International Journal of Systematic and Evolutionary Microbiology (2014), 64, 2625–2636

DOI 10.1099/ijs.0.062885-0

Morphology, morphogenesis and molecular phylogeny of a novel soil ciliate, Pseudouroleptus plestiensis n. sp. (Ciliophora, Oxytrichidae), from the uplands of Colfiorito, Italy Daizy Bharti, Santosh Kumar and Antonietta La Terza Correspondence Santosh Kumar

School of Environmental Science, University of Camerino, Via Gentile III da Varano, 62032 Camerino (MC), Italy

[email protected] Antonietta La Terza [email protected]

The terrestrial oxytrichid ciliate Pseudouroleptus plestiensis n. sp., isolated from soil samples collected from the uplands of Colfiorito (Umbria region, Italy), was investigated using live observation and protargol impregnation. The morphology, morphogenesis and molecular phylogeny inferred from small-subunit (SSU) rRNA gene sequences were studied. The novel species is mainly characterized by the following: a cell size of about 145¾35 mm in vivo; two ellipsoidal macronuclear nodules and two to four micronuclei; adoral zone about 26 % of body length with a mean of 30 membranelles; about 40 cirri in the right marginal row and 38 in the left marginal row; left fronto-ventral row consisting of about 27–40 cirri, right fronto-ventral row of about three to seven cirri forming a short row to the right of the rear portion of the left frontoventral row; one parabuccal cirrus (5III/2), one buccal and one post-peristomial cirrus; and four dorsal kineties with caudal cirri at the end of kineties 1 and 2. The morphogenesis of the novel species is similar to that of Pseudouroleptus caudatus. Phylogenetic analyses based on SSU rRNA gene sequences consistently placed the novel species within the family Oxytrichidae Ehrenberg, 1838, clustering with P. caudatus and the genus Strongylidium. The results from the present study contribute to the expanding knowledge of the diversity of ciliates in Italian soil.

INTRODUCTION The genus Pseudouroleptus Hemberger, 1985 was first assigned to the family Amphisiellidae Jankowski, 1979 by Hemberger (1982) in his unpublished dissertation to include tailed species with fronto-ventral rows and a postperistomial ventral cirrus. Later, it was classified in the family Kahliellidae Tuffrau, 1979 by Tuffrau (1987), Foissner & Foissner (1988) and Tuffrau & Fleury (1994). Eigner (1997), based on morphogenetic data, i.e. the development of the ‘neokinetal 3’ anlagen (a large V-shaped primordium that produces anlagen V and VI of both proter and opisthe), assigned it to the family Oxytrichidae Ehrenberg, 1838. Berger (1999) also classified Pseudouroleptus in the Oxytrichidae based on the dorsal kinety fragmentation as the sole synapomorphy of the family. Presently, Pseudouroleptus is monotypic, and the type species is composed of two subspecies, Pseudouroleptus caudatus caudatus and Pseudouroleptus caudatus namibiensis (Foissner et al., 2002). Abbreviations: BI, Bayesian inference; ML, maximum-likelihood; SSU, small-subunit. The GenBank/EMBL/DDBJ accession number for the SSU rRNA gene sequence of Pseudouroleptus plestiensis n. sp. isolate WPRCP-1 is KJ173910.

062885 G 2014 IUMS

Printed in Great Britain

These subspecies are morphologically similar to some species of the genus Hemiamphisiella; however, the fragmentation of dorsal kinety 3 separates the two genera. Nevertheless, the synonymy of Hemiamphisiella and Pseudouroleptus cannot be excluded, because data on dorsal morphogenesis are unavailable for some species (Berger, 2008). The present paper describes a novel species of the genus Pseudouroleptus isolated from soil samples collected from the regional park of Colfiorito in the province of Perugia, Umbria region (Italy), in a place known as Molinaccio (43u 019 40.720 N 12u 529 39.460 E). This study is the second report on the diversity of ciliates from Italian soils; a novel genus from the Marche region was recently described in detail by Kumar et al. (2014). A detailed description of the morphology, morphogenesis and phylogenetic analyses based on small-subunit (SSU) rRNA gene sequences is presented in this study.

METHODS Sampling and sample processing. Soil samples were collected in

July 2009; for details of the sampling site, see the type locality section. Ten soil samples (0–10 cm deep), including fine plant roots and litter, were collected randomly from an area of approximately 100 m2, 2625

D. Bharti, S. Kumar and A. La Terza mixed to obtain a composite sample (weighing approximately 1 kg) and transferred to the laboratory. Ciliates were made to excyst and emerge from 1-month-dried soil samples (approx. 300 g) by employing the non-flooded Petri dish method (Foissner, 1987a). Twenty to 30 cells were isolated with the help of a glass micropipette

from run-off to raise a culture of Pseudouroleptus plestiensis n. sp. in the laboratory. Cells were found to thrive when maintained at a temperature of 18±2 uC in Pringsheim’s medium with the green alga Chlorogonium elongatum as the food organism (Ammermann et al., 1974).

FC

AZM

BC

PM

RMR DK4

EM DK1 PC

LMR MI

RMR

MA LFVR

RFVR

(d)

II III

(b)

(c) II IV

(a)

III

II

VI

IV

OP

V

VI

(e)

(f)

(g)

Fig. 1. Line diagrams of P. plestiensis from life (a) and after protargol impregnation (b–g). (a) Ventral view of a representative specimen; length 145 mm. (b, c) Ventral (b) and dorsal (c) views of the holotype specimen. Arrowhead in (b) points to cirrus III/ 2. (d–g) Segments showing morphogenetic events on the ventral surface. (d) Oral primordium (OP) formation begins close to 8–13 cirri of the left fronto-ventral row in the mid-body (arrowheads). (e) Anterior end of the OP begins to differentiate and forms two streaks in a Y-shaped pattern; the buccal cirrus begins to disaggregate (arrowhead). Arrow points to the VI anlage. (f) The leftmost streak from the OP gives rise to anlage II of the opisthe; the anterior portion of this streak joins the disaggregating cirrus II/2 and forms anlage II of the proter. The arrowhead points to the disaggregating cirrus III/2, which forms anlage III of the proter. (g) Anlagen II–IV of the proter and opisthe lengthen; the arrowhead points to the base of the Y-shaped streaks, which forms anlage I of the opisthe. Anlagen IV–VI of the proter and opisthe develop from the left fronto-ventral row. AZM, Adoral zone of membranelles; BC, buccal cirrus; CC, caudal cirrus; DK, dorsal kineties; EM, endoral membrane; FC, frontal cirrus; LFVR, left fronto-ventral row; LMR, left marginal row; MA, macronucleus; MI, micronucleus; PC, post-peristomial cirrus; PM, paroral membrane; RFVR, right fronto-ventral row; RMR, right marginal row. Bars, 50 mm (a), 40 mm (b, c, e, g) and 30 mm (d, f). 2626

International Journal of Systematic and Evolutionary Microbiology 64

Pseudouroleptus plestiensis n. sp. Live observations were made using a simple microscope with brightfield illumination. Protargol staining (Kamra & Sapra, 1990) was used to reveal the infraciliature. Biometric characterization was carried out at a magnification of 61000 using the Optika Vision Lite software. An Optika microscope camera was employed for photomicrography and line diagrams were prepared using freehand sketches. To demonstrate changes during morphogenesis, old (parental) structures were depicted by contour, while newly formed structures were shaded in black. The numbering of the frontal-ventral cirri is according to Berger (2008). DNA extraction, PCR amplification and sequencing. Genomic

DNA extraction, gene amplification and gene sequencing of P. plestiensis were conducted according to the methods described by Kumar et al. (2014). Thirty to 40 cells from an overnight-starved culture were collected with the help of glass micropipettes and washed three times with autoclaved distilled water. For DNA extraction, 50 ml Chelex 100 (5 %, w/v) and 2 ml proteinase K (20 mg ml21) solution were added to the sample and the mixture was then incubated at 37 uC for 30 min, followed by incubation at 98 uC for 5 min. The reaction mixture was cooled immediately on ice and centrifuged in a microfuge tube for 2–3 s at 16 000 g. Without disturbing the Chelex 100 beads, 5 ml supernatant was carefully drawn from the top of the sample and stored at 4 uC until PCR amplification or used immediately for PCR. Extracted DNA (5 ml) was dispensed into a PCR tube containing 5 ml distilled water and amplifications were carried out using high-fidelity

Pfx50 DNA polymerase (Invitrogen) in a total volume of 50 ml with the universal eukaryotic primers Euk A (forward, 59-AACCTGGTTGATCCTGCCAGT-39) and Euk B (reverse, 59-TGATCCTTCTGCAGGTTCACCTAC-39) (Medlin et al., 1988). Additionally, primers Eup 18S (forward, 59-TAGAGGGACTTTGTGTGCAACC-39) and Eup 18S (reverse, 59-ATCTCCCTGAAACACACGTTGG-39) were used in combination with the universal primers for amplification and sequencing (Kumar et al., 2014). The PCR program for SSU rRNA gene amplification included an initial denaturation at 94 uC for 3 min, followed by 35 cycles of 94 uC for 1 min, 55 uC for 45 s and 72 uC for 80 s, with a final extension step at 72 uC for 10 min. After confirmation of the presence of a product of the appropriate size, the PCR products were purified using a Nucleospin gel extraction kit (Qiagen) and were then sequenced directly on both strands at StarSEQ (Mainz, Germany). Phylogenetic analyses. For phylogenetic analyses, the newly

sequenced SSU rRNA gene sequence of P. plestiensis n. sp. was aligned with 40 hypotrichid SSU rRNA gene sequences that were retrieved from the GenBank database using the MATTF 7.047 software (choosing the iterative refinement methods L-INS-i) (Katoh & Standley, 2013). The final alignment was then used for subsequent phylogenetic analyses after converting the FASTA (.fas) file to NEXUS (.nex) format using the open web-based tool ALTER (Glez-Pen˜a et al., 2010). A

Table 1. Morphometric data on P. plestiensis Data are based on mounted, protargol-impregnated and randomly selected specimens from an established culture fed with the alga Chlorogonium elongatum. AZM, Adoral zone of membranelles. Means are arithmetic means. Characteristic

Mean

Median

SD

SEM

Body, length (mm) Body, width (mm) Adoral membranelles (n) AZM, length (mm) AZM/body length (%) Macronuclear nodules (n) Macronuclear nodules, length (mm) Macronuclear nodules, width (mm) Micronuclei (n) Micronucleus, length (mm) Micronucleus, width (mm) Frontal cirri (n) Fronto-ventral cirri (III/2) (n) Buccal cirri (n) Post-peristomial cirri (n)* Left fronto-ventral row, number of cirri Right fronto-ventral row, number of cirri Right marginal row, number of cirri Left marginal row, number of cirri Dorsal kineties (DK) (n) DK 1, number of dikinetids DK 2, number of dikinetids DK 3, number of dikinetids DK 4, number of dikinetids Anterior body end to beginning of DK 3, distance (mm) Anterior body end to beginning of DK 4, distance (mm)

141.6 31.2 30.1 36.8 26.1 2.0 15.2 6.9 2.6 3.7 2.6 3.0 1.0 1.0 1.0 36.1 4.9 40.2 37.9 4.0 12.7 23.0 12.2 13.2 7.4 32.2

146.6 32.1 30.0 37.7 25.6 2.0 15.2 6.5 3.0 3.7 2.6 3.0 1.0 1.0 1.0 37.0 5.0 41.0 38.0 4.0 12.0 24.0 12.0 13.0 7.0 30.5

15.4 4.0 1.5 3.0 1.8 0.0 1.5 0.7 0.6 0.4 0.3 0.0 0.0 0.0 0.0 3.3 1.1 4.1 5.2 0.0 1.7 2.9 1.5 2.4 1.7 5.7

3.5 1.0 0.3 0.7 0.4 0.0 0.3 0.2 0.1 0.1 0.1 0.0 0.0 0.0 0.0 0.8 0.3 1.0 1.2 0.0 0.4 0.7 0.4 0.5 0.5 1.5

CV

(%)

10.9 12.8 4.9 8.0 7.1 0.0 9.6 10.4 22.7 10.1 10.7 0.0 0.0 0.0 0.0 9.1 22.5 10.3 13.6 0.0 13.3 12.6 12.6 17.8 23.6 17.7

Min.

Max.

n

97.1 23.8 26 28.2 22.3 2 12.5 5.9 2 3.2 2.0 3 1 1 1 27 3 31 26 4 10 17 9 8 4.0 24.0

159.9 37.4 32 41.5 29.4 2 18.3 8.7 4 4.6 2.9 3 1 1 1 40 7 47 45 4 16 27 15 17 10.0 45.0

19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 15 15

*Two specimens with two and four post-peristomial cirri were observed. http://ijs.sgmjournals.org

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D. Bharti, S. Kumar and A. La Terza Bayesian inference (BI) analysis was performed using MrBayes version 3.2.1 (Ronquist & Huelsenbeck, 2003) and the GTR+I+G model, as selected by the jModel Test version 2.1.3 software (Posada, 2008) under the corrected Akaike information criterion (AICc). Markov chain Monte Carlo simulations were run with two 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 were discarded. The remaining trees were used to generate a consensus tree and to calculate posterior probabilities. A maximum-likelihood (ML) tree was reconstructed using MEGA version 5.2.2 (Tamura et al., 2011) with the GTR+I+G model, and the topology of the trees was inferred by running 1000 bootstrap

CV

(a)

(b)

(c)

(d)

AZM

DK1

MA

DK4 MA LFVR

(e) RMR

LMR

RFVR

(f)

(g)

(h)

Fig. 2. Photomicrographs of P. plestiensis from life (a–f) and after protargol impregnation (g, h). (a–c) Body shape of slightly squeezed (a, b) and freely motile (c) specimens. (d) Optical section showing cytoplasmic granules. (e, f) Optical sections of 5day-old (e) and 2-week-old (f) resting cysts; opposed arrowheads mark the thick cell wall and arrows mark the cyst contents separated from the cyst wall. (g, h) Ventral and dorsal views of a specimen showing the ciliature. The arrow in (g) points to cirrus III/2 and the arrowhead points to the post-peristomial cirrus. AZM, Adoral zone of membranelles; CV, contractile vacuole; DK, dorsal kineties; LFVR, left fronto-ventral row; LMR, left marginal row; MA, macronucleus; RFVR, right fronto-ventral row; RMR, right marginal row. Bars, 50 mm (a–c), 15 mm (d), 20 mm (e), 10 mm (f) and 40 mm (g, h). 2628

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Pseudouroleptus plestiensis n. sp. replicates and was expressed as a percentage. Phylogenetic trees were visualized using the free software package FigTree version 1.4 (written by A. Rambaut; http://tree.bio.ed.ac.uk/software/figtree/).

RESULTS Pseudouroleptus plestiensis n. sp. Diagnosis. Body size about 145635 mm in vivo. Body outline slenderly lanceolate with the posterior portion narrowed in a tail-like manner. Two ellipsoidal macronuclear nodules and two to four micronuclei. Adoral zone about 26 % of body length with a mean of 30 membranelles. Left fronto-ventral row extends to near body end and consists of about 27–40 cirri; right frontoventral cirral row consists of about three to seven widely spaced cirri forming a shortened row right of the rear portion of the left fronto-ventral row. About 40 cirri in the right and 38 in the left marginal row. Four dorsal kineties; caudal cirri at the end of kineties 1 and 2. Type locality. The plains of the Colfiorito and Plestini

uplands, which are situated at an elevation of approximately 800 m above sea-level within the protected areas of the

regional park of Colfiorito (Umbria region, Central Italy), consist of seven karst basins (known as plains) that were occupied by lakes in the past. Soil samples were collected during the dry season (July 2009) from a place known as Molinaccio at an elevation of 760 m. The sampling area is located at the external margins of the Colfiorito wetland, which is generally flooded during the autumn/winter. Geographical coordinates of the sampling site (Colfiorito; 43u 019 40.720 N 12u 529 39.460 E) were taken using an eTrex 30 hand-held GPS unit (Garmin) and are expressed according to the World Geodetic System 1984 (WGS84) following the degrees/minutes/seconds format. Type material. A protargol slide with the holotype

specimen (Fig. 1b, c) circled in black ink is deposited in the Natural History Museum, London, UK, with the registration number NHMUK 2014.3.20.1. One paratype slide is also deposited with the registration number NHMUK 2014.3.20.2. Etymology. The species-group name plestiensis refers to the name of the ancient city of Plestia (fourth century BCE), located in the Plestini uplands within the protected area of the regional park of Colfiorito (Umbria region, central Italy). Masculine gender.

0.88/* 0.82/0.84/-

BI/ML 0.02

1/-

0.95/51 0.95/86 1/99

Paraparentocirrus sibillinensis KF184655 Pattersoniella vitiphila AJ310495 Castrostyla steinii AF508758 1/100 Onychodromus grandis AJ310486 Stylonychia notophora FM209297 0.52/* Stylonychine Steinia sphagnicola AJ310494 0.88/- 1/87 Sterkiella cavicola GU942565 Oxytrichidae Sterkiella histriomuscorum AF164121 Histriculus histrio FM209294 1/96 0.87/46 Pleurotricha lanceolata AF164128 Stylonychia ammermanni FM209295 Stylonychia mytilus AM086661 0.88/43 0.87/73 1/98 Laurentiella strenua AJ310487 0.88/56 Onychodromopsis flexilis AM412764 0.83/62 Paraurostyla weissei AF164127 Cyrtohymena citrina AY498653 1/99 Non1/56 Rubrioxytricha ferruginea AF370027 stylonychine Oxytricha longa AF164125 0.84/Oxytrichidae Oxytricha granulifera AM412772 1/46 1/98 Pseudouroleptus plestiensis KJ173910 Pseudouroleptus caudatus DQ910904 Strongylidium orientale KC153532 0.58/67 1/99 Strongylidium pseudocrassum DQ910903 Spirofilidae 0.71/Oligotrichia Halteria grandinella AF194410 1/- 0.58/* 0.85/61 Kahliellidae Kahliella matisi EU079472 0.73/Gonostomum namibiense AY498655 Gonostomatidae Gonostomum strenuum AJ310493 0.88/Trachelostyla pediculiformis DQ057346 Trachelostylidae 0.99/84 Paruroleptus lepisma AF164132 Uroleptus willii EU399543 Uroleptidae 1/99 Uroleptus piscis AF164131 Pseudokeronopsis carnae AY881633 1/100 Pseudokeronopsidae Pseudokeronopsis flava DQ227798 1/100 Apokeronopsis bergeri DQ777742 1/98 Apokeronopsis crassa DQ359728 Thigmokeronopsis stoecki EU220226 Apourostylopsis sinica EU220227 Metaurostylopsis cheni HM623916 Urostylidae 1/100 Metaurostylopsis salina EU220229 1/99 Metaurostylopsis struederkypkea GU942568 Urostyla grandis EF535731

Fig. 3. Bayesian tree inferred from SSU rRNA gene sequences showing the position of P. plestiensis (bold). GenBank accession numbers are given after species names. Numbers at nodes represent posterior probabilities from BI/bootstrap values from ML analysis (percentages of 1000 replicates). Values lower than 40 % are replaced with asterisks (*). Dashes (-) represent minor differences between the BI and ML tree topologies. Bar, 2 substitutions per 100 nucleotide positions. http://ijs.sgmjournals.org

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Description. Size in vivo 90–160620–40 mm, usually about 145635 mm, mean 140630 mm in protargol preparations.

Body very flexible, slenderly lanceolate with a tail-like posterior body portion, body never spiralled, dorsoventrally flattened about 2 : 1, acontractile (Figs 1a–c and 2a–d, g, h; Table 1). Nuclear apparatus in middle third of cell slightly left of mid-line, composed of two macronuclear nodules and two to four micronuclei (Fig. 1a, b; Table 1). Macronuclear nodules broadly to narrowly ellipsoidal, mean 1567 mm in protargol preparations; contain many small globular nucleoli, 2–5 mm across. Micronuclei usually attached to macronuclear nodules, globular to ellipsoidal, mean 3.762.6 mm in protargol preparations. Contractile

vacuole slightly ahead of mid-body at left cell margin (Fig. 2b). Cortical granules absent. Cytoplasm colourless, filled with cytoplasmic granules about 2 mm in diameter and few fat droplets (Fig. 2d). Specimen in established culture with around 30–50 food vacuoles, mostly in mid-body and posterior region, containing the green alga Chlorogonium elongatum. Glides vividly on and between soil particles with great flexibility (Fig. 2a–c). Adoral zone about 26 % of body length, composed of a mean of 30 membranelles, with cilia about 16 mm long in vivo. Distal end of adoral zone extends 7–12 mm posteriorly, resulting in a DE value of about 0.26 (n511). Paroral

OP

(a)

(b)

VI

V

VI

II

II

I

I

(c)

(d)

Fig. 4. Photomicrographs of protargol-impregnated specimens of P. plestiensis showing morphogenetic events on the ventral surface. (a) Arrowheads point to the proliferating oral primordium (OP). (b, c) The arrow in (b) points to the disaggregating buccal cirrus and the arrow in (c) points to disaggregating cirrus III/2; arrowheads point to anlage VI. (d) Numbers I–VI denote six anlagen; the arrowhead points to the marginal anlagen. For details, see Figs 1(d–g) and 5(a). Bars, 30 mm (a) and 40 mm (b–d). 2630

International Journal of Systematic and Evolutionary Microbiology 64

Pseudouroleptus plestiensis n. sp.

and endoral membranes of about equal length intersect optically in posterior third (Fig. 1b). Invariably three frontal cirri, slightly enlarged, about 15 mm long in vivo; right cirrus posterior to distal end of adoral membranelle, left cirrus anterior of distal end of undulating membranes. One buccal cirrus, about 3 mm distant from the right of anterior end of undulating membranes and one enlarged fronto-ventral cirrus (5III/2) behind right frontal cirrus. One post-peristomial cirrus (5IV/2) behind buccal vertex. Left fronto-ventral row composed of about 36 cirri, commences at the right body margin behind the distal end of adoral zone and extends obliquely to left body margin, terminating at about 74 % of the cell length. Right fronto-ventral row consists of a mean of five cirri forming a shortened row right of rear portion of left fronto-ventral row; distance between cirri decreases posteriorly. Ventral cirri about 10 mm long in vivo. Marginal rows end terminally; right marginal row extends onto dorso-lateral surface anteriorly. Marginal cirri about 14 mm long in vivo.

II

Dorsal bristles 3.5–4.5 mm long in vivo. Four dorsal kineties (Figs 1c and 2h): first and second rows bipolar with 10–16 and 17–27 dikinetids, respectively; the third row begins anteriorly (i.e. about 7 mm from anterior body end), shortened posteriorly and ending in about mid-body, composed of 9–15 dikinetids; fourth row commences at beginning of second quarter of body (i.e. about 32 mm from anterior body end), terminates subapically, composed of 8–17 dikinetids. Caudal cirri inconspicuous in vegetative cells, i.e. easily confused with the last cirri of the marginal rows (Fig. 1c; Table 1). Resting cyst. Four-day-old resting cyst about 40 mm in diameter in vivo; cyst wall about 1.1 mm. Cyst contents separated from the wall by a distance of about 8 mm. Two-week-old resting cysts about 20 mm in diameter in

II I

I III

III

PM+EM

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IV VI

Distance between cirri of marginal rows increases slightly in posterior quarter of body (Figs 1a–c and 2g, h).

VI

PM+EM

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VI

V

IV

II

V

V

I

III II I

III

VI

AZM

V VI

AZM

IV VI

V

(a)

(b)

(c)

Fig. 5. Line diagrams of protargol-impregnated mid- and late-dividers of P. plestiensis showing morphogenetic events on the ventral surface. Numbers I–VI denote six newly formed anlagen for the two daughter cells. (a, b) Two sets of anlagen form, one each for proter and opisthe (a); arrowheads mark the anlagen of the marginal rows at two levels and arrows in (b) mark the right fronto-ventral row formed from anlage VI. Anlagen I–VI produce one, two, two, four, 15 and 17 cirri on average (n57). (c) Anlage V migrates toward the posterior end of the cell. The anterior portion of anlage VI, the anterior two to four cirri of anlage IV and anlage V align to form the left fronto-ventral row, which is thus a mixed row; arrowheads point to the post-peristomial cirrus and arrows point to the right fronto-ventral cirral row. AZM, Adoral zone of membranelles; EM, endoral membrane; PM, paroral membrane. Bars, 40 mm. http://ijs.sgmjournals.org

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vivo; cyst wall about 1.6 mm. Cyst contents attached to the wall from one side, composed of lipid droplets and fused macronucleus (Fig. 2e, f).

the post-peristomial cirrus (Figs 1d and 4a). Two streaks arise from the oral primordium in a Y-shaped pattern and move anteriad. The left streak gives rise to anlage II of the opisthe, and the anterior portion of this streak joins the disaggregating cirrus II/2 and forms anlage II of the proter. The right streak forms anlage III of the opisthe, while the disaggregating cirrus III/2 forms anlage III of the proter (Figs 1e, f and 4b). Anlage I for the opisthe develops from the base of the Y-shaped streaks, just above the new membranelles, while anlage I for the proter is formed from the partially disaggregated parental paroral and endoral membranes (Figs 1g and 4c). Anlagen V and VI for both daughter cells are formed from the two streaks that arise from the left fronto-ventral row, and the streaks extend left and right of the left fronto-ventral row. Anlage IV for both daughter cells develops from the anterior row of basal bodies that split from the left streak (Figs 1f, g and 4b–d). The right fronto-ventral row is not involved in primordium formation. The parental adoral zone is retained for the proter, while the adoral zone of membranelles of the opisthe is formed from an oral primordium. The buccal cirrus,

SSU rRNA gene sequence and phylogeny. The SSU

rRNA gene sequence of P. plestiensis is 1626 bp long and has a DNA G+C content of 45 mol%. Phylogenetic analyses inferred from SSU rRNA gene sequences using BI and ML present similar topologies; therefore, only the BI tree is shown here (Fig. 3). Phylogenetic trees consistently place the novel species within the non-stylonychine oxytrichids; clustering in a clade with P. caudatus and the genus Stronglydium. Divisional morphogenesis. Divisional morphogenesis

resembles that of the type species P. caudatus (for review, see Berger, 1999). Cell division begins with the proliferation of basal bodies behind the parental adoral zone of membranelles, close to 8–13 cirri of the left fronto-ventral row. These basal bodies give rise to the oral primordium, which extends anteriorly from the mid-body and incorporates

VI

IV VI VI

V

V

V VI

VI

VI

V V

(a)

(b)

(c)

Fig. 6. Photomicrographs of protargol-impregnated specimens of P. plestiensis showing morphogenetic events on the ventral surface. (a) Numbers I–VI denote six newly formed anlagen for the two daughter cells. (b, c) Arrowheads point to the postperistomial cirrus and arrows point to the right fronto-ventral cirral row. For details, see Fig. 5. Bars, 40 mm. 2632

International Journal of Systematic and Evolutionary Microbiology 64

Pseudouroleptus plestiensis n. sp.

parabuccal cirrus (5III/2), post-peristomial cirrus, the left fronto-ventral row and the undulating membranes are involved in the formation of six primordial streaks (Figs 1d– g, 4a–d, 5a–c and 6a–c). The left fronto-ventral row originates from the cirri of streaks VI (anterior portion), IV (middle portion) and V (rear portion), which later align to form a mixed row (Figs 5a–c and 6a–c). The marginal primordia arise at two levels by ‘within-row’ anlagen formation by utilizing one or two of the parental cirri. The marginal primordia elongate by utilizing four or five parental cirri and differentiate into new marginal rows. The remaining parental cirri are resorbed (Figs 5a–c and 6a–c). On the dorsal surface, two anlagen are formed within-row from dorsal kineties 1 and 2 at two levels (one set each for the proter and the opisthe). The third dorsal anlage for the proter and opisthe originates de novo, and later fragments in the middle, giving rise to the third and fourth dorsal kineties of nearly equal length. One to two caudal cirri arise

at the posterior end of each of the new kineties 1 and 2. No caudal cirri were observed at the posterior end of dorsal kinety 4 in the post-dividers (n511) (Figs 7a–c and 8a–d). Nuclear division proceeds as usual. The macronuclear nodules fuse to form a single mass in the middle dividers, which divides twice to produce the typical four nodules in the late dividers. The micronuclei undergo typical mitotic division (Fig. 7b, c). Occurrence and ecology. The novel species was identified

in soil samples collected from the ‘Molinaccio’ site during the summer (dry season), where it was moderately abundant in non-flooded Petri dish culture. The main soil physicochemical parameters that were measured at the time of sampling were as follows: soil moisture, 24.3 %; soil temperature, 18.8 uC; soil pH, 7.1. Other ciliate species identified in the same soil sample were Blepharisma hyalinum, Chilodonella uncinata, Colpoda cucullus, Colpoda

RMR

RMR

RMR

DK1

DK1 MI

DK1

MA MA

DK1 DK1

MI

(a)

(b)

(c)

Fig. 7. Line diagrams of protargol-impregnated specimens of P. plestiensis showing morphogenetic events on the dorsal surface. (a) Two anlagen are formed within-row from dorsal kineties 1 and 2 at two levels, one set each for the proter and opisthe, and the third dorsal anlage for the proter and opisthe originates de novo and then fragments to give rise to the third and fourth dorsal kineties. (b, c) Caudal cirri are formed at the posterior end of dorsal kineties 1 and 2 (arrowheads); arrows point to the fragmentation of dorsal kinety 3. Macronucleus and micronucleus division takes place (b) and the macronucleus divides a second time (c). DK, Dorsal kineties; MA, macronucleus; MI, micronucleus; RMR, right marginal row. Bars, 40 mm. http://ijs.sgmjournals.org

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RMR DK1

RMR

RMR DK1

DK1 DK1

(b)

(a)

(c)

(d)

Fig. 8. Photomicrographs of protargol-impregnated specimens of P. plestiensis showing morphogenetic events on the dorsal surface. (a) Within-row formation of the anlagen at two levels for dorsal kineties 1 and 2 and de novo formation of the third anlage for the proter and opisthe. (b) Segment showing the formation of two caudal cirri at the posterior end of dorsal kineties 1 and 2 (arrowheads). (c, d) Arrows point to the fragmentation of dorsal kinety 3. For details, see Fig. 7. DK, Dorsal kineties; RMR, right marginal row. Bars, 40 mm.

steinii, Cyrtohymena muscorum, Gonostomun affine, Halteria grandinella, Notohymena rubescens, Oxytricha lanceolata, Sterkiella cavicola, Sterkiella tricirrata and Tetrahymena rostrata.

and 50 in P. caudatus caudatus) and the location of the caudal cirri (at the posterior end of dorsal kineties 1 and 2 vs also at the posterior end of dorsal kinety 4) (Hemberger, 1985; Foissner et al., 2002; Berger, 2008).

P. caudatus Hemberger, 1985, the only known species of the genus, consists of two subspecies, P. caudatus caudatus Hemberger, 1985 and P. caudatus namibiensis Foissner et al., 2002, which differ in the number of right frontoventral (5transverse) cirri. The present species can be separated from the two subspecies mainly by the absence of cortical granules (vs presence), smaller body (mean 140 mm vs 200 mm), a smaller number of cirri in the right fronto-ventral row (mean 5 vs 25 in P. caudatus namibiensis

P. plestiensis differs from Hemiamphisiella granulifera (Foissner, 1987b) Foissner, 1988 by the absence (vs presence) of cortical granules and by having four (vs three) dorsal kineties. P. plestiensis is distinguished from Hemiamphisiella wilberti (Foissner, 1982) Foissner, 1988 by having 26–32 (vs 42–53) adoral membranelles, 27–40 (vs 55–72) cirri in the left fronto-ventral row and 3–7 (vs 15–27) cirri in the right fronto-ventral row. P. plestiensis differs from Hemiamphisiella quadrinucleata (Foissner, 1984) Foissner, 1988 and Hemiamphisiella terricola Foissner, 1988 mainly in the number of macronuclear nodules (invariably two vs a mean of four in H. quadrinucleata and 8–35 in H. terricola) (for the revision of the genus Hemiamphisiella, see Berger, 2008).

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DISCUSSION Comparison with related species and genera

Pseudouroleptus plestiensis n. sp.

Some species of the genus Hemiamphisiella Foissner, 1988 exhibit a cirral pattern similar to that of Pseudouroleptus species, and they can be easily confused (Berger, 2008). Furthermore, the absence of morphogenetic data on the dorsal ciliature, that is whether dorsal kinety 4 is formed by fragmentation or represents a dorsomarginal row, calls the taxonomic position of some species of Hemiamphisiella into question (Berger, 2008). However, it can be recognized from the illustrations and by taking examples from oxytrichids that, if fragmentation occurs, dorsal kinety 4 starts behind compared to other kineties and terminates close to the posterior body end. In the present species, the distance between the anterior body end and the beginning of dorsal kinety 3 is about 7 mm and that of the dorsal kinety 4 is about 32 mm. This difference in the distance, which results from the fragmentation, can also be established in P. caudatus caudatus and P. caudatus namibiensis from the illustrations provided. In the case of the dorsomarginal row, the leftmost row begins at the same level as or even anterior to the other kineties and generally stops before the posterior body end; therefore, considering the observations mentioned above and the detailed discussion by Berger (2008), we agree that some species of the genus Hemiamphisiella are perhaps misclassified. Phylogenetic analyses The SSU rRNA gene sequence of P. plestiensis matches well with that of P. caudatus caudatus, the only subspecies of the genus for which the sequence is available. In the phylogenetic analyses, P. plestiensis is clustered with P. caudatus caudatus and species of Strongylidium Sterki, 1878 with the full support of 1.0 BI (Fig. 3). Strongylidium orientale Chen et al., 2013 and Strongylidium pseudocrassum Wang and Nie, 1935 are clustered with P. caudatus caudatus with low support of 0.58 BI. The interphase morphology of these two genera is similar, but their ontogenesis is quite different. Specifically, the right fronto-ventral row is formed from the sixth fronto-ventral cirral anlage in Pseudouroleptus, whereas it is formed intrakinetally in Strongylidium (Paiva & Silva-Neto, 2007; Chen et al., 2013). Additionally, the dorsal kinety fragments in Pseudouroleptus, whereas it forms de novo without fragmentation in Strongylidium. Our analysis demonstrates that the genus Pseudouroleptus is currently non-monophyletic; therefore, we anticipate the addition of novel species and related molecular data to lead to a clearer picture of the phylogenetic relationships within this group. Morphologically, the novel species is closely related to the species of the genus Hemiamphisiella. Unfortunately, no molecular data are available for species of this genus.

Minister of University and Research (MIUR) to A. L. T. The authors would like to thank Dr Mauro Tiberi, Dr Giovanni Ciabocco and Dr Cristina Bernacconi from Osservatorio Regionale Suoli (http://suoli. regione.marche.it/) and Dr Emilio Insom and Dr Silvia Marinsalti from University of Camerino for their help in sampling.

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