp., O. pseudofusiformis Dragesco and Dragesco-Kernéis,
344
1986 has a smaller body size (35–46 µm after protargol impregnation vs. 80–110 µm in vivo),
345
fewer cirri in the frontal area (five to seven vs. eight), membranelles (12–18 vs. 25–29), right
346
marginal (7–11 vs. 18–25) and left marginal cirri (seven to nine vs. 18–25) as well as only one
347
(vs. two) micronucleus and transverse cirri that are distinctly (vs. slightly) displaced
348
anteriorly (Berger, 1999).
349
Oxytricha tenella Song and Wilbert, 1989 can be separated from O. paragranulifera n. sp. in
350
having a smaller body (50–70 µm vs. 80–110 µm); cortical granules arranged irregularly (vs.
351
in clusters and short irregular lines); paroral and endoral arranged in the Oxytricha-pattern (vs.
352
in the Stylonychia-pattern), as well as postoral and pretransverse cirri slightly separated (vs. 10
353
well separated) (Berger, 1999).
354
Oxytricha rubripuncta Berger and Foissner, 1987 differs from O. paragranulifera n. sp. in
355
having two to four (vs. two) micronuclei, cirrus III/2 located between cirri IV/3 and VI/3 (vs.
356
between cirri VI/3 and VI/4), paroral and endoral arranged in the Oxytricha-pattern (vs. in the
357
Stylonychia-pattern), reddish (vs. colourless) cortical granules, obviously detached (vs.
358
adjacent) macronuclear nodules and dorsal kinety 5 bipolar (vs. terminating at the mid-body)
359
(Berger, 1999).
360
Oxytricha durhamiensis Berger, 1999 can be separated from O. paragranulifera n. sp. by
361
having cortical granules in linear groups of two to five parallel to the dorsal kineties (vs. in
362
clusters and short irregular lines) and cirrus III/2 located between cirri IV/3 and VI/3 (vs.
363
between cirri VI/3 and VI/4) (Berger, 1999).
364
Oxytricha oxymarina Berger, 1999 can be distinguished from O. paragranulifera n. sp. in
365
having three bipolar dorsal kineties (vs. six dorsal kineties, four of which are not bipolar),
366
distinctly separated (vs. confluent) marginal rows posteriorly, and an adoral zone that is rather
367
long relative to the body length (51% vs. 37% after protargol impregnation) (Berger, 1999).
368
Compared to O. paragranulifera n. sp., Oxytricha alfredi Berger, 1999 has one (vs. two)
369
micronucleus and a rather long posterior-most marginal (or caudal?) cirri (vs.
370
indistinguishable) (Berger, 1999).
371
Compared with O. paragranulifera n. sp., O. acidotolerans Weisse et al., 2013 has its paroral
372
and endoral in the Oxytricha-pattern (vs. in the Stylonychia-pattern), a lack of cortical
373
granules (vs. their presence), distinctly separated (vs. confluent) marginal rows posteriorly
374
and dorsal ciliature (dorsal kineties 1 and 2 shortened anteriorly and each comprising about
375
10 pairs of basal bodies (vs. bipolar and each comprising about 20 pairs of basal bodies) and
376
third dorsal kinety terminating at midline (vs. 4/5) of cell length) (Weisse et al., 2013).
377 378 379
Oxytricha granulifera Foissner and Adam, 1983 (Table 1, Fig. 4a-h)
380
Comparison with Austrian populations. According to the original report (Foissner and
381
Adam, 1983), our population closely resembles the Austrian population of Oxytricha
382
granulifera, the main difference being the number of dorsal kineties (five in Australian
383
population vs. six in the present population. We believe that this difference is intraspecific and
384
therefore not significant for species level separation. Since all other key characters, i.e. body
385
size, shape, macronuclear and micronuclear features, arrangement of cirri, morphometric data
386
(Table 1, Foissner and Adam 1983) and biotope are all consistent with the original description.
387
The identification of the Xi’an population is therefore not in doubt. Further, the similarity
388
between our O. granulifera and O. granulifera (AF164122) is 99.7%, indicating that they are
389
conspecific. 11
390 391
Phylogenetic analyses.
392
In the present SSU rRNA gene trees, Oxytricha granulifera was sister to O. sp. 1 MD-2012,
393
and O. paragranulifera n. sp. clustered with Onychodromopsis flexilis, O. ottowi 1 MD-2012,
394
O. ottowi 2 MD-2012, O. longigranulosa and Urosomoida lanceolata. Each of these species
395
has an 18 frontal-ventral-transverse cirral pattern, dorsomarginal kineties (information in
396
respect to Onychodromopsis unknown) and a flexible body, and has been assigned to the
397
Oxytrichinae based on both morphology and molecular information (Berger, 1999). The
398
fourteen oxytrichids species are distributed among nine clades, and AU test also rejects the
399
monophyly of the genus Oxytricha. This is consistent with the results of many previous
400
studies (for example, Hu et al., 2011; Shao et al., 2011) . The non-monophyly of Oxytricha
401
indicates that the 18-cirri pattern might not be a apomorphy of this genus. Additionally, O.
402
paragranulifera n. sp. was not sister to the two O. granulifera sequences, which demonstrated
403
that O. granulifera and O. paragranulifera n. sp. were different species.
404 405
ACKNOWLEDGEMENTS
406
This work was supported by the Natural Science Foundation of China (Project numbers:
407
31372148, 31172041), the Fundamental Research Funds for the Central Universities and
408
Funded by Research Group Project No. RGP-VPP-083, King Saud University Deanship of
409
Scientific Research.
410 411 412
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Figures and Legends
564 565
Fig. 1. The sample sites and surrounding areas. (a) Map showing the locations of forest beside
566
Ziwu Road, Xi’an, China and Huguang Mangrove Forest, Zhanjiang, China. (b) Showing the
567
location at the Forest beside Ziwu Road where the sample containing Oxytricha granulifera
568
was collected (blue square). (c, d) Showing the location at the Huguang Mangrove Forest
569
where the sample containing O. paragranulifera n. sp. was collected.
570 571 572
Fig. 2. Morphology of Oxytricha paragranulifera n. sp. (a) Ventral view of a representative
573
individual. (b) Distribution of cortical granules on dorsal side. (c) Ventral view of anterior
574
portion, to show the paroral and endoral, and frontoventral cirri. (d, e) Ventral (d) and dorsal
575
(e) view of the holotype, to show the general infraciliature. AZM, adoral zone of
576
membranelles; BC, buccal cirri; CC, caudal cirri; E, endoral; FC, frontal cirri; FVC,
577
frontoventral cirri; LMR, left marginal row; Ma, macronuclear nodules; Mi, micronuclei; P,
578
paroral; PTVC, pretransverse ventral cirri; PVC, postoral ventral cirri; RMR, right marginal
579
row; TC, transverse cirri. 1–6, dorsal kineties. Bars, 35 m (a, d, e); 15 m (b). μ
μ
580 581 582
Fig. 3. Photomicrographs of Oxytricha paragranulifera n. sp. in vivo (a–l) and after protargol
583
impregnation (m–p). (a) Ventral view of a typical cell. (b–e) Ventral views of different
584
individuals, arrow in (d) points to the contractile vacuole. (f) Ventral view, to show the
585
left/right marginal rows (arrows). (g) Ventral view of the anterior portion, showing the frontal
586
cirri (arrowheads). (h, i, k) Dorsal views, to show the arrangement of the small cortical
587
granules and dorsal cilia (arrowheads). (j) Ventral view of the cell end, arrow indicates the
588
five strong transverse cirri. (l) Dorsal view, to show the macronuclear nodules. (m) Ventral
589
view of the anterior portion, showing the frontal, buccal (arrow) and frontoventral (circle)
590
cirri. (n) Ventral view of the middle portion, arrowheads indicate the postoral ventral cirri. (o)
591
Ventral view of the posterior portion, to show the transverse cirri, caudal cirri (arrows) and
592
the posterior ends of the right and left marginal rows. (p) Dorsal view, to show the dorsal
593
kineties. FC, frontal cirri; Ma, Macronuclear nodules; TC, transverse cirri; 2, 3, 5, 6, dorsal
594
kineties. Bars, 35 m (a–e); 15 m (i, m). μ
μ
595 596 597
Fig. 4. Photomicrographs of Oxytricha granulifera from life (a–d) and after protargol
598
impregnation (e–h). (a) Ventral view of a typical cell. (b) Ventral view of a different
599
individual. (c) Ventral view, to show the contractile vacuole and crystals (arrows). (d) Dorsal 17
600
view, to show the arrangement of the cortical granules (arrows). (e) Ventral view of the
601
anterior portion, showing the frontal, buccal (arrowhead) and frontoventral (arrows) cirri. (f)
602
Ventral view, to show the postoral ventral cirri (arrows), macronuclear nodules and
603
micronuclei. (g) Ventral view of the cell end, to show the left (arrowhead) and right
604
(double-arrowhead) marginal, pretransverse and transverse cirri. (h) Dorsal view, to show the
605
dorsal kinetids (arrows). AZM, adoral zone of membranelles; CV, contractile vacuole; FC,
606
frontal cirri; Ma, macronuclear nodules; Mi, micronuclei; PTVC, pretransverse ventral cirri;
607
TC, transverse cirri. Bars, 50 m (a, b); 20 m (d-h). μ
μ
608 609 610
Fig. 5. A maximum-likelihood (ML) phylogenetic tree inferred from the SSU rRNA gene
611
sequences of 53 hypotrichs showing the position of Oxytricha paragranulifera n. sp. and
612
Oxytricha granulifera. The new sequences from the present study are indicated by bold type.
613
Numbers near nodes are nonparametric bootstrap values for ML and posterior probability
614
values for Bayesian inference (BI). “*” refers to a disagreement in the topology of the BI tree.
615
All branches are drawn to scale. The scale bar corresponds to 0.01 expected substitution per
616
site.
18
617
Table 1. Characterization of Oxytricha paragranulifera n. sp. (upper line) and Oxytricha
618
granulifera Foissner and Adam, 1983 (lower line). Character a
HT
Min
Max
Mean
SD
CV
n
Body length
101
71
106
93.4
9.4
10.1
20
90
127
105.5
8.4
7.9
20
39
57
49.4
5.4
11.0
20
45
75
57.7
6.6
11.5
20
28
43
34.6
3.4
9.9
20
35
45
40.7
3.1
7.6
20
25
29
26.7
1.0
3.7
20
28
38
30.7
2.2
7.2
20
1
1
1.0
0
0
20
1
1
1.0
0
0
20
3
3
3.0
0
0
20
3
3
3.0
0
0
20
4
4
4.0
0
0
20
4
4
4.0
0
0
20
3
3
3.0
0
0
20
3
3
3.0
0
0
20
2
2
2.0
0
0
20
2
2
2.0
0
0
20
5
5
5.0
0
0
20
5
5
5.0
0
0
20
18
25
21.5
2.1
9.6
20
23
31
27.9
2.1
7.4
20
18
25
21.6
2.0
9.2
20
26
32
29.1
1.6
5.5
20
3
3
3.0
0
0
20
3
4
3.2
0.4
11.9
20
6
6
6.0
0
0
20
6
6
6.0
0
0
20
15
23
-
-
-
3
-
-
-
-
-
-
16
23
-
-
-
3
Body width
Adoral zone, length
Adoral membranelles, No.
Buccal cirri, No.
Frontal cirri, No.
Frontoventral cirri, No.
Postoral ventral cirri, No.
Pretransverse ventral cirri,
40
42
28
1
3
4
3
2
No. Transverse cirri, No.
Cirri in left marginal row,
5
22
No. Cirri in right marginal row,
21
No. Caudal cirri, No.
Dorsal kineties, No.
Dorsal kinety 1, bristles no.
Dorsal kinety 2, bristles no.
3
6
21
21
19
Dorsal kinety 3, bristles no.
Dorsal kinety 4, bristles no.
Dorsal kinety 5, bristles no.
Dorsal kinety 6, bristles no.
Bristles in total no.
Macronuclear nodules, No.
Micronuclei, No.
Macronuclear nodule, length
Macronuclear nodule, width
14
6
7
2
71
2
2
22
16
-
-
-
-
-
-
13
17
-
-
-
3
-
-
-
-
-
-
6
7
-
-
-
4
-
-
-
-
-
-
6
7
-
-
-
4
-
-
-
-
-
-
2
2
-
-
-
3
-
-
-
-
-
-
60
77
-
-
-
3
-
-
-
-
-
-
2
2
2.0
0
0
20
2
2
2.0
0
0
20
1
2
1.9
0.4
19.8
20
2
5
3.4
0.9
27.9
20
14
26
20.5
3.6
17.8
20
10
14
11.7
1.5
12.4
20
9
16
10.9
1.5
14.1
20
6
8
6.8
0.7
10.2
20
619
a
620
unavailable. Abbreviations: CV, coefficient of variation in %; HT, holotype; Max, maximum;
621
Mean, arithmetic mean; Min, minimum; n, number of cells measured; SD, standard deviation.
All data are based on protargol-impregnated specimens. Measurements in
622
20
m. - data
μ
