Accepted Manuscript Title: Morphology, morphogenesis, and molecular phylogeny of Sterkiella tetracirrata n. sp. (Ciliophora, Oxytrichidae), from the Silent Valley National Park, India Author: Santosh Kumar Komal Kamra Daizy Bharti Antonietta La Terza Neeta Sehgal Alan Warren Gulshan Rai Sapra PII: DOI: Reference:

S0932-4739(14)00085-6 http://dx.doi.org/doi:10.1016/j.ejop.2014.12.002 EJOP 25358

To appear in: Received date: Revised date: Accepted date:

25-3-2014 4-12-2014 5-12-2014

Please cite this article as: Kumar, S., Kamra, K., Bharti, D., La Terza, A., Sehgal, N., Warren, A., Sapra, G.R.,Morphology, morphogenesis, and molecular phylogeny of Sterkiella tetracirrata n. sp. (Ciliophora, Oxytrichidae), from the Silent Valley National Park, India, European Journal of Protistology (2014), http://dx.doi.org/10.1016/j.ejop.2014.12.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Morphology, morphogenesis, and molecular phylogeny of Sterkiella tetracirrata n. sp. (Ciliophora, Oxytrichidae), from the Silent Valley National Park, India Santosh Kumara, b, Komal Kamraa, *, Daizy Bhartib, Antonietta La Terzab, Neeta Sehgalc, Alan

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Warrend, Gulshan Rai Sapraa

Ciliate Biology Laboratory, Sri Guru Tegh Bahadur Khalsa College, University of Delhi, Delhi

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110 007, India b

School of Environmental Science, University of Camerino, Via Gentile III da Varano, 62032

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Camerino (MC), Italy

Department of Zoology, University of Delhi, Delhi 110 007, India

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

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*Corresponding author. Tel.: +91 987 177 1417; Tel./ fax: +91 011 27666220. E-mail address: [email protected] (K. Kamra), [email protected] (S. Kumar).

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Abstract The morphology and morphogenesis during cell division of Sterkiella tetracirrata n. sp., isolated from a soil sample collected from the Silent Valley National Park, Kerala, India, were investigated using live observation, protargol staining and scanning electron microscopy. The new species differs from its congeners by the following combination of features: cell size in vivo 85–110 × 35–

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50 µm, on average 84 × 37 µm in protargol preparations; four ellipsoidal macronuclear nodules; 31 adoral membranelles; 17 frontal-ventral-transverse cirri consisting of three frontal, four

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frontoventral, one buccal, three ventral, two pretransverse and invariably four transverse cirri;

resting cyst with separate macronuclear nodules. Sterkiella tetracirrata differs from the similar

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species S. terricola in the number of transverse cirri (invariably 4 vs. 3) and in the number of adoral membranelles (24–35 vs. 22 or 23). Morphogenesis resembles that of its congeners S. nova and S. histriomuscorum. Phylogenetic analyses based on SSU rRNA gene sequences consistently

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place the new species within the stylonychine oxytrichids, clustering closer to Gastrostyla steinii than to either S. cavicola or S. histriomuscorum. The analyses support the morphological evidence

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(e.g., similarity in the oral apparatus and the dorsal kinety pattern) that Gastrostyla and

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Pattersoniella evolved from a Sterkiella-like ancestor.

Keywords: India; Morphogenesis; Phylogeny; Soil ciliate; Sterkiella tetracirrata n. sp.; Stylonychine oxytrichids

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Introduction According to Berger and Foissner (1997), the family Oxytrichidae comprises two subfamilies – Stylonychinae and Oxytrichinae. In his monograph of the Oxytrichidae, Berger (1999) also recognises these two sub-families, although their validity was rejected by Lynn (2008). The classification of oxytrichids and oxytrichines based on plesiomorphic characters reveals the

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Oxytrichinae to be paraphyletic (Berger 2006, 2008). Based on morphological traits and molecular data, Berger (2008) eliminated the subfamily Oxytrichinae and recognised two groups within the

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Oxytrichidae, namely the Stylonychinae and non-stylonychine oxytrichids. The characteristics of the subfamily Stylonychinae that set it apart from the non-stylonychine oxytrichids, barring some

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exceptions, are primarily the presence of a rigid body, the length of the adoral zone of

membranelles relative to the body length ≥ 40%, the absence of cortical granules, the third postoral ventral cirrus (V/3) distinctly displaced posteriorly and not involved in primordial formation, and

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various morphogenetic peculiarities. Although members of the genus Sterkiella Foissner et al., 1991 belong to the sub-family Stylonychinae, they nevertheless possess a semirigid body, and the

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paroral and endoral optically intersect in the typical Oxytricha pattern. With the transfer of Sterkiella thompsoni to the genus Parasterkiella (Küppers et al. 2011), seven species of Sterkiella Berger 1999; Foissner et al. 1991).

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are currently recognized, only three of which are well-characterized (Berger 1999; Foissner and

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The present study describes a new soil species of the genus Sterkiella, isolated from tropical rain forest tracts of the core zone of the Silent Valley National Park, Kerala, India, a

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biodiversity hotspot whose ciliate fauna has been partially described (Kumar et al. 2010). The new species was brought into clonal culture enabling observations to be made of its morphology based on examination of specimens in vivo, following protargol staining and by scanning electron microscopy (SEM), and of its morphogenetic processes. Sterkiella tetracirrata n. sp. exhibits a unique combination of morphological characters that separates it from other members of the genus. A detailed description of its morphology, morphogenesis, and phylogenetic position based on SSU rRNA gene sequence data is presented.

Material and Methods Collection and cell culture Twelve soil samples (0–10 cm deep) were collected in January 2008 from various ecozones within the core zone of the Silent Valley National Park (11°08′ to 11°13′N; 76°28′ to 76°47′E). Ciliates were made to excyst and emerge from one-month-old dried soil samples (approximately 250 g dry weight) by employing the non-flooded Petri dish method (Foissner 1987). Cells of 3 Page 3 of 22

Sterkiella tetracirrata isolated from one of these samples were used to raise clonal cultures in the laboratory. Cells were found to thrive well when maintained at a temperature of 18oC ± 2oC in Pringsheim’s medium with the green alga Chlorogonium elongatum as the food organism (Ammermann et al. 1974). A single clonal culture was used to obtain data for morphological,

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morphogenetic and molecular analyses.

Morphological and morphogenetic analyses

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Live observations were made using differential interference contrast microscopy. Protargol staining was used to reveal the infraciliature (Kamra and Sapra 1990). Scanning electron

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microscopy (SEM) was performed according to Foissner (1991). Biometric characterization was carried out at a magnification of 40–1000 X directly from the Leica software IM50 image manager. A Leica camera DFC320 was employed for photomicrography. Line diagrams were

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prepared using free-hand sketches and CorelDRAW(R) Graphics Suite – Version 12.0 software. To illustrate the changes during divisional morphogenesis, parental cirri are depicted by contour

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whereas new ones are filled in.

Nomenclature and terminology are according to Berger (1999), Borror (1972), Foissner and Berger (1997), Martin (1982) and Wallengren (1900). The numbering of the frontal-ventral-

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transverse cirri follows Wallengren (1900) and is based on their ontogenetic origins (Fig. 1).

DNA extraction, PCR amplification and sequencing

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Thirty to 40 cells from an overnight-starved clonal culture were collected using glass

micropipettes and washed three times with autoclaved distilled water. DNA was extracted using the DNeasy blood and tissue kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. Extracted DNA was transferred to a fresh microfuge tube and stored at 4°C until PCR amplification.

Extracted DNA (5 μl) was dispensed into a PCR tube containing 5 μl of distilled water and

amplifications were carried out using high-fidelity USB Taq DNA polymerase in a total volume of 50 μl with the universal eukaryotic primers Euk A (FW 5’-AACCTGGTTGATCCTGCCAGT-3’) and Euk B (RV 5’-TGATCCTTCTGCAGGTTCACCTAC- 30) (Medlin et al. 1988). The PCR program for SSU rRNA gene amplification included an initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 1 min, 55°C for 45 s, and 72°C for 80 s, with a final extension step at 72°C for 10 min. After confirmation of the appropriate size, the PCR products were purified using the Nucleospin gel extraction kit (QIAGEN GmbH, Hilden, Germany) and were then directly sequenced on both strands at Ocimum Biosolutions, Bangalore, India. 4 Page 4 of 22

Phylogenetic analyses The newly sequenced SSU rRNA gene sequence of Sterkiella tetracirrata n. sp. was aligned with 36 SSU rRNA hypotrich gene sequences that were retrieved from GenBank database Standley 2013). Eight urostylids were used as the outgroup taxa.

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using the MATTF 7.047 software (choosing the iterative refinement methods L-INS-i) (Katoh and The final alignment was then used for subsequent phylogenetic analyses after converting the

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FASTA (.fas) file to NEXUS (.nex) format using the open web-based tool ALTER (Alignment

Transformation EnviRonment) (Glez-Peña et al. 2010). A Bayesian inference (BI) analysis was

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performed using Mr.Bayes v.3.2.1 (Ronquist and Huelsenbeck 2003) and the GTR+I+G model, as selected by the jModel Test v.2.1.3 software (Posada 2008) under the Akaike Information Criterion corrected (AICc). Markov chain Monte Carlo (MCMC) simulations were run with two

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sets of four chains using the default settings: chain length 10,000,000 generations with trees sampled every 100 generations and with a prior burn-in of 25%, i.e., the first 25,000 sampled trees

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were discarded. The remaining trees were used to generate a consensus tree and to calculate the posterior probabilities (PP).

A Maximum Likelihood (ML) tree was constructed using Molecular Evolutionary Genetic

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Analysis (MEGA) v.5.2.2 (Tamura et al. 2011). The topology of the trees was inferred by running

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1000 bootstrap replicates and was expressed as a percentage. Phylogenetic trees were visualized using the free software package FigTree v1.4 by A. Rambaut at

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http://tree.bio.ed.ac.uk/software/figtree/.

Results

Sterkiella tetracirrata n. sp. (Figs 1A–C, 2A–H; Table 1) Diagnosis: Body oblong, 85–110 × 35–50 µm in vivo; usually 4 macronuclear nodules and 2–4 micronuclei; adoral zone 42% of body length, composed of 31 membranelles on average; 17 frontal-ventral-transverse cirri, composed of 3 frontals, 4 frontoventrals, 1 buccal cirrus, 3 ventral cirri, 2 pretransverse and 4 transverse cirri; left and right marginal rows composed of 21 and 24 cirri on average, respectively; marginal rows not confluent posteriorly; 4 dorsal kineties and 2 dorsomarginal rows Type locality: Soil from a tropical rain forest tract at the Silent Valley National Park, Kerala, India (11°13′N, 76°47′E), about 1 km from the park entrance in the vicinity of the trekking trail.

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Type material: A protargol slide with the holotype specimen (Fig. 2E) encircled in black ink is deposited in the Natural History Museum, London, UK with registration number NHMUK 2011.7.4.1. The same slide has several paratype specimens. Etymology: The species-group name tetracirrata refers to the presence of four transverse cirri.

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Occurrence and ecology: As yet found only from the type locality, where it was moderately abundant in non-flooded Petri dish cultures. A clonal culture was established with the addition of Silent Valley Nation Park are as given in Kumar et al. (2010).

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the green alga Chlorogonium elongatum as the food organism. Other ciliate species recorded from

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Description: Body semirigid, 85–110 × 35–50 µm in vivo, 84 × 37 µm on average in protargol preparations, almost oblong in outline, both ends broadly rounded, dorso-ventrally flattened about 2:1 (Figs 1A–C, 2A–C, E, F). Nuclear apparatus slightly left of midline, about 21

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µm from cell anterior end, composed of four (rarely five) separate macronuclear nodules and 2–4 micronuclei. Macronuclear nodules ellipsoidal, first and fourth macronuclear nodules slightly

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larger; nucleoli 0.5–2.1 µm across. Micronuclei usually attached to macronuclear nodules, on average 2.2 µm across (Figs 2E, H). Contractile vacuole slightly above mid-body at left cell margin. Cortical granules absent, cytoplasm filled with colourless cytoplasmic granules about 1.5–

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2.0 µm across (Figs 1A, 2A–D). Locomotion by rapid crawling over and between soil particles.

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Adoral zone about 42% of cell length after protargol staining, with a DE-value of about 0.25 (n = 13), composed of 31 membranelles on average. Bases of membranelles about 8 µm wide, cilia

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up to 20 µm long. Paroral and endoral about equal length and optically intersect in mid-region. Seventeen frontal-ventral-transverse cirri comprising three slightly hypertrophied frontal cirri, about 14 µm long in protargol preparations, right cirrus posterior of distal end of adoral zone, middle cirrus anterior of buccal cirrus, left cirrus anterior of distal end of undulating membranes; buccal cirrus on average 7 µm behind distal end of paroral membrane. Four frontoventral cirri arranged in a hook-like row, anteriormost cirrus ahead of level of buccal cirrus, slightly right of right frontal cirrus. Three post-oral cirri behind buccal vertex, one of which (V/3) is distinctly displaced posteriorly, about 12 µm long in protargol preparations. Two pre-transverse cirri, distance between postoral cirrus V/3 and anterior pre-transverse cirrus (V/2) about 13 µm in protargol preparations. Invariably four transverse cirri arranged in an oblique-row (in two out of 40 specimens this row is hook-shaped), cirri about 17 µm long in protargol preparations. Distance from last transverse cirrus to posterior end of cell about 8 µm, cirri project beyond posterior cell margin. One left and one right marginal row, cirri about 13 µm long in protargol preparations; anterior ends of left and right marginal row about 26 µm and 14 µm respectively from anterior 6 Page 6 of 22

body end, left row with about 21 cirri, right row with about 24 cirri; marginal rows not confluent posteriorly (Figs 1B, 2E). Four dorsal kineties, dorsal bristles about 3–4 µm long in vivo; first and second dorsal kineties roughly bipolar with about 24 and 21 bristles respectively, third and fourth kineties of almost equal length with about 15 bristles each. Two dorso-marginal rows; first dorso-marginal

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row with about 10 bristles, second dorso-marginal row usually with seven bristles. Three caudal cirri, about 14 µm long in protargol preparations, one each at posterior ends of the dorsal kineties

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1, 2, and 4 (Figs 1C, 2F).

Resting cyst. Resting cysts about 40 μm across in vivo; cyst surface with hyaline ridges, lipid droplets and four globular macronuclear nodules.

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each approximately 2.5 μm high (Fig. 2G). Cyst wall about 1.0 μm thick. Cyst contents includes SSU rRNA gene sequence and phylogeny: The SSU rRNA gene sequence of Sterkiella

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tetracirrata n. sp. was deposited in GenBank with accession number KF668619. The new sequence is 1670 bp in length and has a GC content of 45%. Phylogenetic trees based on SSU

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rRNA gene sequences using BI and ML analyses had similar topologies, therefore only the BI tree is presented here (Fig. 6). Phylogenetic analyses consistently place the new species within the

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stylonychine oxytrichids, clustering in a clade with Gastrostyla steinii.

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Divisional morphogenesis (Figs 3A–J, 4A–G, 5A–C; Table 2): Divisional morphogenesis is in the typical Sterkiella pattern (Foissner and Berger 1999). The parental adoral zone of

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membranelles is retained unchanged for the proter while that of the opisthe is formed from the oral primordium that originates close to transverse cirrus III/1 (Figs 3A, B, 4A, B). Five parental cirri (II/2, III/2, IV/2, IV/3, and V/4) and the paroral and endoral are involved in the formation of six primordial streaks (Figs 3C, D, 4C, D). The paroral and endoral are formed from streak I. Postoral ventral cirrus V/3 is not involved in the primordia formation (Figs 3D–F, 4D–F). The 17 frontalventral-transverse cirri arise from these primordia, splitting in a 1, 2, 3, 3, 4, 4 pattern. No transverse cirrus is formed from streak II (Figs 3G, 4G). The marginal primordia arise at each of two levels by ‘‘within-row’’ primordia formation

utilizing one or two of the parental cirri at each level. The marginal primordia elongate utilizing four or five parental cirri and differentiate into new marginal rows. The rest of the parental marginal cirri are resorbed (Figs 3G–I, 4G, 5A, B). On the dorsal surface, three primordia are formed within-row from dorsal kineties 1, 2 and 3 at two levels (one set for the proter and one for the opisthe). The third dorsal primordium fragments at the middle giving rise to the third and fourth kinety which are of almost equal length. 7 Page 7 of 22

The two dorso-marginal rows arise near the right marginal row. Each caudal cirrus originates at the posterior end of the new dorsal kineties 1, 2, and 4. The caudal cirri are located in the gap between the two marginal rows making them inconspicuous (Figs 3J, 5C). Nuclear division proceeds in the usual manner for oxytrichids. In middle dividers the macronuclear nodules fused to form a single mass which divides twice to produce the typical four

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nodules in late dividers (Fig. 5A). The micronuclei undergo mitotic division in the usual manner.

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Discussion Comparison of S. tetracirrata n. sp. with its congeners

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There are four species of Sterkiella that possess four macronuclei and should therefore be compared with S. tetracirrata, namely S. admirabilis (Foissner, 1980) Berger, 1999, S. quadrinucleatus (Sick, 1933) Berger, 1999, S. cavicola (Kahl, 1935) Foissner et al., 1991 and S.

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terricola Berger, 1999. Sterkiella admirabilis can easily be separated from S. tetracirrata by having a significantly longer body in vivo (350–450 µm vs. 85–110 µm) and more (50–60 vs. 24–

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35) adoral membranelles (Alekperov and Musayev 1988; Foissner 1980). Sterkiella quadrinucleatus differs from S. tetracirrata by having about 40 (vs. ca. 31) adoral membranelles, supernumerary (vs. invariably three) postoral ventral cirri, usually five, occasionally 6–8, (vs.

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invariably four) transverse cirri, and a marine or brackish (vs. soil) habitat (Berger 1999). All

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reported populations of S. cavicola have a larger body size than S. tetracirrata (85–110 µm long in vivo), i.e., 180–250 µm (Kahl 1935), 220–250 µm (Grolière 1969), 140–220 µm (Berger and

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Foissner 1987), and 150–220 µm (Shin and Kim 1994). Furthermore, S. cavicola posseses more transverse cirri (five or six vs. invariably four in S. tetracirrata). The resting cyst of S. tetracirrata differs from that of S. cavicola in size (about 40 µm vs. about 55 µm in diameter) and having separate macronuclear nodules (vs. fused to form single mass). Sterkiella terricola can be separated from S. tetracirrata in having only three (vs. invariably four) transverse cirri (Berger 1999) and 22 or 23 (vs. 24–35) adoral membranelles; minor differences include the macronuclear nodules in a chain (vs. separated), the presence of a small cirrus between the middle and right frontal cirri (vs. no such cirrus present) (however, this feature is likely a misobservation, the small cirrus could be the resorbing parental cirri), and the numbers of right marginal cirri (about 20 vs. about 24), left marginal cirri (about 17 vs. about 21), and micronuclei (four vs. three). Morphogenesis of Sterkiella tetracirrata is very similar to that described for S. histriomuscorum and S. nova (Foissner and Berger 1999). Foissner et al. (2002) mentioned that in Sterkiella cavicola cirrus IV/2, from which opisthe anlage IV originates, also contributes to the oral primordium formation. Although the opisthe anlage IV of S. tetracirrata also originates from 8 Page 8 of 22

cirrus IV/2, this cirrus does not contribute to the oral primordium. Foissner and Berger (1999) mentioned that the fronto-ventral-transverse cirral anlagen of the proter and opisthe are confluent during the early morphogenetic stages in the population of S. histriomuscorum described by Berger et al. (1985), whereas all these anlagen are non-confluent in Sterkiella tetracirrata, S. histriomuscorum and S. nova. Confluent anlagen have also been seen in another poorly known

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species Sterkiella tricirrata (Kumar S., Kamra K. and La Terza A., pers. comm.). We agree with Foissner and Berger (1999) that the population of S. histriomuscorum described by Berger et al.

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(1985) and Sterkiella tricirrata are not conspecific with those having four or five transverse cirri and non-confluent anlagen. Therefore, these taxa should be separated either at the genus or species

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level.

Phylogenetic analyses

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Phylogenetic analyses consistently placed Sterkiella tetracirrata n. sp. close to Gastrostyla steinii, with strong support (0.99 BI; Fig. 6). Although these two genera differ in their cirral

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pattern, it has been reported that the higher number of frontal-ventral-transverse cirri in Gastrostyla (which is due to high numbers of cirri produced in a few anlagen) and the individual cirral groups of 18-FVT cirri hypotrichs (e.g., Stylonychia, Sterkiella, Tetmemena, Histriculus etc.)

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are homologous (Berger 2008). Furthermore, Sterkiella tetracirrata and Gastrostyla steinii exhibit

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similarities in the number (invariably four) and arrangement of transverse cirri as well as the structure of the resting cyst (wrinkled surface). The arrangement and number of transverse cirri is

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probably a more important distinguishing feature than the number of macronuclear nodules in these two genera.

In their phylogenetic analyses of oxytrichid and urostylid stichotrichs, Foissner et al. (2004) showed that Gastrostyla clusters with Pattersoniella, and that the next closest relative was Sterkiella histriomuscorum. With the addition of more gene sequences, e.g. Paraparentocirrus sibillinensis and the present species, Gastrostyla clusters more closely with Sterkiella than with Pattersoniella, the latter clustering with Paraparentocirrus sibillinensis, a sensu lato oxytrichid. These analyses could hint at a possible evolution of Gastrostyla and Pattersoniella from a Sterkiella-like ancestor though molecular data based on single gene markers often do not show true relationships. In the present phylogenetic analyses, Sterkiella tetracirrata clustered in a different clade from the only two congeners (S. cavicola and S. histriomuscorum) for which gene sequence data are available. Clustering of these two species together in a clade (1.0 BI) separate from Sterkiella tetracirrata could be due to under sampling of the genus Sterkiella (Kumar S. and La Terza A., for 9 Page 9 of 22

example, have identified another novel species of Sterkiella, yet to be described, from Italian natural and agricultural soils), and the absence of molecular data of other known species.

Acknowledgements The work is a part of the research conducted under the Project SR/SO/AS-04/2004 awarded

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to the corresponding author (Komal Kamra) by the Department of Science and Technology, Government of India. The encouragement of Prof Rup Lal, Head, Department of Zoology,

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University of Delhi, and a co-investigator in the above project, is greatly appreciated. The authors are grateful to the host institution, Sri Guru Tegh Bahadur Khalsa College for providing the

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necessary infrastructural facilities to carry out the research work.

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Kamra, K., Sapra, G.R., 1990. Partial retention of parental ciliature during morphogenesis of the ciliate Coniculostomum monilata (Dragesco and Njiné, 1971) Njiné, 1978 (Oxytrichidae, Hypotrichida). Eur. J. Protistol. 25, 264–278.

Katoh, K., Standley, D.M., 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780. Kumar, S., Kamra, K., Sapra, G.R., 2010. Ciliates of the Silent Valley National Park, India: urostyloid hypotrichs of the region with a note on the habitat. Acta Protozool. 49, 339–364. Küppers,G.C., Paiva, T.S., Borges, B.N., Harada, M.L., Garraza, G.G., Mataloni, G., 2011. An Antarctic hypotrichous ciliate, Parasterkiella thompsoni (Foissner) nov. gen., nov. comb., 11 Page 11 of 22

recorded in Argentinean peat-bogs: morphology, morphogenesis, and molecular phylogeny. Eur. J. Protistol. 47, 103–123. Lynn, D.H., 2008. The Ciliated Protozoa: Characterization, Classification and Guide to the Literature, 3rd ed. Springer, Dordrecht. Martin, J., 1982. Évolution des patrons morphogénétiques et phylogenése dans le sous-ordre des

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Sporadotrichina (Ciliophora, Hypotrichida). Protistologica 18, 431–447.

Medlin, L., Elwood, H.J., Stickel, S., Sogin, M.L., 1988. The characterization of enzymatically

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amplified eukaryotic 16S-like rRNAcoding regions. Gene 71, 491–499.

Posada, D., 2008. jModelTest: Phylogenetic Model Averaging. Mol. Biol. Evol. 25, 1253–1256. mixed models. Bioinformatics 19, 1572–1574.

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Ronquist, F., Huelsenbeck, J.P., 2003. MRBAYES 3: Bayesian phylogenetic inference under Shin, M.K., Kim, W., 1994. Morphology and biometry of two oxytrichid species of genus

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Histriculus Corliss, 1960 (Ciliophora, Hypotrichida, Oxytrichidae) from Seoul, Korea. Korean J. Zool. 37, 113–119.

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Sick, F., 1933. Die Fauna der Meeresstrandtümpel des Bottsandes (Kieler Bucht). Ein Beitrag zur Ökologie und Faunistik von Brackwassergebieten. Z. Wiss. Zool. 2(B), 54–96. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: Molecular

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evolutionary genetics analysis using Maximum Likelihood, Evolutionary Distance, and

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Maximum Parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Wallengren, H., 1900. Zur Kenntnis der vergleichenden Morphologie der hypotrichen Infusorien.

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Bih. Svensk Vetensk. Akad. Handl. 26, 1–31.

Figure legends

Fig. 1A–C. Line diagrams of Sterkiella tetracirrata n. sp. from life (A) and after protargol staining (B, C). A. A representative cell. B, C. Ventral view of holotype and dorsal view of paratype specimen. AZM, adoral zone of membranelles; CC, caudal cirri; CV, contractile vacuole; DK, dorsal kineties; DM, dorsomarginal rows; E, endoral; FG, fat globules; FV, food vacuole; LM, left marginal row; MA, macronuclear nodule; MI, micronucleus; P, paroral; RM, right marginal row; I/1, II/3, III/3, frontal cirri; II/2, buccal cirrus; III/2, IV/3, VI/3, VI/4, fronto-ventral cirri; IV/2, V/4, V/3, post-oral ventral cirri; V/2, VI/2, pre-transverse ventral cirri; III/1, IV/1, V/1, VI/1, transverse cirri. Scale bars: 25 µm.

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Fig. 2A–H. Photomicrographs of Sterkiella tetracirrata n. sp. from life (A–D), after protargol staining (E, F, H), and in SEM preparation (G). A–D. Body shape of slightly squeezed specimens (A–C) and surface view (D) showing the cytoplasmic granules; arrows point to the contractile vacuole (B), and the four transverse cirri (C). E. Ventral surface of holotype specimen showing, inter alia, cirrus V/3 (double arrowhead), four transverse cirri (arrow) and two pre-transverse cirri

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(arrowheads). F. Dorsal view of paratype specimen, arrow points to dorsal kinety 1. G. Surface view of a resting cyst. H. Ventral view showing nuclear apparatus. AZM, adoral zone of

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membranelles; CC, caudal cirri; FC3, right frontal cirrus; LM, left marginal row; MA,

macronuclear nodule; MI, micronucleus; RM, right marginal row. Scale bars: 30 µm (A–C, E, F);

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10 µm (D, G, H).

Fig. 3A–J. Line diagrams of protargol-stained early (A–G), middle (H) and late dividers (I, J) of

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Sterkiella tetracirrata n. sp. A. Stomatogenesis begins close to cirrus III/1 (arrow). B. Proliferation of this field gives rise to oral primordium (arrow). C–E. Five parental cirri (II/2, III/2, IV/2, IV/3,

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and V/4) disaggregate to give rise to fronto-ventral-transverse streaks for both daughter cells. F–I. Ventral views; no transverse cirrus is formed from streak II. Arrow in (F) points to cirrus V/3, which is not involved in primordia formation. The dorsomarginal rows originate close to the

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anterior ends of the right marginal row primordia (arrow in H). Discontinuous lines in (I) connect

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cirri originating from the same fonto-ventral-transverse anlagen. J. Late divider in dorsal view showing kinety 3 fragmentation (arrows) and dorsomarginal rows (double arrowheads). II/2,

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buccal cirrus; III/1, transverse cirrus; III/2, IV/3, fronto-ventral cirri. Scale bars: 30 µm. Fig. 4A–G. Photomicrographs of protargol-stained dividers of Sterkiella tetracirrata n. sp. depicting morphogenetic events on the ventral surface. Arrows in (C–F) point to intact ventral cirrus V/3. A, B. Arrows point to proliferating oral primordium originating close to transverse cirri. C. Double arrowhead points to first fronto-ventral-transverse streak for opisthe, arrowhead to disaggregating V/4, and short arrow to streaks IV, V and VI for the proter. D. Arrowhead points to disaggregating buccal cirrus. E, F. Arrowheads mark the within-row primordia formation for the marginal rows (E), six cirral anlagen align side by side for both proter and opisthe (F). G. Seventeen fronto-ventral-transverse cirri splitting in 1, 2, 3, 3, 4, 4 pattern. AZM, adoral zone of membranelles; LM, left marginal row; OP, oral primordium; RM, right marginal row; TC, transverse cirri; I, V, VI, fronto-ventral-transverse cirral anlagen. Scale bars: 30 µm.

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Fig. 5A–C. Photomicrographs of protargol-stained late dividers of Sterkiella tetracirrata n. sp. A. Arrowhead show the newly formed dorsomarginal rows of the opisthe. B. Arrows mark the newly formed transverse cirri. C. Short arrow points to dorsal kinety 1, long arrows to newly formed caudal cirri at end of dorsal kineties 1, 2 and 4, double arrowheads to dorsal kinety 4 formed by fragmentation of dorsal kinety 3, and arrowheads to newly formed dorsomarginal rows. AZM,

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adoral zone of membranelles; LM, left marginal row; MA, macronuclear nodule; RM, right

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marginal row; TC, transverse cirri Scale bars: 30 µm.

Fig. 6. Bayesian tree inferred from SSU rRNA gene sequence data showing the position of

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Sterkiella tetracirrata n. sp. (bold). Support values at the nodes represent the posterior probability of Bayesian analysis and the bootstrap values of maximum likelihood out of 1000 replicates. Codes after the species names are GenBank accession numbers. A hyphen (-) represents minor

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disagreements in tree topologies between the BI and ML trees. The scale bar corresponds to two

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substitutions per 100 nucleotide positions.

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Table 1. Morphometric data on Sterkiella tetracirrata n. sp. Characteristic a

x

SD

CV Min Max

n

84.2

6.2

7.4 73.7 101.7 20

Body, width

37.2

3.4

9.2 29.7

43.2 20

Anterior end to proximal end of adoral zone, distance

35.0

2.9

8.3 28.0

39.9 20

Adoral zone, percentage of body length

41.5

2.1

7.7

0.5

Adoral membranelles, number

30.5

2.5

Anterior body end to first macronuclear nodule, distance

20.8

First macronuclear nodule, length

12.7

First macronuclear nodule, width Second macronuclear nodule, width Third macronuclear nodule, length

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Third macronuclear nodule, width Fourth macronuclear nodule, length

35.0 20

2.4 11.3 16.7

25.2 10

1.0

7.7 11.3

14.7

9

5.9

0.5

9.0

5.0

6.9

9

9.1

1.5 16.5

6.6

12.4

9

5.4

0.6 10.3

4.6

6.3

9

8.1

1.0 12.1

6.2

10.1 11

5.5

0.5

4.5

6.7 11

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8.2 24.0

9.4

2.4 16.9 11.8

19.8

9 9

5.6

0.8 14.5

4.4

7.4

4.1

0.3

6.1

4.0

5.0 41

2.2

0.1

2.7

2.1

2.3 20

3.0

0.7 23.4

2.0

4.0 20

Anterior body end to right marginal row, distance

13.7

2.1 15.1

9.9

18.0 10

Right marginal row, number of cirri

23.8

1.0

4.1 21.0

25.0 20

Anterior body end to left marginal row, distance

26.4

1.5

5.8 24.8

30.0 10

Left marginal row, number of cirri

21.4

1.0

4.4 20.0

23.0 20

Frontal cirri, number

3.0

0.0

0.0

3.0

3.0 20

Paroral to buccal cirrus, distance

6.9

1.1 15.6

5.3

9.0 13

Buccal cirri, number

1.0

0.0

0.0

1.0

1.0 20

Fronto-ventral cirri, number

4.0

0.0

0.0

4.0

4.0 20

Postoral ventral cirri, number

3.0

0.0

0.0

3.0

3.0 20

Pre-transverse ventral cirri, number

2.0

0.0

0.0

2.0

2.0 20

Posterior body end to posteriormost transverse cirrus, distance

7.6

2.0 26.6

4.9

Transverse cirri, number

4.0

0.0

0.0

4.0

4.0 40

Dorsal kineties, number

4.0

0.0

0.0

4.0

4.0 20

23.5

1.6

7.0 19.0

26.0 20

Micronucleus, diameter

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Micronuclei, number

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Macronuclear nodules, number

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Fourth macronuclear nodule, width

14.0

6.0

45.0 20 8.6 12

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Second macronuclear nodule, length

5.0 35.5 6.9

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Adoral membranelles, length of largest membranellar basis

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Body, length

Dorsal kinety 1, number of bristles

10.2

8

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Characteristic a

x

SD

CV Min Max

n

Dorsal kinety 2, number of bristles

21.4

1.2

5.8 17.0

23.0 20

Dorsal kinety 3, number of bristles

15.0

1.0

6.1 13.0

16.0 20

Dorsal kinety 4, number of bristles

15.4

1.1

7.1 13.0

18.0 20

2.0

0.0

0.0

10.3

Dorsomarginal row 2, number of bristles Caudal cirri, number

2.0 20

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Dorsomarginal row 1, number of bristles

2.0

1.1 10.5

8.0

13.0 20

6.6

0.6

9.2

6.0

8.0 20

3.2

0.4 11.8

3.0

4.0 20

a

cr

Dorsomarginal rows, number

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Data based on protargol-impregnated non-dividers obtained from a clonal culture fed with green alga Chlorogonium elongatum. Measurements in µm. CV, coefficient of variation in %; Max, maximum; Min, minimum; n, number of individuals investigated; SD, standard deviation; x , arithmetic mean.

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Table 2. Parental ciliary structures associated with the origin of frontal-ventral-transverse anlagen in Sterkiella tetracirrata n. sp. Cirri are numbered according to Wallengren (1900). Daughter cell Anlagen number Parental structure associated with origin of anlagen II

II/2

III

III/2

IV/3

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IV

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Parental paroral and endoral

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Proter

I

Opisthe

V

IV/3

VI

IV/3

I

From base of the five streaks arising from oral primordium

II

Oral primordium

III

Oral primordium

IV

Disaggregation of cirrus IV/2

V

Disaggregation of cirrus V/4

VI

Disaggregation of cirrus V/4

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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